Lionel Verdoucq
email: verdoucq@supagro.inra.fr

Alexandre Grondin

Structure-function analysis of plant aquaporin AtPIP2;1 gating by divalent cations and protons

Keywords: Aquaporin, calcium, proton, proteoliposome, structure-function, regulation aquaporin Ea : activation energy k exp : exponential rate constant PIP : Plasma membrane Intrinsic Protein P f : osmotic water permeability OTG : octyl-β-thioglucopyronoside

Water channel proteins (aquaporins) of the Plasma membrane Intrinsic Protein (PIP) subfamily provide means for fine and quick adjustments of the plant water status. A molecular model for gating of PIPs by cytosolic protons (H + ) and divalent cations was derived from the atomic structure of spinach SoPIP2;1 in an open-and a closed-pore conformation. Here, we produced in Pichia pastoris the Arabidopsis AtPIP2;1 homolog, either wild-type (WT) or with mutations at residues supposedly involved in gating. Stopped-flow spectrophotometric measurements showed that, upon reconstitution in proteoliposomes, all forms function as water channels. First functional evidence for a direct gating of PIPs by divalent cations was obtained. In particular, cadmium and manganese were identified, in addition to calcium (Ca 2+ ) and H + as potent inhibitors of WT AtPIP2;1. Our data further show that His 199 , the previously identified site for H + sensing, but also N-terminal located Glu 31 , and to a lesser extent Asp 28 , are involved in both divalent cations and H + -mediated gating. By contrast mutation of Arg 124 rendered AtPIP2;1 largely insensitive to Ca 2+ while remaining fully sensitive to H + . The role of these residues in binding divalent cations and/or stabilizing the open or closed pore conformations is discussed.

INTRODUCTION

Water is crucial for life and in most organisms its diffusion across membranes is facilitated by aquaporins (AQP), a conserved family of channel proteins [START_REF] Agre | Aquaporin null phenotypes: The importance of classical physiology[END_REF]. AQPs assemble as tetramers, each of the monomers defining an individual pore. These monomers exhibit six membrane-spanning α-helices tilted along the plane of the membrane and connected by five loops (A-E) [START_REF] Fujiyoshi | Structure and function of water channels[END_REF]. In all AQPs, loops B and D, as well as the N-and C-terminal tails, are bathing in the cytoplasm. To achieve constant adjustment of membrane water permeability in a fast, fluctuating environment, cells have developed multiple controls of AQP function. The regulation of AQP gating, i.e. the opening and closing of the pore, allows a rapid and reversible modulation of water transport. In plants, several cellular effectors can affect the intrinsic water permeability of plasma membrane AQPs of the Plasma membrane Intrinsic Protein (PIP) subfamily which, therefore have emerged as a model for studying AQP gating. The water permeability of Arabidopsis plasma membrane vesicles was shown to be reversibly inhibited by protons (H + ), with a half inhibition at pH 7.2 [START_REF] Gerbeau | The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH[END_REF]. A further structure-function analysis identified a His residue conserved in cytoplasmic loop D of all PIPs as central for pH-dependent gating [START_REF] Tournaire-Roux | Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins[END_REF]. This study and the determination of the atomic structure of spinach SoPIP2;1 in an open and a close state [START_REF] Tornroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF] also indicated a possible role for residues of loop D adjacent to the His [START_REF] Tournaire-Roux | Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins[END_REF] or for residues located in the N-terminal tail [START_REF] Tornroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF]. In particular, structural models of SoPIP2;1 suggested that at acidic pH, the channel is maintained in a closed-state by an ionic interaction between the protonated His 193 residue of loop D and the carboxylic sidechain of Asp 28 , located in the N-terminus of the protein. During pHinduced closure, displacement of loop D by 16Å moves the sidechain of Leu 197 into the lumen of the pore, thereby creating an hydrophobic barrier, and a pore constriction that prevents water permeation.

The activity of water channels in the Arabidopsis plasma membrane can also be inhibited by calcium (Ca 2+ ), with an IC50 of 75 µM [START_REF] Gerbeau | The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH[END_REF]. A recent study on plasma membranes from Beta vulgaris storage roots confirmed these observations [START_REF] Alleva | Plasma membrane of Beta vulgaris storage root shows high water channel activity regulated by cytoplasmic pH and a dual range of calcium concentrations[END_REF] and identified both a high (IC50 of 5 nM) and low (IC50 of 200 µM) apparent affinity component for inhibition of membrane water permeability by Ca 2+ . Recent crystallization of SoPIP2;1 in the presence of 0.1 M CdCl 2 and determination of a structure of the protein in a closed state provided further insights into the gating mechanisms induced by divalent cations, such as Cd 2+ and presumably Ca 2+ [START_REF] Tornroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF]. In this structure, Asp 28 and Glu 31 sidechains directly interact with Cd 2+ . Upon binding, Cd 2+ mediates a conformational change of loop D and closure of the pore by a network of hydrogen bonding, involving the cytoplasmic sidechain of Arg 118 in loop B and backbone carbonyl groups of Arg 190 and Asp 191 in loop D. Although Ca 2+ or other divalent cations may substitute for Cd 2+ in this model, the defined effects of divalent cations in terms of apparent inhibition constants have remained undefined. As a consequence, the prevalent role proposed for Ca 2+ in inhibiting water channels in vivo has remained uncertain.

In the present work, we performed a structure-function analysis of PIP gating using water transport assays on a purified protein. AtPIP2;1 represents one of the most highly expressed PIPs in the roots and leaves of Arabidopsis. Wild-type (WT), and site-directed mutants of AtPIP2;1 were produced in the yeast Pichia pastoris, purified and reconstituted into proteoliposomes. The effects of a set of divalent cations on the water transport activity of the various AtPIP2;1 forms provided insights into the molecular basis of cation-dependent gating. Moreover, we showed that pH-and cation-dependent gating can be mechanistically dissociated in some specific mutants.

pastoris expression

The AtPIP2;1 cDNA was amplified by PCR using 2.5 units (U) of the high fidelity Isis DNA polymerase (MP BioMedicals) and the forward EcoRI-AtPIP2;1 and reverse AtPIP2;1-XhoI primers (table 1) containing the ATG and STOP codons, respectively. Site-directed mutants of AtPIP2;1, where Asp 28 , Glu 31 , Arg 124 or His 199 were substituted by an Ala residue, were obtained by PCR-mediated primer extension [START_REF] Ho | Site-directed mutagenesis by overlap extension using the polymerase chain reaction[END_REF] using the forward EcoRI-AtPIP2;1 and reverse AtPIP2;1-XhoI primers, and a pair of complementary mutated oligonucleotides as listed in table 1. PCR products were cloned into the P. pastoris vector pPICZB (Invitrogen) at EcoRI and XhoI restriction sites. After cloning, the sequence of WT or mutated AtPIP2;1 was systematically verified by sequencing (Genoscreen, Lille, France). P. pastoris was transformed by homologous recombination at the chromosomal AOX1 locus. For this, the pPICZB vectors containing WT or mutated AtPIP2;1 cDNAs were linearized with SacI and transferred into the strain X-33 (Invitrogen) using the electroporation method. Transformants were selected on 100 µg/ml zeocin YPDS plates (Invitrogen) according to the manufacter's instructions or on 500 µg/ml zeocin YPDS plates for isolation of "jackpot" clones.

Production of AtPIP2;1 in P. pastoris

Yeast transformants were selected based on an initial small-scale protein production assay [START_REF] Karlsson | Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris[END_REF]. Briefly, after an initial overnight preculture in a glycerol-containing BMGY medium (Invitrogen), cells were inoculated at 22°C (OD 600 = 1.0) in a methanol-containing BMMY medium (Invitrogen) in baffled flasks with shaking at 225 rpm, to induce recombinant protein expression. Cells were harvested after 24 h and total membrane proteins were extracted as previously described [START_REF] Daniels | In vivo functional assay of a recombinant aquaporin in Pichia pastoris[END_REF]. Immunodetection of AtPIP2;1 in the extracts [START_REF] Santoni | Methylation of aquaporins in plant plasma membrane[END_REF] was used to select transformants with the highest protein expression level. For large scale production, membranes were extracted using a procedure adapted from [START_REF] Gerbeau | The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH[END_REF] and [START_REF] Karlsson | Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris[END_REF]. All steps were realized at 4°C with all buffers supplemented by 1 % of a cocktail of yeast protease inhibitors (Sigma). Cells were resuspended in 5 ml/g cells of an extraction buffer (500 mM saccharose, 10 % glycerol, 20 mM EDTA, 20 mM EGTA, 50 mM NaF, 5 mM β-glycerophosphate, 1 mM phenantroline, 0.6 % PVP, 10 mM ascorbic acid, 5 mM DTT, 1 mM sodium orthovanadate, 50 mM Tris, pH 8) and homogenized by high pressure cavitation at 1200 bars in a Cell Disruptor (Constant System Ltd, Warwick, UK). The lysate was centrifuged for 10 min at 10000 g and the resulting surpernatant was further centrifuged for 35 min at 57000 g. The final microsomal pellet was resuspended in 330 mM saccharose, 2 mM DTT, 10 mM NaF, 5 mM potassium phosphate buffer, pH 7.8. Membranes were stripped by an urea/alkali treatment [START_REF] Karlsson | Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris[END_REF], resuspended in 10 % glycerol, 2 mM DTT, 10 mM NaF, 20 mM piperazine, pH 9.5, at a protein concentration of about 20 mg/ml and stored at -80°C until use.

Solubilization and purification of AtPIP2;1

Stripped membranes were diluted to a protein concentration of 2 mg/ml and solubilized in 3 % octyl-β-thioglucopyronoside (OTG; Anatrace, Maumee OH, USA). After 1 h at 28°C, unsolubilized material was pelleted at 100000 g for 30 min. AtPIP2;1 was purified by anion exchange chromatography using 1 ml HiTrap Sepharose HP columns (Amersham). After a first equilibration step in buffer A (0.5 % OTG, 10 % glycerol, 2 mM DTT, 20 mM piperazine, pH 9.5), solubilized proteins were injected into the column and unbound proteins were washed out by 5 ml of buffer A. The retained proteins were then eluted by a linear gradient from 0.1 to 0.65 M NaCl in 20 ml of buffer A. Fractions that were the most enriched in AtPIP2;1 were pooled and AtPIP2;1 was further concentrated by ultrafiltration on 50 kDa filters (Microcon, Millipore). Proteins concentration was determined by a BCA titration assay (Pierce).

Liposome and proteoliposome reconstitution

Purified AtPIP2;1 was reconstituted into vesicles of Escherichia coli polar lipid extract (Avanti Polar Lipids, Alabaster AL, USA). The lipids were dissolved in a reconstitution buffer (30 mM KCl, 20 mM Tris-Mes, pH 8.3) to a concentration of 2.5 mg/ml and OTG was added at a concentration of 1 % (w/v) to the preformed liposomes. Proteins were added to the liposome solution, at a lipid to protein ratio (LPR) between 16 and 66. After incubation at 28°C for 30 min, proteoliposomes were formed at room temperature by a detergent removal procedure using SM2 polystyrene beads (Biorad). The mass of beads was 30-times the mass of OTG present in the lipid solution [START_REF] Rigaud | Reconstitution of membrane proteins into liposomes[END_REF]. Vesicle size was normalized at 40°C to a mean diameter of 0.2 µm by serial extrusion of the proteoliposomes suspension through a polycarbonate filter (Mini-extruder, Avanti Polar Lipids). Control liposomes were prepared in the same way as the proteoliposomes, except that a pure reconstitution buffer instead of a protein sample was added. Probably due to variable experimental conditions (room temperature during liposome formation, quality/age of the Biobeads used for detergent removal, quality/age of detergent and lipids, …) we were not able to obtain the same specific activity for every reconstitution, even when working at the same LPR and with the same protein extract (Supplemental Table 1). This variability yielded an apparent discrepancy in calculated osmotic water permeability (P f ) values. We checked on a set of 6 independent reconstitutions of AtPIP2;1, all at LPR 33, with P f measurements done at pH = 8.3 and 6.0, that the calculated percentage of AtPIP2;1 P f inhibition is not dependent on P f , that is on the reconstitution efficiency of the protein into proteoliposomes. Therefore, we decided to express in the manuscript the results as percentage of initial P f . Because divalent cations are not diffusible through lipid bilayers, a modified procedure was required to study their effects on vesicle water transport. In brief, the newly reconstituted vesicles were loaded with 0.6 M glycerol for 2 h and diluted 20-fold in a reconstitution buffer complemented with the indicated concentration of divalent cations. The hypo-osmotic shock associated with vesicle dilution induces a transient opening of vesicles and equilibration of their interior with the extravesicular solution [START_REF] Biber | Phosphorylation of rat kidney proximal tubular brush border membranes. Role of cAMP dependent protein phosphorylation in the regulation of phosphate transport[END_REF]. Resealed vesicles were then extruded at 0.2 µm as described above.

Stopped flow spectrophotometry

Kinetics of vesicle volume adjustment were followed by 90° light scattering at λ ex = 515 nm. Measurements were performed at 15°C, in a SFM3 stopped-flow spectrophotometer (Biologic, Claix, France) essentially as previously described [START_REF] Maurel | Purified vesicles of tobacco cell vacuolar and plasma membranes exhibit dramatically different water permeability and water channel activity[END_REF]. Briefly, membranes were diluted 10-fold into the reconstitution buffer (105 mosmol.kg -1 H 2 O). Alternatively, membranes were diluted in the same medium but adjusted at a modified pH and incubated for 2 h at room temperature. Vesicles were then loaded in the stopped-flow device and mixed (dead time <3 ms) with an equal volume of the reconstitution buffer but with a concentration of 270 mM mannitol (392 mosmol.kg -1 H 2 O). This resulted in a 144 mosmol.kg -1 H 2 O inward osmotic gradient. The traces from ≥10 individual stopped flow acquisitions were averaged and the curves were fitted to single exponential equations to determine an exponential rate constant k exp . The osmotic water permeability coefficient (P f ) was computed from the light scattering time course and the size of membrane vesicles according to the following equation: where V o is the initial mean vesicle volume, A v is the mean vesicle surface, V w is the molar volume of water, and C out is the external osmolality [START_REF] Maurel | Purified vesicles of tobacco cell vacuolar and plasma membranes exhibit dramatically different water permeability and water channel activity[END_REF]. Because they were extruded, all vesicle preparations were considered to have a mean diameter of 0.2 µm.

P f = k exp .V o /A v .V w . C out

Statistics

Statistical analyses were performed using a Statistica 7 software (StatSoft). Data were analysed using parametric (ANOVA followed by Newman-Keuls tests) and, in case of small samples (n ≤6), non parametric (Mann and Whitney) statistical tests. Results are means ± S.E.M. Differences were considered significant at p <0.05.

RESULTS

Production of AtPIP2;1 in P. pastoris

In order to get hundreds of micrograms of purified AtPIP2;1, production in P. pastoris had first to be optimised. We first observed that when P. pastoris transformed strains were grown in the presence of methanol, maximum AtPIP2;1 levels as monitored by western blots on total protein extracts were reached after 24 h of growth while biomass still increased for an extra day (results not shown). In addition, standard selection of P. pastoris strains on 100 µg/ml zeocin led to 60-70 % clones being positive for protein expression. Yet, we selected "jackpot" strains at 500 µg/ml zeocin which, although much more rare, showed a much higher level of AtPIP2;1 expression than clones selected in standard conditions (results not shown).

Because of our interest in the mechanisms of AtPIP2;1 gating, we produced a native, untagged form of the protein. Treatment of stripped membranes with OTG was able to fully solubilize AtPIP2;1 and yielded a protein extract with very few apparent contaminant proteins, as shown by Coomassie blue staining (results not shown). The solubilized protein sample was then fractionated by anionic exchange, and fractions eluted with 0.36-0.42 M NaCl contained most of the AtPIP2;1 protein which was further concentrated (Figure 1). Mass spectrometry analysis of a tryptic digest of the 28 kDa band shown in Figure 1 revealed that the 8 most abundant peptides corresponded to AtPIP2;1 (results not shown). An overall yield of ~65 µg AtPIP2;1 per liter of culture was obtained using this optimized expression procedure.

Functional reconstitution of AtPIP2;1 in proteoliposomes

AtPIP2;1 was reconstituted in proteoliposomes at LPRs of 16, 33 and 66 and the water transport properties of the proteoliposomes were compared to those of control E. coli liposomes by stopped-flow spectrophotometry. Figure 2 shows light-scattering recordings of the kinetics of liposome volume adjustment in response to a hypertonic challenge imposed at t =0 in a buffer at pH 8.3. The rate constant (k exp ) of the exponential curve fitted to the experimental light scattering recordings was 89.1 ± 2.6 s -1 , 60.6 ± 4.0 s -1 and 21.3 ± 0.9 s -1 for proteoliposomes with LPRs of 16, 33 and 66, respectively (Figure 2). Corresponding osmotic water permeability (P f ) values of 691.6 ± 20.2 µm.s -1 , 470.2 ± 31.0 µm.s -1 and 165.0 ± 7.0 µm.s -1 were calculated for the three types of proteoliposomes. Control liposomes were reconstituted, either with the reconstitution buffer alone or with proteins tentatively purified, in the same conditions as AtPIP2;1, but from untransformed control yeasts. The two control preparations displayed similar k exp values of 3.9 ± 0.1 s -1 and 4.3 ± 0.2 s -1 , respectively (Figure 2). These correspond to P f values of 30.6 ± 0.5 µm.s -1 and 33.7 ± 1.3 µm.s -1 , respectively.

Temperature dependence of water transport indicated activation energy (Ea) values of 17.4 kcal.mol -1 and 4.0 kcal.mol -1 for control liposomes and for AtPIP2;1 proteoliposomes reconstituted at LPR = 33, respectively (Supplemental Figure 1) The low P f and high Ea of control liposomes are typical of a lipid-mediated water transport. By contrast, the increased P f associated to a lower Ea of proteoliposomes containing AtPIP2;1 establish the water channel activity of the reconstituted aquaporin.

pH regulation of AtPIP2;1 water transport activity

To evaluate the H + -dependency of AtPIP2;1 mediated water transport, proteoliposomes reconstituted at pH 8.3 were diluted 10-fold with an iso-osmotic buffer at pH 6.0, and vesicles were osmotically challenged using an hyper-osmotic buffer also equilibrated at pH 6.0. While a short incubation time (~1 min) in the iso-osmotic buffer at pH 6.0 led to a 50 % decrease in P f , longer incubations (up to 2 h) gradually decreased P f to 8 % of its initial value (Supplemental Figure 2). These results suggest that AtPIP2;1 was inserted in the proteoliposomes with about one half of the proteins in an inside-out (cytoplasmic loop D outside) configuration which therefore could be instantaneously blocked by an extravesicular acidification. The timedependent inhibition of P f was interpreted as reflecting the time required for H + diffusion through the lipid bilayer and blockade of AtPIP2;1 proteins in an outside-out configuration. pHdependency of AtPIP2;1 activity was then tested after a 2 h incubation at 25°C to allow H + equilibration on both sides of the membrane. Figure 3 shows the effect of pH ranging from 3.8 to 9.5 on water transport of vesicles, either control or containing AtPIP2;1. P f of AtPIP2;1 proteoliposomes was maximal at pH >8 and was decreased by 99 % at pH 3.8, with a half inhibition at pH = 7.15 (± 0.15; n = 2). Hill coefficients deduced from fitted experimental curves were close to unity (h = 1.22 ± 0.23), suggesting a lack of cooperativity between individual monomers during pH-induced channel closure. The pH-dependency of the reconstituted AtPIP2;1 is consistent with measurements realized on plant plasma membrane vesicles or on Xenopus oocytes expressing AtPIP2;2 [START_REF] Gerbeau | The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH[END_REF][START_REF] Tournaire-Roux | Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins[END_REF][START_REF] Alleva | Plasma membrane of Beta vulgaris storage root shows high water channel activity regulated by cytoplasmic pH and a dual range of calcium concentrations[END_REF].

Regulation of AtPIP2;1 water transport activity by divalent cations

To evaluate the ability of divalent cations, and Ca 2+ in particular, to directly gate AtPIP2;1, 1 mM CaCl 2 was added directly on proteoliposomes in an iso-osmotic solution 3 min prior to the water transport assay. This treatment resulted in a ~50 % inhibition of P f (data not shown). To ensure that Ca 2+ had access to the interior of the vesicles and could block AtPIP2;1 in either orientation, proteoliposomes were submitted to a sudden hypo-osmotic shock in the presence of 1 mM CaCl 2 . This shock induces a transient opening of the vesicles and equilibration of their interior with the extra-vesicular solution [START_REF] Biber | Phosphorylation of rat kidney proximal tubular brush border membranes. Role of cAMP dependent protein phosphorylation in the regulation of phosphate transport[END_REF]. Under these conditions, the P f of AtPIP2;1 vesicles was reduced by 86 % (results not shown). We also checked that a similar hypotonic treatment in the presence of Ca 2+ did not alter the P f of control liposomes (results not shown). In experiments where the concentration of free Ca 2+ was varied between 0 and 5 mM (Figure 4), we observed a monophasic dose-response curve with an apparent IC50 of 42 ± 25 µM (n = 5). The fitted Hill coefficient was close to unity (h = 0.83 ± 0.11) suggesting a lack of cooperativity during the process of Ca 2+ -dependent P f inhibition. These data provide evidence that Ca 2+ can act directly on AtPIP2;1 to induce pore closure. Effects of a series of divalents cations on P f of AtPIP2;1 vesicles were compared at a fixed concentration of 150 µM (Figure 5). We tested divalent cations that either belong to the same alkaline earth metal series as Ca 2+ (Mg 2+ , Ba 2+ , Sr 2+ ), display a similar atomic radius (Mn 2+ ), or are known to bind to plant SoPIP2;1 (Cd 2+ ) or to regulate activity of animal AQPs (Ni 2+ ) [START_REF] Tornroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF][START_REF] Zelenina | Nickel and extracellular acidification inhibit the water permeability of human aquaporin-3 in lung epithelial cells[END_REF]. In addition, we tested the effects of mercury ions (Hg 2+ ) which have been reported as common AQP blockers [START_REF] Yukutake | Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes[END_REF]. Surprisingly, Hg 2+ was not able to inhibit the P f of AtPIP2;1 vesicles (Figure [START_REF] Tornroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF]. Mg 2+ did not alter significantly water transport in AtPIP2;1 vesicles either whereas Ca 2+ inhibited P f by 60 %. Sr 2+ , Ba 2+ and Ni 2+ reduced the P f by 40 %, 37 % and 48 %, respectively. Mn 2+ induced an inhibition of P f by 60 %, similar to Ca 2+ . Cd 2+ was the most effective cation with an inhibition of P f by 70 % (Figure 5). Dose-response experiments for inhibition of P f in AtPIP2;1 vesicles by Cd 2+ , Mn 2+ and Ni 2+ indicated apparent IC50 values of 27 ± 13 µM, 85 ± 37 µM and 178 ± 46 µM, respectively (Figure 6). This set of data clearly indicates that AtPIP2;1 can be blocked by a wide range of divalent cations with distinct sensitivities, Ca 2+ being one of the most efficient blockers.

Cytoplasmic determinants for regulation of AtPIP2;1 by divalent cations and pH

To examine the molecular bases of AtPIP2;1 sensitivity to H + or divalent cations, we investigated the effects of point mutations of four residues that may be involved in these regulations. We selected Asp 28 , Glu 31 , Arg 124 and His 199 , based on the possible role of the corresponding residues of SoPIP2;1, as was proposed from the atomic structures of the protein in an open and closed states [START_REF] Tornroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF]. The four residues were individually mutated to Ala residues and the resulting D28A, E31A, R124A and H199A forms were expressed in P. pastoris and purified as described for wild-type AtPIP2;1 (WT) (Supplemental Figure 4). In this series of experiments, functional reconstitution of the mutant or WT proteins sometimes failed at the highest protein content (LPR range: 16-33). Therefore, we analysed P f of proteoliposomes reconstituted at a lower protein content (LPR range: 33-40), which however ensured a very significant contribution of AQPs to P f (Supplemental Figure 3). When compared at similar LPR, the increased water channel activity of proteoliposomes containing the WT or mutant proteins indicated that no major change in intrinsic water permeability had been induced by either one of the introduced mutations (results not shown). The sensitivities of mutant and WT proteins to H + were compared at pH = 6.0 (Figure 7A) and at intermediate blocking conditions by divalent cations (Ca 2+ , Cd 2+ and Mn 2+ , all at 150 µM), to discriminate more easily between weakly and strongly affected mutants (Figure 7B). The R124A mutant showed an inhibition of P f by H + (84.4 ± 1.5 %), not significantly different to that of WT (83.7 ± 2.8 %) (Figure 7A). By contrast, the H199A mutant was almost insensitive to H + (5.5 ± 4.0 %) while the D28A and E31A forms showed intermediate P f inhibitory responses by 54.3 ± 4.4 % and 56.5 ± 4.6 %, respectively. These results confirm a central role for His 199 in H + -induced pore closure and provide the first functional data for a role of the N-terminal tail in this process.

In parallel experiments, the WT form showed a P f inhibition of 39.8 ± 8.1 %, 61.0 ± 5.8 % and 50.0 ± 7.7 % in response to 150 µM of Ca 2+ , Cd 2+ , and Mn 2+ , respectively. With respect to the WT form, the D28A mutant showed a tendency to lower inhibition by the three divalent cations (27.1 ± 8.8 %, 43.8 ± 9.8 % and 41.3 ± 6.8 %). By contrast, the E31A mutation had significant effects on Ca 2+ and Cd 2+ sensitivities, with a P f inhibition of 6.4 ± 7.1 % and 21.6 ± 14.2 %, respectively. Yet, the inhibition of P f by Mn 2+ (36.8 ± 7.9 %) was similar to that of the WT form. The R124A form was significantly affected in its P f sensitivity to all divalent cations and the percentages of inhibition by Ca 2+ (10.0 ± 5.7 %), Cd 2+ (18.7 ± 11.2 %) and Mn 2+ (26.3 ± 7.0 %) were all reduced by >2-fold with respect to those of the WT form. Finally, the H199A mutant also showed a very poor sensitivity to divalent cations with a P f inhibition of 3.9 ± 7.9 %, 11.6 ± 13.7 % and 4.9 ± 14.0 % in response to 150 µM of Ca 2+ , Cd 2+ and Mn 2+ , respectively. Dose-dependent effects of Ca 2+ , Cd 2+ and Mn 2+ on P f of liposomes (control) and proteoliposomes reconstituted with AtPIP2;1, either WT or mutant (D28A, E31A, R124A and H199A) forms are shown in Supplemental Figure 4. These experiments showed a relative insensitivity of the four mutants to high Ca 2+ concentration (1 mM). By contrast, a similar concentration of Cd 2+ and Mn 2+ was able to fully inhibit the R124A mutant form while being poorly active on D28A, E31A, and H199A. In conclusion, these analyses showed that all tested residues (Asp 28 , Glu 31 , Arg 124 , and His 199 ) are, although to different extent, involved in divalent cation-dependent gating of AtPIP2;1. Furthermore, substitution of Glu 31 by an Ala residue reveals a striking ability of the mutant protein to discriminate between distinct divalent cations.

DISCUSSION

The present work reports on the reconstitution in proteoliposomes of AtPIP2;1, one of the most highly expressed PIPs in Arabidopsis. The protein conferred on liposomes extremely high water permeability with P f levels exceeding 1000 µm.s -1 . Noticeably, these values are similar to those obtained with animal AQPs [START_REF] Liu | Purification and functional characterization of aquaporin-8[END_REF] and are the highest obtained for a reconstituted plant aquaporin [START_REF] Karlsson | Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris[END_REF][START_REF] Nyblom | 1042/BJ20080275 S t a g e 2 ( a ) P O S T -P R I N T Exceptional overproduction of a functional human membrane protein[END_REF]. PIP aquaporins have been described to be post-translationally regulated by several factors or modifications, such as H + , free Ca 2+ or Ser phosphorylation, all acting on the cytosolic side of the protein [START_REF] Gerbeau | The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH[END_REF][START_REF] Tournaire-Roux | Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins[END_REF][START_REF] Alleva | Plasma membrane of Beta vulgaris storage root shows high water channel activity regulated by cytoplasmic pH and a dual range of calcium concentrations[END_REF]18]. While functional expression of PIPs in Xenopus oocytes was instrumental for deciphering the regulation of water channel activity by H + and phosphorylation, structure-function studies addressing the regulation of PIPs by divalent cations have been lacking.

Here, we demonstrated that the water channel activity of AtPIP2;1 can be fully blocked by H + , with a half-inhibition at pH 7.15. In addition, AtPIP2;1 was shown to be inhibited by divalent cations, with the highest efficiency for Ca 2+ , Cd 2+ , and Mn 2+ . With respect to previous studies on plasma membrane vesicles purified from plants, the present work represents the first description of the direct inhibition of an individual PIP isoform by divalent cations. In particular, AtPIP2;1 showed an IC50 of 42 µM (± 25 µM) for Ca 2+ -mediated P f inhibition which is in good agreement with the IC50 of 75 µM observed for plasma membrane vesicles from Arabidopsis suspension cells [START_REF] Gerbeau | The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH[END_REF]. By contrast, Ca 2+ -dependent inhibition of P f in plasma membranes from Beta vulgaris storage roots [START_REF] Alleva | Plasma membrane of Beta vulgaris storage root shows high water channel activity regulated by cytoplasmic pH and a dual range of calcium concentrations[END_REF] showed both a high (IC50 ~ 5 nM) and a low (IC50 ~ 200 µM) apparent affinity component. Because of their conserved structure, all PIP isoforms are likely to be blocked by Ca 2+ but may differ in their sensitivity. The response of Beta vulgaris plasma membrane, which composition in AQPs is unknown, may reflect this heterogeneity. Because PIPs can be divided in two subclasses, PIP1 and PIP2, it will be interesting to determine the sensitivity of PIP1s to Ca 2+ inhibition. Surprisingly, AtPIP2;1 was not inhibited by mercury ions although it has the same conserved Cys residues as most other PIPs. Nevertheless, our observations are consistent with previous studies in Xenopus oocytes showing that AtPIP2;1 was insensitive to 1 mM HgCl 2 whereas four other Arabidopsis PIP isoforms (AtPIP1;1, AtPIP1;2, AtPIP1;3 and AtPIP2;2) were significantly blocked [START_REF] Kammerloher | Water channels in the plant plasma membrane cloned by immunoselection from a mammalian expression system[END_REF].

Because the in vitro inhibition of PIPs by divalent cations such as Ca 2+ or Cd 2+ exhibits a relatively low sensitivity, the physiological significance of this phenomenon has been questioned. Peak free Ca 2+ concentrations of a few µM have been measured in the plant cytosol [20]. However, free Ca 2+ may locally reach higher concentrations, especially in the close vicinity of Ca 2+ channels. In these respects, PIPs were found in detergent resistant membranes [START_REF] Mongrand | Lipid rafts in higher plant cells: purification and characterization of Triton X-100-insoluble microdomains from tobacco plasma membrane[END_REF][START_REF] Morel | Proteomics of plant detergent-resistant membranes[END_REF] where they may cluster with other membrane transport proteins. Exposure of plants to trace concentrations of Cd 2+ leads to accumulation of this highly toxic ion in plant cells and can affect the plant-water relations [START_REF] Poschenrieder | Influence of Cadmium on Water Relations, Stomatal Resistance, and Abscisic Acid Content in Expanding Bean Leaves[END_REF][START_REF] Perfus-Barbeoch | Heavy metal toxicity: cadmium permeates through calcium channels and disturbs the plant water status[END_REF]. Our results suggest that one basis of these effects may be a direct gating of PIPs by the ion. The atomic structure of spinach SoPIP2;1 in an open and a closed conformation [START_REF] Tornroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF] has provided a landmark advance for investigating the molecular determinants of PIP gating. The present structure-function approach provides a step forward and allows to confirm and refine the proposed model [START_REF] Tornroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF]. The atomic structure of SoPIP2;1 in the closed state showed a Cd 2+ ion in a close vicinity of the Asp 28 and Glu 31 residues [START_REF] Tornroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF]. Although the D28A mutant shows a tendency to a lower inhibition by divalent cations, our data establish a clear functional role for Glu 31 only. The data suggest that the latter residue is the main actor of this process, probably due to a higher flexibility of the Glu than the Asp sidechain [START_REF] Cates | Molecular mechanisms of calcium and magnesium binding to parvalbumin[END_REF]. The role of Glu 31 in primarily binding the ion is further supported by the finding that its mutation selectively altered Ca 2+ , and to a lesser extent Cd 2+ inhibition of P f , whereas it did not altered significantly Mn 2+ inhibition. This is in agreement with the notion that Ca 2+ usually displays higher coordination numbers than Cd 2+ or Mn 2+ [START_REF] Oberhauser | Activation by divalent cations of a Ca 2+ -activated K + channel from skeletal muscle membrane[END_REF]. Similar to Glu 31 , substitution of Arg 124 drastically altered gating of PIPs in response to Ca 2+ and Cd 2+ . Here, our results support the proposed role of this residue in bridging the Nterminal binding site of ions with loop D, in order to stabilize the closed conformation of the pore (Figure 8). We note however that, since Glu 31 and Arg 124 likely form hydrogen bonds [START_REF] Tornroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF], each of the two residues may actually contribute to both ion binding and stabilization of the closed-pore conformation. More unexpectedly, our analysis uncovered a role for His 199 in loop D in divalent cation dependent-gating. Here, the analysis of the open and the closed conformations is not giving a direct clue to explain this result. We propose that the mutation of this or neighbouring residues may alter loop D local structure and unstabilize the closed conformation, leading to a constitutively open pore.

The present work also provides insights into the mechanism of PIP gating by H + . Firstly, we confirmed, by an independent approach, that a conserved His in the loop D of all PIPs is a major H + sensor [START_REF] Tournaire-Roux | Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins[END_REF]. In view of the results discussed above, we cannot, however, rule out the hypothesis that substitution of His199 alters loop D local conformation, thus leading to an apparent H + -insensitivity. However, the half inhibition of P f at pH = 7.15 supports the hypothesis that residue with a near neutral pKa, that is a His residue, acts as sensor. From the atomic structure of SoPIP2;1 in a closed conformation, it was proposed that the protonated His would interact through a salt bridge with the carboxylic group of residue 28, found as a Glu or an Asp, in PIP1s and in PIP2s, respectively [START_REF] Tornroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF]. Our structure-function analysis confirms this interaction but also unravels a role for Glu 31 . Therefore, our study indicates a flexibility for the salt bridge which again was not deduced from the atomic structure. Interestingly, conserved charged residues adjacent of the loop D His (namely Arg 196 and Asp 197 in AtPIP2;1) can confer, after mutation to Ala, H + -insensitivity to the protein [START_REF] Tournaire-Roux | Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins[END_REF]. A closed-conformation model of AtPIP2;1 suggest that Asp 197 could make a salt bridge with the N-terminal Lys 33 , conserved in most PIP2s (Figure 8). Noticeably, this positively charged residue is not found in PIP1s and these AQPs may possibly exhibit a sensitivity to H + slightly different from that of PIP2s.

Changes in H + and free Ca 2+ concentrations in the cytosol are used by plant cells, independently or in combination to transduce developmental, hormonal or stress signals [27-30]. One well identified downstream effect of Ca 2+ is the stimulation of Ca 2+ -dependent protein kinases, which have been reported to act on PIPs [18]. Therefore, PIP gating is the converging point of multiple cell signalling mechanisms. The atomic structure of PIPs together with the present structure/function analysis show that the gating of PIPs by H + and divalent cations relies at least in part on common molecular mechanisms. The present work also allowed to identify mechanisms that are specific for divalent cation-dependent gating. In particular, we showed that, although Arg 124 is not directly involved in ion binding, a R124A mutant was largely insensitive to Ca 2+ while remaining fully sensitive to H + . The expression in transgenic plants of this type of Figure 5: Effects of a series of divalent cations on P f of liposomes (control) and proteoliposomes reconstituted with AtPIP2;1 (AtPIP2;1). Vesicles were equilibrated, as explained in the legend of Figure 4, with 150 µM of the indicated divalent cations, all provided as chloride salts, and at pH 8.3. P f was measured using the indicated procedure. Ø represents measurements in a buffer in the absence of any divalent cation. The figure shows (except for HgCl 2 ) representative data obtained on a same protein reconstitution experiment, at a LPR of 33. The absolute P f value of proteoliposomes in control conditions was 380.4 ± 41.5 µm.s -1 . Stopped-flow measurements were made in triplicate (n=3). Results as expressed as mean % (± SEM) of P f of untreated proteoliposomes. In parallel experiments (not shown), we checked that all ions investigated had no significant effect on the P f of control liposomes.

Biochemical

  Journal Immediate Publication. Published on 21 Jul 2008 as manuscript BJ20080275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080275 S t a g e 2 ( a ) P O S T -P R I N T EXPERIMENTAL Cloning of WT and site-directed mutants of AtPIP2;1 for P.

Biochemical

  Journal Immediate Publication. Published on 21 Jul 2008 as manuscript BJ20080275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080275 S t a g e 2 ( a ) P O S T -P R I N T

  Biochemical Journal Immediate Publication. Published on 21 Jul 2008 as manuscript BJ20080275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080275 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 21 Jul 2008 as manuscript BJ20080275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080275 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 21 Jul 2008 as manuscript BJ20080275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080275 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 21 Jul 2008 as manuscript BJ20080275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080275 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 21 Jul 2008 as manuscript BJ20080275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080275 S t a g e 2 ( a ) P O S T -P R I N T mutant will be instrumental to test for the physiological significance and specific contribution of divalent cations in AQP gating and regulation of the whole plant water status. Phosphorylation of PIP2s in loop B and in the C-terminal tail are also supposed to exert a control of pore opening and closing [18, 31]. Therefore, a next step to add to our approach will be to understand the interplays between the multiple phosphorylated states of PIPs and their direct gating by H + and Ca 2+ . Biochemical Journal Immediate Publication. Published on 21 Jul 2008 as manuscript BJ20080275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080275 S t a g e 2 ( a ) P O S T -P R I N T used for the mutation and cloning of WT and mutated forms of AtPIP2;1 in an pPICZ-B vector (Invitrogen), as described in the experimental section. All primers are given in the 5' to 3' orientation. Cloning sites are indicated in bold characters, mutated/inserted residues are indicated in italics and the start/stop codons are underlined. Biochemical Journal Immediate Publication. Published on 21 Jul 2008 as manuscript BJ20080275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080275 S t a g e 2 ( a ) P O S T -P R I N T

Figure 1 :

 1 Figure 1 : SDS-PAGE profile of purified AtPIP2;1. Protein extracts eluted from Hitrap Q HP columns were obtained from P. pastoris strains, either control or expressing AtPIP2;1. Proteins were stained by Coomassie brilliant blue R250. The two arrows represent monomeric and dimeric forms of AtPIP2;1.

Figure 2 :

 2 Figure 2: Stopped-flow light scattering recordings of water transport in liposomes and proteoliposomes containing AtPIP2;1 at different LPRs. All measurements were performed with solutions at pH 8.3. Imposition of a hyperosmotic gradient at t = 0 causes a shrinkage of the vesicles that can be monitored by a time-dependent change in light scattering. Control liposomes reconstituted with the liposome buffer alone (control) or with proteins tentatively purified from control yeasts (empty plasmid) show typically slow light scattering kinetics. Proteoliposomes reconstituted with increasing amounts of AtPIP2;1 (LPRs of 66, 33 and 16) show a dosedependent increase in the rate of vesicle volume adjustment. For each type of reconstitution, the traces from ≥10 individual stopped flow acquisitions were averaged and the curves were fitted to single exponential equations to determine an exponential rate constant k exp . Mean values are indicated in the text. The inset shows a close up of the recordings between 0 and 0.4 s.

Figure 3 :

 3 Figure3: Effects of pH on P f of liposomes and proteoliposomes containing AtPIP2;1. Control liposomes and proteoliposomes were equilibrated for 2 h at the indicated pH and stopped-flow measurements were performed, as exemplified in Figure2. P f was calculated from the exponential rate constants (k exp ) fitted from the time course of scattered light intensity and from the size of the extruded vesicles (diameter of 0.2 µm). The figure shows representative data obtained on a same protein reconstitution experiment at a LPR of 16, with stopped-flow measurements made in triplicate (n=4). Results are mean ± SEM.

Figure 4 :

 4 Figure4: Effects of Ca 2+ on P f of liposomes (control) and proteoliposomes containing AtPIP2;1 (AtPIP2;1). Vesicles were equilibrated for 3 h in 0.65 M glycerol, and subsequently submitted to a sudden hypo-osmotic shock in the presence of the indicated Ca 2+ concentrations at pH 8.3. This shock induces a transient opening of the vesicles and equilibration of their interior with the extravesicular solution. P f was determined by stopped-flow recordings as exemplified in Figure2. The figure shows representative data obtained on a same protein reconstitution experiment at a LPR of 33, with stopped-flow measurements made in triplicate (n=3). Results are mean ± SEM.

BiochemicalFigure 6 :

 6 Figure6: Dose-dependent effects of Cd 2+ , Mn 2+ and Ni 2+ on P f of proteoliposomes containing AtPIP2;1. Procedures and conventions are as in Figure4. The curve fitted from concentrationdependent effects of Ca 2+ on P f (see Figure4) is shown as a dotted line. Results are mean ± SEM.

Figure 7 :

 7 Figure7: Effects of H + and divalent cations on P f of proteoliposomes containing AtPIP2;1, either WT (WT) or with the indicated mutations (D28A, E31A, R124A, H199A). For all AtPIP2;1 forms a P f value was measured at pH =8.3, in the absence of divalent cations, and subtracted by the P f value of control liposomes (P f = 28.7 ± 1.8 µm.s -1 ). The derived P f values, which reflect the full water transport activity of each of the AtPIP2;1 forms in the reconstituted proteoliposomes were: WT, P f = 542.0 ± 56.1 µm.s -1 ; D28A, P f = 390.5 ± 104.1 µm.s -1 ; E31A, P f = 329.4 ± 92.1 µm.s -1 ; R124A: P f = 461.7 ± 71.3 µm.s -1 ; H199A P f = 270.8 ± 39.7 µm.s -1 . These P f values were used as a reference to calculate the relative P f inhibition observed in the indicated conditions. Proteoliposome equilibration and stopped-flow measurements were performed at pH 6.0 (A) or in the presence of 150 µM of indicated cations (B). Numbers indicate the number of independent proteoliposome reconstitutions, from at least three independent protein productions. For each reconstitution, H + -or divalent cation-dependent inhibition was characterized in triplicate stopped-flow assays. Results are mean SEM. *: differs from WT at p <0.05.

Figure 8 :

 8 Figure 8: Interpretative model of PIP2 gating by H + and divalent cations. Close-up view of the H + and divalent cation-binding site of a closed-conformation model of AtPIP2;1 generated by homology modelling from the SoPIP2;1 closed-conformation structure (PDB code 1Z98). The location of the divalent cation is shown in purple. Putative salt bridges between His 199 and Asp 28 , Glu 31 and Arg 124 , and between Asp 197 and Lys 33 side chains are represented. Numbers are distances in Å.

Figure 1 CFigure 2 AtPIP2Biochemical

 12 Figure 1

1 ) 1 )

 11 Figure 3

2

 2 Figure 5

Figure 8 Biochemical

 8 Figure 8 Biochemical Journal Immediate Publication. Published on 21 Jul 2008 as manuscript BJ20080275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080275

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Authors are grateful to Dr Véronique Santoni for MS analyses performed during the study. This work was supported in part by a grant from the French Ministry of Research (ACI2003 'Biologie du Développement et Physiologie Intégrative').


Maja Jensen

Jane Palsgaard

Rehannah Borup

Pierre De Meyts

Lauge Schäffer

Pierre De

Activation of the insulin receptor (IR) by insulin and a synthetic peptide has different effects on gene expression in IR-transfected L6 myoblasts

Keywords: Insulin mimetic peptide, gene expression profiling, L6 myoblasts, mitogenicity, MAPK pathway, signaling

Single chain peptides have been recently produced that display either mimetic or antagonistic properties against the insulin and IGF-1 receptors. We have earlier shown that the insulin mimetic peptide S597 leads to significant differences in receptor activation and initiation of downstream signaling cascades despite similar binding affinity and in vivo hypoglycemic potency. It is still unclear how two ligands can initiate different signaling responses through the insulin receptor (IR). To further investigate how the activation of the IR by insulin and S597 differentially activates post-receptor signaling, we here studied the gene expression profile in response to IR activation by either insulin or S597 using microarray technology. We found striking differences between the patterns induced by these two ligands. Most remarkable was that almost half of the genes differentially regulated by insulin and S597 were involved in cell proliferation and growth. Insulin either selectively regulated the expression of these genes or was a more potent regulator. Furthermore we found that half of the differentially regulated genes interact with the genes involved with the MAPK pathway. These findings support our signaling results obtained earlier and confirm that the main difference between S597 and insulin stimulation resides in the activation of the MAPK pathway. In conclusion we show that insulin and S597 acting via the same receptor differentially affect gene expression in cells resulting in a different mitogenicity of the two ligands, a finding which has critical therapeutic implications.

INTRODUCTION

The insulin receptor (IR) is a member of the large family of receptor tyrosine kinases and has a covalent dimeric α 2 β 2 -organization [START_REF] De Meyts | Structural biology of insulin and IGF1 receptors: implications for drug design[END_REF]. Activation of the IR initiates a cascade of intracellular signaling events, which regulate multiple biological processes such as glucose and lipid uptake/metabolism, gene expression/protein synthesis and cell growth, division and survival [START_REF] Taniguchi | Critical nodes in signalling pathways: insights into insulin action[END_REF]. The binding of insulin to its receptor is complex and binding models for insulin integrating the available data from structural and kinetic evidence have been proposed [3;4]. The complexity of the insulin binding mechanism makes the design of IR agonists especially difficult. Investigation of phage display libraries (ranging in size from 20 to 40 amino acids) have identified several peptides that bind to the IR [START_REF] Pillutla | Peptides identify the critical hotspots involved in the biological activation of the insulin receptor[END_REF]. These peptides bound three non-overlapping hot spots on the IR (Sites 1, 2 and 3). Site 1 and 2 seem to correlate with the two insulin binding sites on the IR, whereas the site 3 IR binding peptides seem to bind somewhere near the site 2 peptides possibly influencing the binding of these peptides. Whether or not these insulin mimetic peptides actually bind to the same sites as insulin on the IR is not known, but they are located close enough to be able to compete with insulin for binding [START_REF] Pillutla | Peptides identify the critical hotspots involved in the biological activation of the insulin receptor[END_REF]. Recently homodimers and heterodimers of the site 1 and 2 mimetics have been generated to create molecules that activate the insulin receptor with high potency and specificity -possibly through a mechanism similar to the binding of insulin [START_REF] Schäffer | Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks[END_REF]. From this selection a heterodimer (site 2-site1 mimetic) was found to lower the blood glucose level in rats with a potency similar to insulin's. Due to the smaller size and simpler structure of these insulin mimetic peptides, they are good candidates for drug development with potential use in the treatment of diabetes as a replacement for,-or in combination with, insulin, and as templates for the design of peptidomimetics. We have now studied such an optimized peptide (S597) in detail with regard to binding to the IR (isoform A) (manuscript in preparation) as well as the effect on IR phosphorylation and downstream signaling molecules [START_REF] Jensen | Activation of the insulin receptor by insulin and a synthetic peptide leads to divergent metabolic and mitogenic signaling and responses[END_REF]. S597 was found to bind selectively to the IR and not the closely related IGF-1 receptor. The binding of S597 to the IR is similar to insulin with respect to association rates, affinity and pH dependence of binding. However S597 dissociates very slowly from the IR, suggesting that it interacts with the receptor in a different way than insulin ( [START_REF] Jensen | Activation of the insulin receptor by insulin and a synthetic peptide leads to divergent metabolic and mitogenic signaling and responses[END_REF] and manuscript in preparation). Moreover we found that binding of S597 and insulin leads to significant differences in receptor activation and initiation of downstream signaling pathways [START_REF] Jensen | Activation of the insulin receptor by insulin and a synthetic peptide leads to divergent metabolic and mitogenic signaling and responses[END_REF]. S597 phosphorylates the same number of insulin receptors as insulin but to a lower degree. S597 can fully activate the PI3K/Akt pathway compared to insulin but only weakly activates the Shc/ERK pathway [START_REF] Jensen | Activation of the insulin receptor by insulin and a synthetic peptide leads to divergent metabolic and mitogenic signaling and responses[END_REF]. Furthermore S597 is able to stimulate glycogen synthesis to almost the same extent as insulin but has a much lesser effect on cell proliferation. It is still unclear how two ligands can initiate different signaling responses through the IR. To further investigate how the activation of the IR by insulin and S597 differentially activates post-receptor signaling, we here studied the gene expression profile in response to IR activation by either insulin or S597 using microarray technology.

EXPERIMENTAL The peptide, S597

The insulin mimetic peptide, S597, with the sequence Ac-SLEEEWAQIECEVYGRGCPSESFYDWFERQL-amide, was synthesized by standard solid phase peptide synthesis using Fmoc chemistry and purified by RP-HPLC after formation of the disulphide bridge. Insulin was from Novo Nordisk, Denmark

Cell culture

The rat L6 myoblast cell line stably transfected with the human insulin receptor (hIR) isoform A (L6-hIR) used in this study was kindly provided by Bo Falck Hansen, Novo Nordisk, Denmark. The cells were cultured in DMEM 21885-025 supplemented with 10% Fetal Bovine Serum, 100 U/ml Penicillin, 100 µg/ml Streptomycin and 0.5 mg/ml Geneticin. The cells were grown at 37 o C in 5 % CO 2 humidified atmosphere. Cells were passaged 2-3 times a week by trypsination and resuspended in fresh media at an appropriate dilution -usually 1:6 (all from Gibco, USA).

Preparation of cells, RNA and microarrays

L6-hIR cells were harvested, seeded out in 12 large Petri dishes and allowed to recover for 3 days. The cells were then starved for 24 hours and stimulated for 3 hours with 100 nM insulin, S597 or left unstimulated. RNA isolation, reverse transcription and cRNA synthesis, labeling and hybridization with Affymetrix Rat genome 230 2.0 GeneChip ® array (Affymetrix Microarray Suite; Affymetirx, Santa Clara, CA) were conducted following the standard affymetrix protocol (available at http://www.affymetrix.com/support/technical/manual/expression_manual.affx). In summary four GeneChip microarrays were hybridized with cells from the same passage for each experimental condition (insulin-, S597-or non-stimulated cells).

Microarray analysis

Quality control of the arrays was performed using the R package from Bioconductor and confirmed that the arrays were of good quality. The probe level data (CEL files) were transformed into expression measures using R (http://cran.at.r-project.org) and the gcrma package from BioConductor (http://www.bioconductor.org). Gene list comparisons were made in DChip [START_REF] Li | Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection[END_REF] (available at http://www.dchip.org) using a P value < 0.005, a fold change > 1.5 fold or 2 fold, and a lower confidence bound of fold changes for filtering. Furthermore the threshold for absolute difference between the two group means was set to 35. The P value was chosen as it gave a false discovery rate -of max. 3.3% calculated by running 100 permutations in dChip. Transcripts preferentially regulated by insulin (i.e. the genes that met the above mentioned selection criteria for insulin treated samples compared to untreated samples but not for S597 treated samples compared to untreated) (these are the results shown in figure 2) were determined using dChip by comparing the gene expression affected more than 1.5 and 2 fold by insulin and excluding transcripts that also were affected more than 1.5 and 2 fold by S597 (compared to untreated). Transcripts preferentially regulated by S597 were determined in the same way. Transcripts responsive to both ligands were identified using dChip by only including transcripts that were affected more than 1.5 fold by both ligands compared to untreated samples. As many genes are represented on arrays by multiple probesets the number of transcripts regulated by insulin and S597 given in figure 1 and 2 and, table 1 and 2 refer to the number of probesets found on the microarray and not number of unique genes. The transcripts responsive to insulin, S597 or both ligands are listed in supplemental table 2. Functional analyses of these transcripts were subsequently generated using Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com). Transcripts that met the fold change cut off of 1.5 and were associated with biological functions in the Ingenuity Pathways Knowledge Base were considered for the analysis. Fischer's exact test was used to calculate a p-value determining the probability that each biological function assigned to that data set is due to chance alone. The differentially regulated transcripts were identified in a direct comparison of the insulin-and S597-treated samples using dChip (listed in supplemental table 3). The transcripts regulated similarly by insulin and S597 were determined by comparing the gene expression affected by insulin and S597 compared to untreated, excluding transcripts already categorized as differentially regulated (listed in supplemental table 4).

Validation of genes using qRTPCR on biological replicates

Two-step qRT-PCR was performed on 13 selected genes to validate the microarray data. Cells from four different passages were stimulated at different days with 100 nM insulin, S597 or nothing and the RNA extracted as described above. The RNA was reverse transcribed into single-stranded cDNA according to the manufacturer's protocol using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science). The real-time RT-PCR assay was performed using FastStart TaqMan Probe Master (Rox) (Roche Applied Science). Probes were purchased from Universal ProbeLibrary and primer sequences designed using the ProbeFinder software (Universal ProbeLibrary, Roche). The primers used are listed in supplementary table 1 and were purchased from DNA-technologies, DK. Standard curves were generated using serial dilutions of cDNA. TaqMan PCR assays for each gene target were performed in triplicate on cDNA samples on an ABI 7900HT Prism sequence detection system (Applied Biosystems) according to manufacturers protocol (Roche Applied Science). 18S was used as an internal control and included on all plates. The data were analyzed using Sequence Detector Software (Applied Biostystems), the C T values normalized to S18 and fold changes calculated.

RESULTS

To analyze gene expression profiles following IR activation by either insulin or S597, L6 rat myoblasts stably transfected with the human IR were stimulated with either ligand (100 nM) for 3 hours. This cell line was chosen as skeletal muscle is one of the major targets for insulin action. The same concentration was used as S597 binds specifically and with a similar affinity as insulin to the IR isoform A [START_REF] Jensen | Activation of the insulin receptor by insulin and a synthetic peptide leads to divergent metabolic and mitogenic signaling and responses[END_REF]. Cells from the same passage were stimulated and harvested in quadruples. RNA was isolated and hybrized to Affymetrix GeneChip Rat Genome 230 2.0 Arrays according to Affymetrix procedure -four arrays per condition.

Cellular and molecular functions of transcripts affected by insulin and S597

We identified transcripts (i.e. probe sets) that were regulated between the unstimulated, insulin-or S597-stimulated samples, using a P value < 0.005 and fold changes > 1.5 fold or 2 fold as selection criteria (see Experimental for more detail). Using these selection criteria we found that insulin affected two (> 1.5 fold change) to four (> 2 fold change) times as many transcripts as S597 (figure 1). As many genes are represented on arrays by multiple probesets, the number of transcripts regulated by insulin refer to the number of probesets found on the microarray and not to the number of unique genes. Subsequently we focused on the transcripts that were regulated by insulin and S597 compared to untreated. These transcripts were divided into three groups: transcripts preferentially regulated by insulin, by S597 or by both insulin and S597 (figure 2, all transcripts are listed in supplemental table 2). The transcripts regulated by both ligands were defined as transcripts that met the above mentioned selection criteria for both insulin and S597 treated samples when comparing with untreated. In contrast transcripts preferentially regulated by insulin were defined as transcripts that only met the selection criteria when comparing insulin treated with untreated samples and not S597 treated samples. In this way transcripts preferentially regulated by insulin are transcripts that are significant regulated by insulin compared to untreated However this does not mean that there exists a significant difference in expression between insulin and S597 samples, only that S597 is not able to induce a fold change that is significant compared to untreated. Transcripts preferentially regulated by S597 were defined in the same way. An overview of the gene lists used in this study is shown in table 1. Subsequently the five most significant molecular and cellular functions associated with the three groups were investigated by performing a functional analysis using the Ingenuity Pathway Analysis software (Ingenuity® Systems, www.ingenuity.com) as described in Experimental. Not all the transcripts are annotated in Ingenuity and furthermore only a subset of these genes is eligible for functional analysis. The differences in significance as well as the number of unique genes associated with the cellular and molecular functions between the three groups are shown in table 2. The five most significant functions with which the transcripts preferentially regulated by insulin were associated were: Gene expression, cellular development, cellular growth and proliferation, cell cycle and cell death. Transcripts preferentially regulated by S597 were most significantly associated with: molecular transport, gene expression, amino acid metabolism, cell cycle and cell morphology. The most significant functions with which the third group, transcripts responsive to both insulin and S597, was associated were: cellular growth and proliferation, cell cycle, gene expression, cell death and cell morphology. This analysis indicates that the major functional themes associated with insulin and S597 stimulation are related to cellular growth and proliferation, gene expression and cell cycle.

Transcripts differentially regulated by insulin and S597

It is difficult to define if genes are differentially or similarly regulated by two ligands as no real biological selection criteria exist. As the aim of this study was to investigate in more detail how S597 and insulin initiate different responses through the IR we started by defining the selection criteria for the differentially regulated transcripts. We defined differentially regulated transcripts as transcripts where there is a significant (P value < 0.005) difference and a fold change in expression values of more than 1.5 fold between insulin and S597 samples. In this way we are certain that a significant difference exists between the insulin and S597 treated samples but without making assumptions about the effect compared to untreated. 131 transcripts (i.e. probesets) were found to be differentially regulated by insulin and S597. The annotated transcripts are listed in table 3 (the complete list (i.e. inclusive the ESTs) is in supplemental table 3). 77 of the differentially regulated transcripts responded (>1.5 fc) only to insulin. One transcript, an EST (affymetrix probe set 1377092_at) responded (>1.5 fc) only to S597 and was down-regulated. Using Blast (http://www.ncbi.nlm.nih.gov/) we searched for similar sequences. We found that this EST has a high sequence identity (93%) to a part of the mRNA of suppressor of cytokine signaling 3 (SOCS3) in mouse. However we could not confirm the presence of this transcript in the samples using qRT-PCR. Accordingly 58% of the differentially regulated transcripts are transcripts regulated by insulin > 1.5 fold but not by S597. No transcripts were found to be regulated in opposite directions by insulin and S597 compared to untreated. For the remaining transcripts S597 has a similar effect as insulin compared to untreated but to a lesser extent. The differentially regulated transcripts are involved in many functions but as many as 33 of the annotated (unique) genes are involved in growth and proliferation as found by using the Ingenuity Pathway Analysis software, dChip and PubMed searches (marked in table 3 and supplementary table 3). Twenty-two of these genes are selectively regulated by insulin. The rest of the genes involved in growth and proliferation are up-or down-regulated by insulin and to a lesser extent S597 compared to untreated. Analyzing the differential regulated annotated genes (using the Ingenuity Pathway Analysis software) we see that the changes in gene expression have both positive and negative effects on cell growth and cell proliferation (shown in table 3). The number of genes of which up-or down-regulation has a positive effect on cell growth and cell proliferation is larger than the ones having a negative effect. The analysis does not, however, provide information regarding the importance of individual genes for the resulting effect (positive or negative) on the function. Using the KEGG pathway database we also identified five genes involved in the MAPK signaling pathway: v-jun sarcoma virus 17 oncogene homolog (JUN), activating transcription factor ATF-4 (ATF4), dual specificity phosphatase 5 (DUSP5), dual specificity phosphatase 6 (DUSP6) and v-myc avian myelocytomatosis viral oncogene homolog (MYC). The proteins encoded by DUSP5 and DUSP6 are phosphatases that negatively regulate members of the MAPK superfamily (MAPK/ERK, SAPK/JNK, p38). The expression of DUSP5 is induced by VEGF and further regulated by KRAS, which both are found to be up-regulated in this study [START_REF] Fu | Growth retardation alters the epigenetic characteristics of hepatic dual specificity phosphatase 5[END_REF]. The expression of DUSP6 has been found to be regulated by MYC and IL-6 [START_REF] Zhao | The mechanism of dephosphorylation of extracellular signalregulated kinase 2 by mitogen-activated protein kinase phosphatase 3[END_REF] which both are up-regulated by insulin and to a lesser extent S597 in this study. The genes hexokinase 2 (HK2) and MYC are involved in the expression level of glucose-6phosphatase, a key-enzyme in glycolysis and gluconeogenesis [11;12]. HK2 and heparin-binding EGF-like growth factor (HBEGF) are involved in the metabolism of D-glucose [START_REF] Robey | Regulation of mesangial cell hexokinase activity and expression by heparin-binding epidermal growth factor-like growth factor: epidermal growth factors and phorbol esters increase glucose metabolism via a 15[END_REF]. These genes were all more up-regulated by insulin than by S597.

Transcripts regulated similarly by insulin and S597

We defined transcripts regulated similarly by insulin and S597 as transcripts of which expression was affected in the same direction and to a similar extent by both ligands compared to untreated samples (as described in Experimental). In this way only 139 transcripts were similarly regulated out of the 176 transcripts found above to be affected by both insulin and S597. The annotated transcripts (44 unique genes) are listed in table 4 (the complete list (i.e. inclusive the ESTs) is in supplemental table 4). The expression of the gene heme oxygenase 1 (HMOX1) was found to be much more up-regulated by insulin (14.84 fold) than by S597 (4.65 fold). Heme oxygenase is a microsomal enzyme which catalyzes the oxidation of heme to biliverdin and carbon monoxide [14]. The reason that it is not excluded from the similarly regulated genes in the microarray analysis is that the P value was not below 0.005. However, the fold changes were validated using qRT-PCR and showed that HMOX1 is indeed differentially regulated, with insulin being a much more potent inducer of HMOX1 expression than S597 (figure 4). The role of heme oxygenase in skeletal muscle is still unclear but it is involved in cellular protection against oxidative stress and regulation of heme turnover [15]. The MAPK signaling pathway is one of the major signaling pathways initiated after activation of the IR and is highly associated with cellular proliferation and differentiation and we have recently shown that S597 is not able to activate the MAPK pathway to the same extent as insulin [START_REF] Jensen | Activation of the insulin receptor by insulin and a synthetic peptide leads to divergent metabolic and mitogenic signaling and responses[END_REF]. Using the KEGG pathway database (http://www.genome.jp/kegg/pathway.html) we found two upregulated genes that are involved in the MAPK signaling pathway: RAS p21 protein activator 1 (RASA1) and Kirsten rat sarcoma viral oncogene homolog (KRAS). Using dChip, Ingenuity Pathway Analysis and PubMed Central searches we furthermore found that many of the transcripts that were regulated similarly by insulin and S597 are involved in growth and proliferation. Using the Ingenuity software it is possible to distinguish if the changes in expression influence a given function in a positive and negative sense. In many instances however is the effect of a specific gene unclear or unknown. Analyzing the similarly regulated transcripts we saw that the changes in gene expression have both positive and negative effects on cell growth and cell proliferation (shown in table 4). The analysis does not, however, provide information regarding the importance of individual transcripts for the resulting effect on the function.

Interactions between genes involved in the MAPK pathway

To investigate if the lack of activation of the MAPK pathway when stimulating with S597 [START_REF] Jensen | Activation of the insulin receptor by insulin and a synthetic peptide leads to divergent metabolic and mitogenic signaling and responses[END_REF] could explain some of the differences in gene expression induced by insulin and S597 we took a closer look at the similarly and differentially regulated genes that were involved in the MAPK pathway to see how they interact with the rest of the genes. We imported the similarly regulated genes KRAS and RASA1 as well as the differentially regulated genes JUN, ATF4, DUSP5, DUSP6 and MYC into the Ingenuity Pathway Analysis software as they were identified to belong to the MAPK pathway (using the KEGG database). Using the Ingenuity Pathway Analysis software we analyzed for interactions between these genes and the rest of the similarly or differentially regulated genes. The interactions are shown in figure 3. Only two of the similarly regulated genes (KRAS and RASA1) belonged to the MAPK pathway and only KRAS interacts (indirectly) with one similar regulated gene (PLAGL1). In contrast the five differentially regulated MAPK pathway genes (JUN, ATF4, DUSP5, DUSP6 and MYC) interact with 21 of the rest of the differentially regulated genes. These genes were either only regulated by insulin or insulin was a more potent regulator than S597 indicating that insulin is more potent in affecting the MAPK pathway than S597.

Validation of microarray data using quantitative real-time PCR

Quantitative Real-Time PCR (qRT-PCR) was performed on 13 genes using four biological replicates (cells from different passages stimulated and harvested at different days) to confirm the microarray data. The C T values were normalized to the internal control of 18S and fold changes calculated (figure 5). The direction (up-or down-regulated) of the expression of all 13 genes compared to untreated were the same obtained with qRT-PCR and microarray analysis. However due to variation between the biological replicates it is unclear whether S597 and insulin induces the expression of TRIB3 and MYC to a different or similar degree.

DISCUSSION

The molecular mechanisms responsible for how different ligands activating the same receptor can initiate different biological responses in the same cell are still not completely known. Selectivity in IR signaling has been suggested to be gained by differences in binding properties as well as signal transduction from different cellular compartments [16;17]. This is consistent with our recent findings that activating the IR with the slow-dissociating and non-internalizing synthetic peptide S597 and insulin led to significant differences in receptor activation and initiation of downstream signaling pathways and biological responses [START_REF] Jensen | Activation of the insulin receptor by insulin and a synthetic peptide leads to divergent metabolic and mitogenic signaling and responses[END_REF]. We have in this study further investigated how the activation of the IR by insulin and S597 differentially activates post-receptor signaling by studying the gene expression profile in response to IR activation by either insulin or S597. Using microarray technology we have found striking differences in the effect of these ligands on gene expression.

Out of more than 30,000 transcripts present on the microarrays, a total of 738 transcripts were regulated by one or both ligands (insulin and S597). The majority of these transcripts were regulated in the same direction by insulin and S597. 131 transcripts were differentially expressed in response to either insulin or S597. No genes were found to be regulated in opposite directions by insulin and S597 compared to untreated.

Microarray analysis of insulin-induced gene expression in IR-transfected cells has previously been done, however in these studies different cell models (that were not classical insulin target cells) and different stimulation and analysis modalities were used. Dupont et al. [18;19] used NIH-3T3 cells transfected with IR and stimulated for 90 min. Mulligan et al., 2002 [20] used 3T3 fibroblasts transfected with a TrkC/IR chimeric receptor and stimulated for 4 hours. Pandini et al. [START_REF] Pandini | Differential gene expression induced by insulin and insulin-like growth factor-II through the insulin receptor isoform A[END_REF] used IGF-I receptor-deficient murine fibroblasts transfected with the human IR. In this study they stimulated the cells with 10 nM insulin for 30 min, 3 or 8 hours. All of these studies gave different lists containing genes responsive towards insulin stimulation. The different setups make it difficult to compare the results between these and our studies. However, the insulin responsive genes that are found in both these and this study are regulated in the same direction between studies. These are: early growth response 1 (EGR1), zinc transporter 1 (SLC30A1), HBEGF, cyclin G2 (CCNG2), Kruppel-like factor 9 (KLF9), JUNB and IL-6. Pandini et al. [START_REF] Pandini | Differential gene expression induced by insulin and insulin-like growth factor-II through the insulin receptor isoform A[END_REF], also found many genes involved in proliferation, cell cycle and apoptosis up-regulated by insulin as we do. Other genes found in this study to be insulinresponsive have also previously been shown to respond to insulin using other methodologies; EGR1, Vascular Endothelial Growth Factor (VEGF), GLUT1, Basic Helix-loop-helix domain B (BHLHB2) [START_REF] Miele | Insulin and insulin-like growth factor-I induce vascular endothelial growth factor mRNA expression via different signaling pathways[END_REF][START_REF] Zelzer | Insulin induces transcription of target genes through the hypoxia-inducible factor HIF-1alpha/ARNT[END_REF][START_REF] Keeton | Insulin-regulated expression of Egr-1 and Krox20: dependence on ERK1/2 and interaction with p38 and PI3-kinase pathways[END_REF][START_REF] Shen | Basic helix-loop-helix protein DEC1 promotes chondrocyte differentiation at the early and terminal stages[END_REF]. This concordance together with the qRT-PCR validation experiments on biological replicates indicates that the microarray data generated here are a valid picture of the gene expression profile.

To investigate if insulin and S597 induced different gene expression profiles we used fold changes and P values as a way to define transcripts that were differentially regulated by the two ligands. In this way the genes lists obtained with similarly and differentially regulated genes are not two discrete biological classes defined by biological criteria but opposite ends of a continuum, in which assignment is very dependent on arbitrary cut-offs set for fold change and statistical significance. Furthermore we chose very stringent selection criteria. It is therefore likely that the number of differentially regulated genes is higher. There is no good way of defining selection criteria for when genes are similarly regulated. We chose to focus on genes that were significantly regulated by both insulin and S597 compared to untreated and then exclude all genes that also fulfilled the selection criteria we made for differentially regulated genes. The transcripts included as similarly regulated are thereby not included based on statistically significant similarity but lack of statistically significant difference. This is why some of the genes included in the similarly regulated genes have very different fold changes (like HMOX1) and in reality are differentially regulated. Which genes and how many genes are included in these lists will thereby change accordingly to different cut offs.

The genes induced by insulin and S597 were involved in many different regulatory functions. As the definition of these classes is somewhat arbitrary it is difficult to make clear conclusions when comparing the cellular and molecular functions of genes in different regulatory classes. It was, however striking that almost half of the genes differentially regulated by insulin and S597 were involved in cell proliferation and growth. Insulin either selectively regulated the gene expression of these genes or was a more potent regulator than S597. Using the Ingenuity software we found that many of the changes in gene expression induced (selectively or more potently) by insulin had a positive effect on cell growth and proliferation. This is consistent which the recent finding that S597 was less potent than insulin in stimulating cell proliferation using a thymidine incorporation assay [START_REF] Jensen | Activation of the insulin receptor by insulin and a synthetic peptide leads to divergent metabolic and mitogenic signaling and responses[END_REF]. Furthermore we find that half of the (annotated) differentially regulated genes interact directly or indirectly with the genes involved with the MAPK pathway (figure 3). This fits with the earlier finding that S597 stimulates activation of Shc and ERK which are members of the MAPK pathway [START_REF] Jensen | Activation of the insulin receptor by insulin and a synthetic peptide leads to divergent metabolic and mitogenic signaling and responses[END_REF] much more weakly than insulin, and supports the idea that the main difference between insulin and S597 stimulation is the lack of activation of the MAPK pathway (14). How much the different signaling pathways contribute to the proliferation effect of insulin is not completely clear, but the MAPK pathway has been shown to play a major role in inducing the mitogenic response of insulin and the lack of activation of this pathway in our study is most likely the main reason why S597 is less potent than insulin in stimulating cell proliferation [START_REF] Jensen | Activation of the insulin receptor by insulin and a synthetic peptide leads to divergent metabolic and mitogenic signaling and responses[END_REF]. This is further supported by studies that show that activation of genes like EGR1 (that is massively up-regulated by insulin compared to S597 and interacts with the MAPK pathway genes) is critical for insulin-inducible DNA synthesis [START_REF] Gousseva | Early growth response factor-1 mediates insulin-inducible vascular endothelial cell proliferation and regrowth after injury[END_REF]. The difference in mitogenicity of insulin and S597 results most likely from different activation of the IR and not simply from activation of IGF-1 receptors or hybrid receptors by insulin. Insulin has been shown to have a low affinity for the IGF-1 receptor and hybrid receptors [27;28]. Therefore the effect of insulin through the IGF-1 receptor or receptor hybrids should be minimal at the concentration of 100 nM insulin used in the microarray studies. This is further supported by the finding that insulin only had a small effect on phosphorylation of the IGF-1 receptor and proliferation in untransfected L6 cells which express only few IRs, while S597 had no effect [START_REF] Jensen | Activation of the insulin receptor by insulin and a synthetic peptide leads to divergent metabolic and mitogenic signaling and responses[END_REF]. This supports that it is not cross-talk with IGF-1 receptors that results in the higher mitogenicity of insulin.

One of the other genes involved in cell proliferation that was dramatically regulated by insulin (32.73 fold down-regulated) compared to S597 (3.95 fold down-regulated) is cyclin G2 (CCNG2). This massive fold change was also validated using qRT-PCR. This gene has also earlier been found [START_REF] Pandini | Differential gene expression induced by insulin and insulin-like growth factor-II through the insulin receptor isoform A[END_REF], using microarray analysis and qRT-PCR, to be down-regulated by insulin in IGF-I receptor-deficient murine fibroblasts transfected with the human IR. Cyclin G2 is an unconventional cyclin highly expressed in postmitotic cells. Unlike classical cyclins that promote cell cycle progression, cyclin G2 blocks cell cycle entry [START_REF] Horne | Cyclin G2 is up-regulated during growth inhibition and B cell antigen receptor-mediated cell cycle arrest[END_REF][START_REF] Bennin | Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G1/S phase cell cycle arrest[END_REF][START_REF] Martinez-Gac | Control of cyclin G2 mRNA expression by forkhead transcription factors: novel mechanism for cell cycle control by phosphoinositide 3-kinase and forkhead[END_REF]. It has been shown that ectopic expression of cycling G2 inhibits proliferation of several cell types [START_REF] Horne | Cyclin G2 is up-regulated during growth inhibition and B cell antigen receptor-mediated cell cycle arrest[END_REF][START_REF] Yue | Cyclin G1 and cyclin G2 are expressed in the periimplantation mouse uterus in a cell-specific and progesterone-dependent manner: evidence for aberrant regulation with Hoxa-10 deficiency[END_REF][START_REF] Bennin | Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G1/S phase cell cycle arrest[END_REF][START_REF] Rachchige-Don | Cyclin G2 is a centrosome-associated nucleocytoplasmic shuttling protein that influences microtubule stability and induces a p53-dependent cell cycle arrest[END_REF][START_REF] Liu | Effect of cyclin G2 on proliferative ability of SGC-7901 cell[END_REF][START_REF] Le | Roles of human epidermal growth factor receptor 2, c-jun NH2-terminal kinase, phosphoinositide 3-kinase, and p70 S6 kinase pathways in regulation of cyclin G2 expression in human breast cancer cells[END_REF] and that stable down-regulation of CCGN2 by short interfering RNAs resulted in enhanced cell growth. So the big difference in CCNG2 expression induced by insulin and S597 may play a critical role in the difference in mitogenicity of the two ligands. This is currently being investigated further in our laboratory. However the expression of this gene has been shown not to be affected by inhibition of ERK in human breast cancer cells [START_REF] Le | Roles of human epidermal growth factor receptor 2, c-jun NH2-terminal kinase, phosphoinositide 3-kinase, and p70 S6 kinase pathways in regulation of cyclin G2 expression in human breast cancer cells[END_REF] and this suggests that other signaling pathways that the MAPK pathway may be affected differently by S597 and insulin. Furthermore it has been shown that CCGN2 dysregulation may play an important role in epithelial transformation and the early stages of human oral cancer development [START_REF] Kim | Cyclin G2 dysregulation in human oral cancer[END_REF]. Development of insulin mimetic peptides like S597 that do not lead to dysregulation of such genes could turn out to have profound therapeutic implications as enhanced mitogenicity associated with some insulin analogues designed for optimized therapy of diabetes has been a matter of major concern [START_REF] Kurtzhals | Correlations of receptor binding and metabolic and mitogenic potencies of insulin analogs designed for clinical use[END_REF] The finding that two ligands activating the IR give different mitogenic responses is reminiscent of earlier studies investigating the effect of insulin and IGF-2 on the IR, isoform A [21;40-42]. Like in this study, the two ligands (insulin and IGF-2) bind with a similar affinity to the IR but result in different biological effects. Insulin led primarily to metabolic effects (measured by glucose uptake), whereas IGF-2 led primarily to mitogenic effects (measured by 3H-thymidine incorporation assays). These differences in the biological effects were associated with differential recruitment and activation of intracellular substrates as well as selective changes in gene expression. However, in our present work, it is the noncognate ligand that is much less mitogenic than insulin.

The molecular mechanisms responsible for how different ligands activating the same receptor can initiate different biological responses in the same cell are still not completely known. Selectivity in insulin signaling has been suggested to be gained by differences in binding properties as well as signal transduction from different cellular compartments [16;17]. Previous work showed that insulin analogues dissociating very slowly from the IR have an increased mitogenic activity, indicating that an increased duration of the insulin signal at the receptor level result in a shift towards a more mitogenic insulin response [39;43-45]. This is in contrast to what is found in this study as S597 dissociates slowly from the IR but has a much lower mitogenic potency compared to insulin. However, we have currently shown that S597 phosphorylates the same number of insulin receptors as insulin but to a lower degree. This suggests that even though S597 is dissociating much more slowly than insulin it is not able to activate the insulin receptor to the same degree as insulin. As the duration of the phosphorylation is similar to insulin stimulation, it seems that it is not the duration that is responsible for the differences in biological properties of S597 and insulin. We believe that the reason for the differential signaling is due to this lesser activation of the insulin receptor as well as lack of internalization of the S597/insulin receptor complex seen in the study, which in the end results in full activation of the PI3K/Akt pathway and almost no activation of the Shc/ERK pathway (explaining the lower mitogenicity) [START_REF] Jensen | Activation of the insulin receptor by insulin and a synthetic peptide leads to divergent metabolic and mitogenic signaling and responses[END_REF]. The lesser activation of the MAPK pathway is most likely the main reason for the differences observed in the insulin/S597 stimulated gene expression in this study. The low phosphorylation degree of the IR is most likely part of the reason for the lack of internalization as internalization is dependent on phosphorylation of several of the tyrosines residues on the IR [START_REF] Wilden | The role of insulin receptor kinase domain autophosphorylation in receptor-mediated activities. Analysis with insulin and anti-receptor antibodies[END_REF]. That selective signaling transduction occurs from different cellular compartments has been suggested by several studies (reviewed by [16;17]). It has been shown that activation of Shc is more dependent on internalization of the IR then IRS1 [47;48]. These studies support the general concept, that insulin/IGF-1 receptors initiate metabolic signaling from the plasma membrane whereas mitogenic signaling originates from inside the cells following internalization of activated receptors. Recently another study was published that investigates how different signaling cascades in beta cells can be activated through the IR [START_REF] Uhles | Selective gene activation by spatial segregation of insulin receptor B signaling[END_REF]. The authors showed that selective activation of the glucokinase and cfos genes was achieved through the IR-B by signaling from different cellular compartments. This supports our findings in this study that in insulin-target cells i.e. muscle cells and through the IR isoform A we can obtain differential signaling and effects on gene expression through the same receptor. That S597 stays on the cell surface for much longer than insulin is likely an important part of the reason for the differences in signaling, gene expression profiles and mitogenicity.

Activation of the MAPK pathway and Akt signaling pathway seemingly differ in the dependence of the activation degree of IR [START_REF] Jensen | Activation of the insulin receptor by insulin and a synthetic peptide leads to divergent metabolic and mitogenic signaling and responses[END_REF]. It is likely that gene-regulatory events also are differently dependent on the level of activation of signaling pathways. Thus differences in the biological responses (signaling, internalization, gene expression) could be due to different dependences for receptor activation rather than distinct signaling mechanisms. If this is the case weak agonists might be the more selective therapeutic agents, in terms of balancing metabolic and mitogenic effects. Our work adds to a growing body of evidence suggesting that activation of the IR is not simply a matter of flipping an on/off switch, and that qualitatively as well as quantitatively the biological consequences of receptor activation are dependent on the nature, binding kinetics and spatial localization of the activating ligand.

Gene lists used in this study

Comparison of insulin-and S597-treated samples to untreated (supplemental table 2):

Regulated by both ligands (176 transcripts)

Transcripts that are significantly regulated by insulin and S597 compared to untreated.

Preferentially regulated ranscripts that are only significantly regulated by insulin compared to untreated.

Similarly regulated

Transcripts that are significantly regulated by insulin and S597 (139 transcripts) untreated (as above) excluding transcripts where there is a significant difference between insulin and S597 samples

  expressed when compared rectly to insulin samples) parison of insulin ulated (131 transcripts) ranscripts where there is a significant difference in expression values between sulin and S597 samples. these transcripts are not (significantly) differently expressed when compared directly to S597 samples) Transcripts that are only significantly regulated by S597 compared to untrea (None of these transcripts are (signific Preferentially regu by S597 di -and S597-untreated samples (supplemental tables 3 and 4): Direct com Differentially reg T in

Figure 4 .

 4 Figure 4. QRT-PC on biological replicates. in biologica eplicates Foldchanges for insulin-mple pared to (UT) sample s were m asured for A: Similarly regulated gene

Figure 1 Figure 2 18

 12 Figure 1

Figure 3

 3 Figure 3

Table 1 .

 1 Definitions of th study.Significantly regulated is fined as: P value < 0.005 and a difference in expression fold change > 1.5

e gene lists used in this de

  Genes preferentially regulated by insulin (465 transcripts in total -189 unique genes eligible for functional analysis)

	Molecular and cellular function	Significance	Number of genes
	Gene expression	1.37E-11-4.42E-03		64
	Cellular development	1.30E-11 -4.42E-03	56
	Cellular growth and pro	1.74E-07 -4.42E-03	73

Table 2 .

 2 Molecular and cellular functions associated with tran

				liferation
	Cell cycle					5.72E-07 -4.64E-03	33
	Cell death					1.18E-06 -4.62E-03	60
	ferentially regulated by S597 (97 transcripts in total -42 unique Genes pre	genes eligible for function	al analy	sis)
	ansport Molecular tr			4.53E-04 -4.75E-02	7
	Gene expression				2.79E-03 -3.51E-02	11
	Amino acid metabolism		3.24E-03 -4.68E-02	5
	Cell cycle					3.24E-03 -4.53E-02	4
	Cell morphology				3.24E-03 -4.92E-02	7
	Genes responsive to both i	nsulin and S597 (176 transcripts in total-66 unique	genes eligible for functional analysis)
	eration Cellular growth and prolif
					functions. Comparison of the	P values for the	most significant
					scripts preferentially regulated by	insulin, S597 or by	both	ds. The ligan
	five most	significant functions associated with each of the th ree groups were id	entified using the
	Ingenuity Pathwa	y Analysis software. Several of top five functio ns where the same	betwe en t	he three
	groups (eight dif	ferent functions were found in total). The signifi cance (P value) as w	ell as	the	number
	of unique	genes associated with a given function for each of th e three groups is sh	own.	See	text for
	more details.

Table 4 . Ge simil ted by insulin and S597.

 4 number of probesets found on the microarray and not the number of unique genes.

	1.88E-10 -6.63E-03	33

Biochemical Journal Immediate Publication. Published on 05 Mar 2008 as manuscript BJ20080279 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080279 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 05 Mar 2008 as manuscript BJ20080279 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080279 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 05 Mar 2008 as manuscript BJ20080279 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080279 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 05 Mar 2008 as manuscript BJ20080279 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080279 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 05 Mar 2008 as manuscript BJ20080279 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080279 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEGDEMENTS

We thank B. F. Hansen for providing the IR transfected L6 cell line. We also thank S. Smed for her help with scanning the microarrays. M.J. thanks CORA/Novo Nordisk A/S, The University of Copenhagen and The Danish Ministry of Science, Technology and Innovation for a scholarship.

The Receptor Systems Biology Laboratory and the Hagedorn Research Institute are independent basic research components of Novo Nordisk A/S.