Andrew Botham

Xiaodan Guo

Yupei Pei Xiao

Alyn H Morice

Steven J Compton

Laura R Sadofsky
email: l.r.sadofsky@hull.ac.uk

Palmitoylation of Human Proteinase-Activated Receptor-2 Differentially Regulates Receptor Triggered ERK1/2 Activation, Calcium Signalling, and Endocytosis

Keywords: ERK1/2, GPCR, Palmitoylation, PAR 2 α-MEM, Dulbecco's α-modified essential medium, ANOVA, analysis of variance, AP, activating protein, CHO, Chinese hamster ovary, ERK1/2, extracellular signal-regulated kinases, FCS, foetal calf serum, GPCR, G-Protein coupled receptor, HA11, influenza haemagglutinin, hPAR 2 , human proteinase activated receptor, MAPK, mitogen activated protein kinase, PTX, pertussis toxin, RT, room temperature

published or not. The documents may come

INTRODUCTION 1

Post-translational modifications of GPCRs can extensively regulate numerous aspects of their biology including cell surface expression, trafficking, folding, signalling, and ligand binding. Examples of post-translational modifications known to regulate GPCR function include N-linked glycosylation, phosphorylation and palmitoylation [1][START_REF] Qanbar | Role of palmitoylation/depalmitoylation reactions in G-protein-coupled receptor function[END_REF]. Phosphorylation of GPCRs is well known to induce receptor desensitisation through the action of second messenger-activated and GPCR kinases on serine and threonine residues located on the intracellular and carboxyl tail of the receptors [START_REF] Krupnick | The role of receptor kinases and arrestins in G protein-coupled receptor regulation[END_REF]. Such phosphorylation results in uncoupling of the receptor from G-proteins through the involvement of arrestins which bind to the phosphorylated receptors and target them towards clatherin coated pits where the receptor undergoes endocytosis [START_REF] Krupnick | The role of receptor kinases and arrestins in G protein-coupled receptor regulation[END_REF]. Following endocytosis, -arrestin can target the receptor to the mitogen-activated protein kinases (MAPK) signalling pathway [START_REF] Ma | Beta-arrestin signaling and regulation of transcription[END_REF]. Thus, processes that influence -arrestin binding to a GPCR may govern which signalling pathways are triggered by the receptor.

Conflicting reports concerning PAR 2 activation by tryptase were found to be a result of Nlinked glycosylation [1,[START_REF] Compton | Glycosylation and the activation of proteinase-activated receptor 2 (PAR(2)) by human mast cell tryptase[END_REF], thus post-translational modifications of PARs can have a profound influence on receptor function and have to date been little investigated. The post-translational covalent attachment of lipid moieties is essential for many proteins. The most common lipid modification is the post-translational addition of palmitic acid, which also seems to have the most wide-ranging effects [START_REF] Bijlmakers | The on-off story of protein palmitoylation[END_REF]. Palmitoylation is a thioesterification which often occurs with cyteine residues 10-14 amino acids downstream of the last transmembrane domain [START_REF] Escriba | Lipid-protein interactions in GPCR-associated signaling[END_REF]. Using this principal we have identified a C-terminal cysteine residue in hPAR 2 (C361) as a putative palmitoylation site (Figure 1). The importance of palmitoylation in GPCRs is wide-ranging [START_REF] Qanbar | Role of palmitoylation/depalmitoylation reactions in G-protein-coupled receptor function[END_REF] and has been implicated in numerous processes including agonist-induced internalisation [START_REF] Kawate | Palmitoylation of luteinizing hormone/human choriogonadotropin receptors in transfected cells. Abolition of palmitoylation by mutation of Cys-621 and Cys-622 residues in the cytoplasmic tail increases ligand-induced internalization of the receptor[END_REF], and agonist induced downregulation [START_REF] Eason | The palmitoylated cysteine of the cytoplasmic tail of alpha 2A-adrenergic receptors confers subtype-specific agonist-promoted downregulation[END_REF], but its role varies depending on the GPCR considered. Proteins can be constitutively palmitoylated or may require agonist activation for the modification to occur [START_REF] Ponimaskin | 5-Hydroxytryptamine 4(a) receptor expressed in Sf9 cells is palmitoylated in an agonist-dependent manner[END_REF][11] In this study we investigate the role of C361 in various aspects of hPAR 2 expression and function.

EXPERIMENTAL

Materials.

Human plasma thrombin was purchased from Calbiochem, Merck Chemicals Ltd, (Nottingham, UK). Anti-HA11 antibody was purchased from Covance (Harrogate, UK). Sam11 antibody was purchased from Zymed (Invitrogen, Paisley, UK). Foetal calf serum (FCS), Dulbecco's α-modified essential medium (α-MEM, with ribonucleosides and deoxyribonucleosides), non-enzymic cell-dissociation solution, geneticin, penicillin, streptomycin, PBS (without calcium and magnesium) pcDNA 3.1(+) and fluo-3 acetoxymethyl ester (fluo-3 AM) were from Invitrogen (Paisley, UK). All oligonucleotides were synthesized by the peptide synthesis facility, University of Calgary (Alberta, Canada) or purchased from peptides international (Kentucky, USA). µMACS™ HA11 Epitope Tag Protein Isolation Kit were supplied by Miltenyi Biotech (Bergisch, Germany). Hybond C PVDF membrane, hyperfilm x-ray film, peroxidase conjugated goat anti-mouse IgG, Amplify™ and ECL was purchased from Amersham Biosciences (Buckinghamshire, U.K.). All other chemicals and reagents were purchased from Sigma (Dorset, UK.) unless otherwise stated.

Expression Vectors.

The wt-hPAR 2 cDNA possessing a C-terminal HA11 epitope (YPYDVPDYA) used in this study has been described in detail previously [1]. Human PAR 2 C361A was generated using the QuikChange site-directed mutagenesis kit (Stratagene, Amsterdam, Netherlands), according to the manufacturer's instructions. The engineered mutation in PAR 2 was then confirmed by automated DNA sequencing (MWG, Germany).

Cell culture and transfection

The Pro5 Chinese Hamster Ovary (CHO) cell line, that permanently expressed either wild-type or mutant hPAR 2 receptors, were propagated in selective growth media (α-MEM containing 10% FCS, 100 units/ml penicillin, 100 ng/ml streptomycin, 250 ng/ml amphotericin B, and 800 µg/ml geneticin) 5% CO 2 at 37 o C. All cell lines were harvested using non-enzymatic cell dissociation solution.

Permanent receptor expressing cell lines cells were generated by transfection using the LipofectAMINE2000 (Invitrogen, Paisley, U.K.) method, according to the manufacturer's protocol. Transfected Pro5 cells were propagated in selective growth medium for two weeks to allow selection. Permanently expressing cell-lines were selected by single cell cloning and assessed for receptor expression by flow cytometry.

Determination of cell surface expression of hPAR 2 wild-type, and hPAR 2 C361A receptors.

It has previously been found that in the cell lines used "receptor expression (is) sensitive to confluence" [1]. Therefore flow cytometry was used to assess receptor expression. Cells were grown to different levels of confluence and then labelled with Sam11 mouse anti-human PAR 2 antibody and anti-mouse FITC conjugate before analysing with flow cytometry to assess and match receptor expression.

Localisation of Receptor by Confocal Imaging

Confocal microscopy was used to determine the location of PAR 2 and PAR 2 C361A within the cells. Sam11 shows cell surface expression and HA11 shows total cell expression. For Sam11 staining cells grown on coverslips were incubated with Sam11 anti-PAR 2 antibody (1μg/ml) at RT for 60 min before incubation with FITC conjugated anti-mouse antibody (1μg/ml). For HA11 staining, coverslips were pre-incubated in PBS (1% BSA, 3% Rabbit Serum) for 15 min prior to addition of anti-HA11 mouse monoclonal antibody (1μg/ml) and incubated at RT for 60 min. Coverslips were then incubated with FITC conjugated anti-mouse antibody (1μg/ml). The slides were then analysed on a confocal microscope using an argon laser exciting at 488 nm and detecting fluorescence emissions above 500 nm. Each experiment consisted of two pictures from each of three coverslip repeats for each treatment and a final consensus photo being chosen.

[ 3 H]-palmitate labelling and PAR 2 immunoprecipitation. The constitutive palmitoylation of PAR 2 was investigated by detecting [ 3 H]-palmitate labelling of the receptor. Wt-hPAR 2 and hPAR 2 C361A expressing Pro5 cell lines were seeded in 100 mm dishes, pre-incubated overnight in serum-free medium, and labelled with 1 mCi [ 3 H] palmitic acid for 4 h at 37 °C. After labelling, PAR 2 protein from the cell lines was isolated by immunoprecipitation using the µMACS™ HA11 Epitope Tag Protein Isolation Kit (Miltenyi Biotech, Bergisch, Germany) as per manufacturers protocol.

Proteins were then resolved on SDS-PAGE in non-reducing conditions and then fixed for 30 min (isopropanol:water:acetic acid, 25:62:10). Gels were then treated with Amplify™ for 15 min at RT before drying under vacuum at 80 o C. The dried gel was then exposed to Hyperfilm™ x-ray film at -80 o C for 24 hours, before developing.

In order to confirm equal loading of radiolabelled PAR 2 protein, immunoblots were performed with half of the protein sample used in the palmitate experiments. Briefly immunoprecipitated samples were separated on a 10% SDS/PAGE gel before transfer to Hybond C PVDF membrane. The membrane was blocked with 5% non-fat milk powder in PBS/Tween 20 (0.1%) for 1 h before incubation overnight at 4 °C with the mouse monoclonal anti-HA.11 antibody (1 in 1000 dilution in PBS/Tween-20 (0.1%) containing 2% non-fat milk) then incubated with peroxidase conjugated goat anti-mouse IgG (1 in 1000) for 1 h. The epitope tagged hPAR 2 receptor was then visualized using enhanced chemiluminescence (ECL2), and captured on a UVP Epi Chem II Darkroom with fluorescence camera and analysed using Labworks v4.5 software.

Calcium signalling assay

Calcium signalling was performed as described previously [START_REF] Compton | A polymorphic protease-activated receptor 2 (PAR2) displaying reduced sensitivity to trypsin and differential responses to PAR agonists[END_REF]. Cells were harvested nonenzymically and incubated in 1 ml of -MEM containing 0.25 mM sulphinpyrazone and 22 μM Fluo-3 AM for 25 min at RT on an orbital mixer. Cells were then washed and resuspended in calcium assay buffer, (150 mM NaCl, 3 mM KCl, 1.5 mM CaCl 2 , 10 mM glucose, 20 mM Hepes and 0.25 mM sulphinpyrazone) pH 7.4. Fluorescence measurements were performed on a Photon Technology International Fluorospectrophotometer (West Sussex, UK), with an excitation wavelength of 480 nm and emission recorded at 530 nm. The signal produced by the addition of a test agonist was measured as a percentage of the fluorescence peak height, yielded by the addition of 2 µM calcium ionophore (A23187). ERK1/2 phosphorylation and immunoblots. Cells were grown in 6 well plates to a confluence of 40-50% in normal media. Media was then replaced with serum-free media and cells incubated overnight. Cells were then treated with agonist for a specified time (0, 5, 10, 30, 60 min) before halting treatment and harvesting cells in Laemlli's Sample Buffer (30% glycerol, 140mM Tris pH 6.8, 5% SDS, 7 nM Bromophenol blue, 5 mM EDTA, 1/20 2-mercaptoethanol). Samples were then incubated at 100 o C for 5 min prior to analysis by western blotting (as described in [ 3 H]-palmitate labelling and PAR 2 immunoprecipitation) using p44/42 MAP kinase Antibody (1 in 1000) and Phospho-p44/42 MAP Kinase Antibody (1 in 1000) (Cell Signaling Technology, NEB, Herts, UK).

Receptor Internalisation Assay

Semi-confluent cells (approx. 40% confluence) were harvested and treated with trypsin (100 nM) or the synthetic peptide SLIGKV-NH 2 (100 μM) which corresponds to the tethered ligand exposed by trypsin cleavage of PAR 2 , for 0-30min at RT before being placed on ice. Cells were then washed and centrifuged. Pelleted cells were resuspended in cold-PBS and placed on ice for 10 min before incubation with Sam11 mouse anti-PAR 2 monoclonal antibody (3 μg/mL) for 1 h on ice followed by incubation with anti-mouse FITC-conjugated antibody (10 μg/mL) for a further 45 min. Cells were then analysed for PAR 2 cell-surface expression by flow cytometry (Becton Dickinson, Oxford, U.K.).

Statistical Analysis.

Graphs were produced using Prism Graphpad. For a two dataset comparison of group mean a twotailed test was used, when this was to compare two different treatments on the same cell line, or when this was to compare two different cell lines subjected to the same treatment a paired t-test was adopted. In order to assess a change over a period of time using a specific treatment, or to assess a change over a concentration range for a specific cell line a repeated measures one-way ANOVA table was used. When a comparison of two different time courses or concentration ranges was required a two-way ANOVA table was adopted.

RESULTS

Expression and palmitoylated state of wt-PAR 2 and PAR 2 C361A.

In order to study the potential role of palmitoylation in regulating PAR 2 function, Pro5 cells were stably transfected with HA11-tagged wt-hPAR 2 or hPAR 2 C361A where the putative palmitoylation site (C361) had been replaced with an alanine. Receptor expressing cell lines were established and used at confluences which resulted in similar receptor expression as determined by flow cytometry (Figure 2). These were subsequently employed for further studies.

Confocal imaging was used to identify the cellular localisation of receptor expression (Figure 3). When stained with Sam11 anti-PAR 2 antibody empty vector transfected cells (Figure 3A) showed no staining. Wt-hPAR 2 (Figure 3B) and hPAR 2 C361A (Figure 3C) cells showed staining at the cell membrane. However, low levels of fluorescence were seen using this method and a high photomultiplier gain was required to observe any signal. Cells were therefore permeabilised and stained with anti-HA11 antibody. Wt-hPAR 2 (Figure 3E) displayed distinct, uniform and robust immunostaining around the cell membrane. Some punctate cytoplasmic staining was also observed. No staining was observed in the empty vector transfected cells (Figure 3D). The staining pattern observed for hPAR 2 C361A (Figure 3F) was strikingly different to that of wt-hPAR 2 (Figure 3E). The ring staining for hPAR 2 C361A was not uniform and distinct pockets of intense staining were observed at the cell surface. In addition considerable punctate staining was observed in the cytoplasmic region of the cells (Figure 3F). However, the staining for hPAR 2 C361A appeared brighter than that of wt-PAR 2 .

The palmitoylation state of the wt-hPAR 2 and hPAR 2 C361A was then determined by metabolic labelling of the cells with [ 3 H] palmitic acid followed by immunoprecipitation of the receptors. Figure 4A shows that detectable levels of tritium were incorporated into wt-PAR 2 . Two broad smears were observed, one between ~50-75kDa and a second from ~25-30kDa. In contrast, PAR 2 C361A displayed an undetectable level of tritium incorporation. Western blot analysis of the same samples that were employed for the palmitoylation gel, demonstrated that approximately equal amounts of PAR 2 protein were present in the wt-PAR 2 and PAR 2 C361A samples (Figure 4B).

Role of palmitoylation in regulating intracellular calcium signalling.

To assess the importance of palmitoylation in regulating hPAR 2 coupling to calcium, we compared the ability of wt-hPAR 2 and hPAR 2 C361A to trigger increases in intracellular calcium in response to the hPAR 2 agonists trypsin and SLIGKV-NH 2 . Trypsin and SLIGKV-NH 2 concentration effect curves for hPAR 2 and hPAR 2 C361A cell lines with matched expression (Figure 2) were constructed (Figure 5). For wt-hPAR 2 , both trypsin and SLIGKV-NH 2 stimulated robust and similar maximal responses (~60% of A23187) at 316 nM, and 316 μM respectively (Figure 5). The EC 50 for trypsin and SLIGKV-NH 2 in activating wt-PAR 2 was 11.44 nM and 15.69 μM respectively. In contrast to wt-hPAR 2 , trypsin and SLIGKV-NH 2 displayed significantly reduced efficacy towards hPAR 2 C361A (P=0.0003 and P=0.0058 respectively). The maximal obtainable responses to both trypsin and SLIGKV-NH 2 were only ~50% and ~30% of that observed for wt-hPAR 2 respectively (Figure 5). The hPAR 2 C361A EC 50 s for trypsin and SLIGKV-NH 2 were 40.76 nM and 103.45 M respectively, displaying a 4 and 6 fold increase compared to that obtained for wt-hPAR 2 .

Inhibition of Trypsin Mediated Calcium Signal by Pertussis Toxin.

Pertussis toxin was used to ascertain what proportion of the total IP 3 induced Ca 2+ flux is due to signalling through G i in wt-hPAR 2 and the palmitoylation deficient mutant hPAR 2 C361A. PTX dose dependently inhibited wt-hPAR 2 and hPAR 2 C361A mediated calcium signalling to trypsin. wt-hPAR 2 generated a maximum calcium signal of ~70% of A23187 decreasing to ~50% of A23187 at the maximum PTX concentration of 316 ng/ml (Figure 6). Whereas, hPAR 2 C361A produced a maximum calcium signal of ~30% of A23187 reducing down to <10% of A23187 at the maximum PTX concentration of 316 ng/ml. As such both curves reduce by ~20% of their A23187 signal (Figure 6).

Role of Palmitoylation in Regulating ERK1/2 Phosphorylation.

Having established that the palmitoylation deficient hPAR 2 C361A mutant receptor coupled less efficiently to calcium, we asked whether receptor palmitoylation possibly regulated hPAR 2 signalling to ERK1/2 (p44/42). Significant differences in the pattern of the ERK1/2 stimulation were observed for hPAR 2 C361A compared to wt-hPAR 2 (p<0.001) (Figure 7A). SLIGKV-NH 2 stimulated a significantly greater degree of ERK1/2 phosphorylation in hPAR 2 C361A cells at the 5 and 10 min time points, and the ERK1/2 activation levels remaining significantly elevated up to the 60 min time point tested. Similarly, trypsin was also found to stimulate a significantly greater degree of ERK1/2 phosphorylation through hPAR 2 C361A compared to wt-hPAR 2 (P< 0.001) (Figure 7B). Trypsin stimulated a significantly greater degree of ERK1/2 phosphorylation in hPAR 2 C361A cells at the 5, 10 and 20 min time points, and a more sustained ERK1/2 activation levels remaining significantly elevated up to the 60 min time point tested. All phosphorylated ERK1/2 blots were stripped of antibodies and re-probed with anti-total ERK1/2 and the phosphorylated ERK1/2 were then normalised against total ERK1/2 and expressed as a fold change of no treatment (NT).

Inhibition of MAP Kinase Signalling by Pertussis Toxin

To ascertain what proportion of the ERK signalling is due to signalling through G i and to determine whether the lack of palmitoylation altered this signalling, wt-hPAR 2 , hPAR 2 C361A and empty vector control cells were preincubated with PTX before treating with PAR 2 agonists (trypsin and SLIGKV).

Trypsin and SLIGKV-NH 2 evoked negligible changes in ERK1/2 signalling compared to NT in the empty vector cells (data not shown). Treatment with PTX caused no significant change in ERK1/2 agonist induced signalling. For wt-hPAR 2 (Figure 8A) SLIGKV-NH 2 and trypsin caused a 2 and 1.5 fold increase in ERK1/2 signal over NT respectively. Following pre-treatment with PTX there appeared to be a slight decrease in ERK1/2 signalling with all treatments though no significant difference was noted. SLIGKV-NH 2 and trypsin evoked a 2 and 3 fold increase in ERK1/2 signalling over NT respectively in hPAR 2 C361A expressing cells (Figure 8B). When pre-treated with PTX ERK1/2 signalling in hPAR 2 C361A decreased a small amount following no treatment and trypsin treatment in comparison to samples not pre-treated with PTX. However, a large drop in ERK1/2 signalling can be seen in the PTX pre-incubated SLIGKV-NH 2 treated samples compared those not pre-incubated with PTX, though the changes are not statistically significant.

Agonist Triggered Internalisation of PAR 2 .

Flow cytometry was employed to determine the agonist induced internalisation of PAR 2 using the monoclonal Sam11 antibody which recognises a sequence of the N-terminal domain that is located Cterminal to the tethered-ligand [START_REF] Molino | Interactions of mast cell tryptase with thrombin receptors and PAR-2[END_REF](Figure 9). Thus, receptors that have been activated by trypsin can still be readily detected by this antibody [START_REF] Molino | Interactions of mast cell tryptase with thrombin receptors and PAR-2[END_REF]. As shown in figure 9, trypsin (100 nM) promoted rapid and significant (~50%) loss of cell surface wt-PAR 2 by 10 min post agonist challenge. At the 60 min time point ~42% of wt-PAR 2 was still detectable at the cell surface. Curiously, SLIGKV-NH 2 (100 M) only stimulated a modest loss of cell surface receptor at 30 and 60 min post agonist challenge. In contrast to wt-hPAR 2 , trypsin stimulated a significantly greater loss of hPAR 2 C361A from the cell surface at all time points tested, with over 81 ± 1.3% lost from the cell surface by 10 min post agonist challenge (P=0.016). Maximal loss of cell surface receptor was observed by 20 min (95 ± 2.6%) which thereafter remained unchanged up to the 60 min time point. As with wt-hPAR 2 cells, we also observed only a minor degree of receptor internalisation with hPAR 2 C361A following treatment with SLIGKV-NH 2 with maximal internalisation observed at 60 min (16 ± 2%). The data presented here provides the first direct biochemical and molecular evidence that hPAR 2 is palmitoylated and that cysteine 361 is likely to be the primary site of palmitoylation. In addition evidence is presented here which demonstrates that palmitoylation of hPAR 2 is a dynamic process that is influenced by agonist activation. Further, palmitoylation of hPAR 2 was shown to regulate constitutive receptor expression, agonist triggered internalisation and more importantly inversely regulate receptor signalling to two major signalling pathways, namely calcium and MAPK.

DISCUSSION

The hPAR 2 C361A cell line displayed greater receptor expression than wt-hPAR 2 . Removal of palmitoylation often results in loss of cell surface expression as seen with the V 2 vasopressin receptor and CCR5 [START_REF] Sadeghi | Palmitoylation of the V2 vasopressin receptor[END_REF][16][START_REF] Blanpain | Palmitoylation of CCR5 is critical for receptor trafficking and efficient activation of intracellular signaling pathways[END_REF]. To our knowledge, this is the first study to find an increase in receptor expression resulting from the mutagenic removal of a palmitoylated cysteine. Possible reasons for increased receptor expression are covered later.

Confocal microscopy was next employed to compare the cellular distribution of wt-hPAR 2 and hPAR 2 C361A. Confocal imaging shows that PAR 2 is located at the plasma membrane along with some internal localisation (which is greater in hPAR 2 C361A), this is presumably, at least in part, PAR 2 contained in golgi stores, which is confirmatory of previous confocal imaging of PAR 2 [START_REF] Defea | beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2[END_REF][START_REF] Dery | Trafficking of proteinase-activated receptor-2 and beta-arrestin-1 tagged with green fluorescent protein. beta-Arrestin-dependent endocytosis of a proteinase receptor[END_REF][START_REF] Bohm | Mechanisms of desensitization and resensitization of proteinase-activated receptor-2[END_REF].

Immunoprecipitation of wt-hPAR 2 and hPAR 2 C361A was carried out using the HA11 epitope tag, and western blotting carried out using anti-HA11 antibody as Sam11 does not work well with western blotting. Immunoprecipitation was required as half of the sample was used to detect palmitoylation, it was therefore essential to remove unwanted (non-HA11 tagged) proteins. The banding pattern seen is in keeping with the previous immunoblotting of hPAR [START_REF] Qanbar | Role of palmitoylation/depalmitoylation reactions in G-protein-coupled receptor function[END_REF] [1] and the C361A appears to have no perceptible effect on the receptor molecular weight compared to wild-type. Despite flow cytometry data showing higher hPAR 2 C361A expression than the wild type, three times more cells were required to achieve matching quantities of receptor. However, if hPAR 2 is palmitoylated at C361, the C361A mutation will result in a reduced C-terminal association with the cell membrane and potentially leave the C-terminus available to be more easily cleaved by endogenous proteases so removing the HA11 tag. Alternatively, the epitope may be obscured by the C-terminus becoming associated with different receptor domains, or sterically by an accessory protein binding to a nearby region on the C-terminus [START_REF] Bockaert | GPCR interacting proteins (GIP)[END_REF][START_REF] Bockaert | GPCR-interacting proteins (GIPs): nature and functions[END_REF].

Using direct biochemical labelling we provide compelling evidence that hPAR 2 is palmitoylated. There is evidence that GPCRs can be palmitoylated in regions other than the Cterminal tail [START_REF] Hawtin | Palmitoylation of the vasopressin V1a receptor reveals different conformational requirements for signaling, agonist-induced receptor phosphorylation, and sequestration[END_REF]. However, since no [ 3 H]-palmitate signal is evident in hPAR 2 C361A it seems likely this is the sole palmitoylation site for hPAR 2 .

PAR 2 palmitoylation has a significant effect on receptor mediated calcium signalling. The decreased signal and agonist sensitivity observed for the hPAR 2 C361A mutant may be due to the revealing of phosphorylation sites within the C-terminus, which may normally be obscured by association with the membrane when palmitoylation is present. This is similar to the β-adrenergic receptor where substitution of the palmitoylated cysteine appears to allow agonist-independent phosphorylation of receptor PKA and GRK sites which would normally only phosphorylate upon activation [START_REF] Moffett | The palmitoylation state of the beta(2)-adrenergic receptor regulates the synergistic action of cyclic AMPdependent protein kinase and beta-adrenergic receptor kinase involved in its phosphorylation and desensitization[END_REF]. In other GPCRs desensitisation/internalisation can be driven by phosphorylation dependent interations with β-arrestin [START_REF] Moffett | The palmitoylation state of the beta(2)-adrenergic receptor regulates the synergistic action of cyclic AMPdependent protein kinase and beta-adrenergic receptor kinase involved in its phosphorylation and desensitization[END_REF][START_REF] Munshi | Palmitoylation of the luteinizing hormone/human chorionic gonadotropin receptor regulates receptor interaction with the arrestin-mediated internalization pathway[END_REF][26]. Mutagenic replacement of the hPAR 2 palmitoylation site may alternatively cause conformational change in the receptor leaving the tethered ligand and AP incapable of correctly binding to the ligand binding site. Although this has not previously been seen in the mutagenic replacement of palmitoylation, removal and mutagenesis of areas of the C-terminal tail in some GPCR have been shown to affect receptor folding [START_REF] Piersen | A carboxyl-terminally truncated mutant and nonglycosylated A2a adenosine receptors retain ligand binding[END_REF][START_REF] Klinger | Removal of the carboxy terminus of the A2A-adenosine receptor blunts constitutive activity: differential effect on cAMP accumulation and MAP kinase stimulation[END_REF]. The reduction in receptor mediated calcium signalling may be as a result of altered G-protein coupling. The C-terminal tail of many GPCRs are associated with G-protein coupling and as such removal of the palmitoylation site affects G-protein coupling in many GPCRs [START_REF] Moffett | The palmitoylation state of the beta(2)-adrenergic receptor regulates the synergistic action of cyclic AMPdependent protein kinase and beta-adrenergic receptor kinase involved in its phosphorylation and desensitization[END_REF]26,[START_REF] Okamoto | Palmitoylation of human endothelinB. Its critical role in G protein coupling and a differential requirement for the cytoplasmic tail by G protein subtypes[END_REF][START_REF] Hayashi | Palmitoylation of muscarinic acetylcholine receptor m2 subtypes: reduction in their ability to activate G proteins by mutation of a putative palmitoylation site, cysteine 457, in the carboxyl-terminal tail[END_REF]. In some this is most likely because of increased phosphorylation [START_REF] Moffett | The palmitoylation state of the beta(2)-adrenergic receptor regulates the synergistic action of cyclic AMPdependent protein kinase and beta-adrenergic receptor kinase involved in its phosphorylation and desensitization[END_REF][START_REF] Munshi | Palmitoylation of the luteinizing hormone/human chorionic gonadotropin receptor regulates receptor interaction with the arrestin-mediated internalization pathway[END_REF][26][START_REF] Palmer | Identification of threonine residues controlling the agonist-dependent phosphorylation and desensitization of the rat A(3) adenosine receptor[END_REF] but some mutagenically depalmitoylated GPCRs have decreased G-protein coupling without displaying increased phosphorylation [START_REF] Okamoto | Palmitoylation of human endothelinB. Its critical role in G protein coupling and a differential requirement for the cytoplasmic tail by G protein subtypes[END_REF][START_REF] Doi | Interactions of endothelin receptor subtypes A and B with Gi, Go, and Gq in reconstituted phospholipid vesicles[END_REF] suggesting another mechanism.

Experiments assessing MAPK activation revealed that palmitoylation of PAR 2 inversely regulates MAPK activation. This increased ERK1/2 signalling seen with the non-palmitoylated mutant may be due to increased β-arrestin association. It is well known that phosphorylation of the Cterminus of PAR 2 results in an increased affinity for β-arrestin binding which in turn uncouples the receptor from it's G-protein by a steric mechanism [START_REF] Defea | beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2[END_REF][START_REF] Dery | Trafficking of proteinase-activated receptor-2 and beta-arrestin-1 tagged with green fluorescent protein. beta-Arrestin-dependent endocytosis of a proteinase receptor[END_REF][START_REF] Bohm | Mechanisms of desensitization and resensitization of proteinase-activated receptor-2[END_REF]. Additionally, PAR 2 expressed alongside a β-arrestin mutant incapable of receptor binding shows diminished ability to activate ERK1/2 [START_REF] Defea | beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2[END_REF]. Studies on a number of GPCRs have now demonstrated that β-arrestins can mediate a number of signalling pathways independent of G-proteins [START_REF] Shenoy | beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor[END_REF][START_REF] Wei | Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2[END_REF]. Recently PAR 2 activation has also been shown to promote β-arrestin actions-independent of G-proteins by direct inhibition of Gα q/11 , as well as receptor coupling to Gα q/11 [START_REF] Zoudilova | beta -arrestin-dependent regulation of the cofilin pathway downstream of protease-activated receptor-2[END_REF]. There is additional evidence to suggest that residues in the Cterminus of PAR 2 define specificity of β-arrestin binding and the duration of ERK1/2 association with β-arrestin [START_REF] Defea | beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2[END_REF][36][37]. This combined with β-arrestin 1 and 2 association with ERK1/2 activation in the early and intermediate phases [38] supports the hypothesis that ERK1/2 signalling in hPAR 2 C361A maybe enhanced due to an increase in β-arrestin association. This increased β-arrestin association may also go to explain the increased receptor expression seen in hPAR 2 C361A compared to wt-hPAR 2 as studies have shown a role for β-arrestins in trafficking of PAR 2 from the golgi [START_REF] Zoudilova | beta -arrestin-dependent regulation of the cofilin pathway downstream of protease-activated receptor-2[END_REF]38] Further studies to investigate the role of β-arrestins in hPAR 2 C361A signalling, expression and internalisation are required. Alternatively, palmitoylation is linked with the targeting of receptors and signalling molecules alike to specific membrane microdomains such as lipid rafts [39-40]. It has been shown that Gα q interacts with caveolin, where as Gα i does not, suggesting they may therefore be targeted to different lipid regions [START_REF] Oh | Segregation of heterotrimeric G proteins in cell surface microdomains. G(q) binds caveolin to concentrate in caveolae, whereas G(i) and G(s) target lipid rafts by default[END_REF]. The oxytocin receptor (OTR) is known to couple to both G q and G i but differentially activates one or the other depending on it's membrane localisation [START_REF] Rimoldi | Oxytocin receptor elicits different EGFR/MAPK activation patterns depending on its localization in caveolin-1 enriched domains[END_REF]. When located within lipid rafts OTR activation results in cell growth through a G q mediated pathway, when outside lipid rafts it results in G i mediated inhibition of cell growth. This effect on cell growth is shown to be as a result of a different temporal pattern of EGFR and ERK1/2 phosphorylation, and is shown to be more persistent when receptors are located outside of lipid raft microdomains [START_REF] Rimoldi | Oxytocin receptor elicits different EGFR/MAPK activation patterns depending on its localization in caveolin-1 enriched domains[END_REF]. As such palmitoylation in hPAR 2 may target the receptor to specific lipid raft microdomains, removal of which results in movement outside of these lipid rafts resulting in greater G i association and prolonged ERK1/2 signalling.

Palmitoylation has been shown to affect a GPCR ability to couple to its respective G-protein α subunit. For some GPCRs palmitoylation has been shown to selectively effect coupling to specific Gα subunits [START_REF] Okamoto | Palmitoylation of human endothelinB. Its critical role in G protein coupling and a differential requirement for the cytoplasmic tail by G protein subtypes[END_REF][START_REF] Doi | Interactions of endothelin receptor subtypes A and B with Gi, Go, and Gq in reconstituted phospholipid vesicles[END_REF] removing coupling to one or more subunits whilst maintaining coupling with another. PTX, an inhibitor of signalling through G i was used to further understand the effect of palmitoylation on PAR 2 G-protein coupling calcium signalling. Interestingly calcium signalling of wt-hPAR 2 and hPAR 2 C361A was reduced by a similar magnitude, however in the mutant receptor, this reduction almost completely ablated the trypsin evoked calcium response suggesting signalling in hPAR 2 C361A is due to coupling with G i . Although previous GPCRs have shown selective uncoupling of Gα subunits [START_REF] Okamoto | Palmitoylation of human endothelinB. Its critical role in G protein coupling and a differential requirement for the cytoplasmic tail by G protein subtypes[END_REF][START_REF] Doi | Interactions of endothelin receptor subtypes A and B with Gi, Go, and Gq in reconstituted phospholipid vesicles[END_REF] these have preferentially lost G i and maintained G q coupling. This was suggested by Okomoto et al as being due to G i requiring a higher level of palmitoylation. However, if palmitoylation of hPAR 2 targets the receptor to G q containing lipid rafts, as with the OTR [START_REF] Rimoldi | Oxytocin receptor elicits different EGFR/MAPK activation patterns depending on its localization in caveolin-1 enriched domains[END_REF], and removal of palmitoylation results in the receptor locating outside of the raft then association with G q would be disrupted. A recent study suggested that PAR 2 , unlike PAR 1 , does not signal through G i [START_REF] Mccoy | PAR1 and PAR2 couple to overlapping and distinct sets of G proteins and linked signaling pathways to differentially regulate cell physiology[END_REF], however in our cell system, it appears that in order to provoke calcium signalling hPAR 2 C361A couples to G i. Like DeFea et al we showed that hPAR 2 activates ERK1/2 through a PTX insensitive pathway, so having little or no involvement by G i [START_REF] Defea | beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2[END_REF]. In contrast, the non palmitoylated hPAR 2 C361A displayed reduced ERK phosphorylation post SLIGKV-NH 2 activation, suggesting a G i mediated response. Numerous studies have shown ERK activation to be independent of G-protein signalling and PAR 2 ERK signalling has been shown to be mediated by β-arrestin association [START_REF] Zoudilova | beta -arrestin-dependent regulation of the cofilin pathway downstream of protease-activated receptor-2[END_REF]38]. It maybe the increased association with G i results in a shift in the preferred pathway for ERK1/2 activation. The signalling pathway responsible for activation of ERK in PAR 2 has previously been shown to "switch" in the case of PAR 2 δST363/366A, a phosphorylation deficient mutant, which activated ERK1/2 via a proline-rich tyrosine kinase-2 (PYK2) mediated pathway [START_REF] Defea | beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2[END_REF]. It was hypothesized that the interaction with the PYK2 pathway was a result of the prolonged Ca 2+mobilisation observed in this mutant. As stated earlier Ca 2+ -mobilisation following hPAR 2 C361A activation takes longer to reach a signalling plateau so it maybe that the G i -dependant activation of ERK1/2 activation observed is PYK2-mediated. In addition to G i and G q , PAR 2 is also thought to signal through G 12/13 and Rho pathways [START_REF] Mccoy | PAR1 and PAR2 couple to overlapping and distinct sets of G proteins and linked signaling pathways to differentially regulate cell physiology[END_REF]. Although we have not investigated these pathways here it would be interesting to determine the effect of PAR 2 palmitoylation status on these pathways.

The rate and extent of internalisation in response to trypsin is increased in the palmitoylated mutant compared to wt-hPAR 2 . However, neither mutant nor wild-type receptor showed internalisation following treatment with SLIGKV-NH 2 . This is contrary to what has previously been reported for PAR 2 following SLIGKV-NH 2 treatment [START_REF] Defea | beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2[END_REF][START_REF] Dery | Trafficking of proteinase-activated receptor-2 and beta-arrestin-1 tagged with green fluorescent protein. beta-Arrestin-dependent endocytosis of a proteinase receptor[END_REF][START_REF] Bohm | Mechanisms of desensitization and resensitization of proteinase-activated receptor-2[END_REF]. Internalisation rather than receptor degradation of PAR 2 was confirmed by permeabolising cells and using endocytotic and PKC inhibitors concanavalin A and hyperosmolar glucose as well as fixing the membrane (data not shown). Hypertonic sucrose is known to prevent the recruitment of clathrin and interferes with normal coatedpit formation and endocytosis [START_REF] Hansen | Clathrin and HA2 adaptors: effects of potassium depletion, hypertonic medium, and cytosol acidification[END_REF][45], and concanavalin A is known to prevent the formation of coated pits [46][START_REF] Jiang | Regulation of membrane-type matrix metalloproteinase 1 activity by dynamin-mediated endocytosis[END_REF]. As such we have also demonstrated that hPAR 2 internalises by clathrin-coated pits, and hPAR 2 C361A internalises by the same mechanism albeit more rapidly. Although this study is the first to report a differential effect of receptor cell surface expression and post-agonist endocytosis with trypsin and SLIGKV-NH 2 , the internalisation seen post-SLIGKV-NH 2 treatment in previous publications maybe due to over-expression of β-arrestin in the cell lines used. A recent study has shown over-expression of β-arrestin to result in its constitutive association with cellular components [START_REF] Zoudilova | beta -arrestin-dependent regulation of the cofilin pathway downstream of protease-activated receptor-2[END_REF].

Despite increased expression of hPAR 2 C361A compared to PAR 2 in the resting state, with the addition of agonists the endocytosis of the mutant receptor appears increased over the wild type. This enhanced post-activation endocytosis may be for a number of different reasons. If as previously discussed hPAR 2 C361A is constitutively phosphorylated then β-arrestin may associate with the receptor C-terminus. For the conformational change to occur in β-arrestin that allows high-affinity binding of AP-2 and clathrin, resulting in endocytosis, it must be bound to a receptor that is both phosphorylated and activated [START_REF] Moore | Regulation of receptor trafficking by GRKs and arrestins[END_REF]. Since β-arrestin is already associated with the receptor upon activation, endocytosis can occur with fewer binding steps. If hPAR 2 functions similarly to the OTR, then following activation the receptor depalmitoylates and needs to translocate from the caveolae domain before being endocytosed [START_REF] Rimoldi | Oxytocin receptor elicits different EGFR/MAPK activation patterns depending on its localization in caveolin-1 enriched domains[END_REF]. The removal of hPAR 2 palmitoylation would mean the receptor was already located outside of the caveolae domain allowing clathrin binding and endocytosis without the need for receptor translocation. The increase in the amount of receptor endocytosed post-activation may occur as a result of uncoupling from G q , phospholipase C (PLC) activity has previously been shown to inhibit endocytosis via clathrin-coated pits [START_REF] Carvou | Signalling through phospholipase C interferes with clathrin-mediated endocytosis[END_REF]. Since hPAR 2 C361A has reduced/abolished coupling to G q , PLC activity would be significantly reduced/abolished so allowing increased endocytosis via clathrin-coated pits in the absence of inhibition from PLC.

Upon activation the receptor must become depalmitoylated and phosphorylated before βarrestin binding. In order to mediate receptor endocytosis β-arrestin must undergo a conformational change which increases binding affinity of clathrin and AP-2 [START_REF] Moore | Regulation of receptor trafficking by GRKs and arrestins[END_REF]. For this conformational change to occur the receptor must be both in an activated confirmation and phosphorylated [START_REF] Moore | Regulation of receptor trafficking by GRKs and arrestins[END_REF]. Additionally, Receptor phosphorylation has previously been shown to be the rate limiting step for β-arrestin association [50]. As stated earlier it may be that the removal of palmitoylation results in the receptor being phosphorylated due the increased availability of the C-terminal phosphorylation sites, thus allowing β-arrestin binding but endocytosis does not occur until the receptor is activated. Since βarrestin is already associated with the receptor endocytosis can occur immediately without initial βarrestin association. It would be interesting to prove this by further experimentation. This is the first study investigating the palmitoylation status of hPAR 2 and its effect on receptor function. We have demonstrated that hPAR 2 is palmitoylated pre-dominantly, if not entirely on C361. We have further demonstrated the multifaceted importance of palmitoylation on receptor expression, agonist sensitivity, densensitisation and internalisation.

50 Krasel, C., Vilardaga, J. P., Bunemann, M. and Lohse, M. J. ( 2004) Kinetics of Gprotein-coupled receptor signalling and desensitization. Biochemical Society transactions. 32, 1029-1031 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 31 May 2011 as manuscript BJ20101958 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101958A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 31 May 2011 as manuscript BJ20101958 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101958A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 31 May 2011 as manuscript BJ20101958 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101958A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1 .

 1 Figure 1. Model in order to illustrate placement of haemagglutinin epitope tag (HA.11) and the four cysteine residues of interest. C361 is shown as a putative palmitoylation site and C148 and C226 are shown as putative sites for a disulphide bridge.

Figure 2 .

 2 Figure 2. Assessment of receptor cell surface expression at varying cell confluences. Pro5 cells stably expressing wt-hPAR 2 and hPAR 2 C361A, were grown to confluence levels of 20 -70% before harvesting and labelling with Sam11 anti-PAR 2 antibody and anti-mouse FITC conjugate and analysing using flow cytometry. Results are expressed as the mean ± SEM of 4 separate experiments.

Figure 3 .

 3 Figure 3. Confocal images of wt-hPAR 2 and hPAR 2 C361A localisation within the cell. Cells were incubated with Sam11 anti-PAR 2 primary antibody and anti-mouse FITC conjugated secondary antibody. Coverslips were then mounted and analysed by confocal microscopy. (A) -pcDNA3.1 empty vector. (B) -wt-hPAR 2 . (C) -hPAR 2 C361A. Fixed and permeabilised cells were incubated with anti-HA11 primary antibody and anti-mouse FITC conjugated secondary antibody before analysing by confocal microscopy. (D) -pcDNA3.1 empty vector. (E) -Wt-hPAR 2 . (F) -hPAR 2 C361A. Presence of PAR 2 at the plasma membrane is indicated with arrowheads . PAR 2 contained internally is indicated with arrows . Images shown are representative of three experiments

Figure 4 .

 4 Figure 4. [ 3 H]-palmitate incorporation into wt-hPAR 2 and hPAR 2 C361A. HA11-epitope immunoprecipitated protein preparations from empty vector, wt-hPAR 2 and hPAR 2 C361A cells each labelled with 1 mCi [ 3 H]-palmitate for 4 hours run on a 10% SDS-PAGE. Gel dried under vacuum and exposed to Hyperfilm™ x-ray film at -80 o C for 6 weeks (A). A portion of the same samples were analysed on western blot using anti-HA11 antibody to assess receptor loading (B) and a 20 min exposure time. Blots shown are representative of three experiments.

Figure 5 .

 5 Figure 5. Agonist concentration effect curves for wt-hPAR 2 and hPAR 2 C361A. Fluo3 loaded Pro5 cells stably expressing for wt-hPAR 2 and hPAR 2 C361A were stimulated with increasing concentrations of trypsin or SLIGKV-NH 2 and increases in intracellular calcium concentrations were measured. Results were expressed as a percentage of the maximum attainable response (response to A23187, calcium ionophore). Results are expressed as the mean ± SEM of 4 separate experiments. Each preformed in duplicate.

Figure 6 .

 6 Figure 6. Graph showing inhibition of trypsin mediated Ca 2+ release in wt-hPAR 2 and hPAR 2 C361A. Changes in intracellular calcium levels were measured using a fluorospectrometer in fluo3 loaded wt-hPAR 2 and hPAR 2 C361A Pro5 cells incubated with different concentrations of PTX (for 18 hours) prior to addition of 100 nM trypsin. Values shown as a percentage of the maximum attainable response (response to A23187).

Figure 7 .

 7 Figure 7. Agonist stimulated MAPK phosphorylation curves and representative phosphorylated and total p44/42 western blots. Graphs showing densitometric values for bands produced by western blot for phosphorylated p44/42 post-SLIGKV-NH 2 treatment [A] and post trypsin treatment [B]. Normalised values are expressed as a fold change over no treatment (NT). Results are expressed as the mean ± SEM of 3 separate experiments, while the bands above are representative of the blots.

Figure 8 .

 8 Figure 8. Western blots and bar charts showing inhibition of MAPK with PTX. wt-hPAR 2 (A), and hPAR 2 C361A (B) cells were incubated with and without 100 ng/ml PTX (for 18 hours) prior to addition of 20 nM trypsin (Tryp) or 100 μM SLIGKV-NH 2 (KV) or no treatment (NT) for 10 mins.

Biochemical

  Journal Immediate Publication. Published on 31 May 2011 as manuscript BJ20101958 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101958 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited Cells were lysed using laemlli buffer before separating by 10% SDS-PAGE and immunoblotting. Normalised values are expressed as a fold change over no treatment (NT). A representative total p44/42 and anti-phospho p44/42 blot is shown above the respective histogram. Results expressed as the mean ± SEM of 3 separate experiments.

Figure 9 .Figure 1

 91 Figure 9. Time course of receptor cell surface expression post-agonist addition. Pro5 cell stably expressing wt-hPAR 2 and hPAR 2 C361A were incubated with either 100 nM trypsin or 100 µM SLIGKV-NH 2 for increasing periods of time before washing and labelling with Sam11 anti-PAR 2 antibody and anti-mouse FITC conjugate and analysed using flow cytometry. Results are expressed as the mean ± SEM of 3 separate experiments

  Figure 2

  Figure 3

  Figure 4

Figure 5 wt

 5 Figure 5

Figure 6 wt

 6 Figure 6

Figure 7 AB

 7 Figure 7

Figure 8 AB

 8 Figure 8

Figure 9 Time

 9 Figure 9

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

FUNDING

This study was supported by a British Heart Foundation PhD Studentship [grant number FS/03/078/15918]. REFERENCES 1 Compton, S. J., Sandhu, S., Wijesuriya, S. J. and Hollenberg, M. D. (2002) Glycosylation of human proteinase-activated receptor-2 (hPAR2): role in cell surface expression and signalling. Biochem J. 368, 495-505


Markus Kaufmann
email: kaufmann@kth.se

Dan Zenkert

Christophe Mattei

Cost optimization of composite aircraft structures including variable laminate qualities

Keywords: A. Structural Composites, D. Non-destructive testing, B. Strength, C. Finite element analysis (FEA), Optimization

Composite structures can lower the weight of an airliner significantly. The increased production cost, however, requires the application of cost-effective design strategies in which cost, weight and the desired laminate quality are taken into account. This paper proposes an optimization framework for composite aircraft structures that minimizes the direct operating cost on a part level. In addition to previous models, a non-destructive testing model is implemented that calculates design allowables of a laminate based on the ultrasonic scan parameters. In a case study, the effect of the laminate quality on the direct operating cost is discussed. It is investigated how the flaw size and therefore the scan pitch of a composite laminate can influence the optimal solution in terms of cost and weight; thus, the manufacturing cost, the non-destructive testing cost and the weight of a component can be balanced by optimizing the laminate quality in an early design phase.

Introduction

Today's performance requirements necessitate the full application of carbon fibers to primary structures of airliners. The shift to composite materials lowers the structural weight significantly; the drawback, however, is the increased manufacturing cost.

Through cost-effective design, tradeoffs between the minimum weight and the minimum ACCEPTED MANUSCRIPT cost solution are found, two extremes that often contradict each other. Weight savings are directly associated with a reduced fuel consumption and an increased payload, while the reduced manufacturing cost has an impact on the acquisition cost. Therefore, the focus should not only be on pure weight reduction, but rather on a combined minimization of manufacturing cost and structural weight.

Attempts for a combined cost/weight optimization have been done by Geiger and Dilts [START_REF] Geiger | Automated design-to-cost: Integrating costing into the design decision[END_REF], Heinmuller and Dilts [START_REF] Heinmuller | Automated design-to-cost: Application in the aerospace industry[END_REF], Kassapoglou [START_REF] Kassapoglou | Simultaneous cost and weight minimization of composite-stiffened panels under compression and shear[END_REF][START_REF] Kassapoglou | Minimum cost and weight design of fuselage frames. part a: Design constraints and manufacturing process characteristics[END_REF][START_REF] Kassapoglou | Minimum cost and weight design of fuselage frames. part b: Cost considerations, optimization, and results[END_REF], Marx et al. [START_REF] Marx | A knowledge-based system integrated with numerical analysis tools for aircraft life-cycle design[END_REF], Gantois [START_REF] Gantois | The multi-disciplinary design of a large-scale civil aircraft wing taking account of manufacturing costs[END_REF], the Rolls-Royce's DATUM project [START_REF] Scanlan | Datum project: Cost estimating environment for support of aerospace design decision making[END_REF], Park et al. [START_REF] Park | Simultaneous optimization of composite structures considering mechanical performance and manufacturing cost[END_REF], Edke and Chang [START_REF] Edke | Shape optimization of heavy load carrying components for structural performance and manufacturing cost[END_REF], and Curran et al. [START_REF] Curran | Integrated digital design for manufacture for reduced life cycle cost[END_REF]. Further, Kelly and Wang [START_REF] Kelly | A guided tradeoff for cost and weight for generating optimal conceptual designs[END_REF], Wang et al. [START_REF] Wang | Multi-objective optimisation of composite aerospace structures[END_REF], Curran et al. [START_REF] Curran | A numerical method for cost-weight optimisation of stringer-skin panels[END_REF], and Kaufmann et al. [START_REF] Kaufmann | Integrated cost/weight optimization of composite skin/stringer elements[END_REF] incorporated the idea of a weight penalty; this weight penalty was applied to balance the tradeoff between manufacturing cost and structural weight. Each kilogram of structural weight was given a lifetime fuel burn cost and the sum of fuel burn cost and manufacturing cost formed the objective function. Apart from embedding cost and weight, not much research has been performed on optimizing aircraft structures for life-cycle cost (LCC) or direct operating cost (DOC), as defined by Roskam [START_REF] Roskam | Airplane Design Part VIII: Airplane Cost Estimation Design Development and Manufacturing and Operating[END_REF].

Further, no attempts have been done to include the material quality of composite laminates into these optimization frameworks.

In the studies cited above it is assumed that the quality of the raw material is equal and no isolated inspection cost were identified in the cost models. This is due to the fact that the models have been built up for metallic structures, where in-service inspection plays a minor role in the design process or that the cost model was not detailed enough to capture these costs. Unlike their metallic counterparts, however, composite parts are fabricated in-situ and the quality of these structures is highly dependent on the process robustness and the workmen skills. As a result, every composite part has to undergo rigorous ultrasonic testing. Here, non-destructive testing (NDT) currently is the key to maintain the quality assurance of the structure. Typical manufacturing generated defects in composite parts are voids, porosity, fiber misalignment, wrinkling, poor cure, resin-rich or resin-poor areas and the inclusion of foreign bodies.

Both the manufacturing of autoclave prepreg parts and the NDT methods have continuously been developed and refined during the last years. Nevertheless, the design is still based on rather conservative presuppositions, such as two percent through-the-thickness porosity and flaws of a size of typically 6mm in diameter. In this work, these presuppositions are challenged. In particular, the optimization framework developed in [START_REF] Kaufmann | Integrated cost/weight optimization of composite skin/stringer elements[END_REF] has been enhanced by an NDT cost and strength reduction model.

The first time NDT cost has been mentioned in the literature was in 1937 by Schmid [START_REF] Schmid | Costs involved in non-destructive testing of materials[END_REF]. Schmid investigated the cost of x-ray, gamma and magnetic testing of metal parts in terms of recurrent and non-recurrent cost. Although the test methods and components were not related to aircraft structures, this article is noteworthy. Schmid pointed out that the choice of an appropriate testing method has to be in proportion to its cost, the cost of the structural part and the cost of possible flaws and damages.

Depending on the complexity of the part, different technical skills were needed, thus leading to different labor cost. Simple, similar components, for example, need a less qualified operator than complex, forged components. The cost was further determined by the inspection method, the part size and the accuracy in flaw detection.

Since then, most of the relevant literature concentrated on the cost of aircraft maintenance and not on in-production testing. For instance, the life-cycle cost of aircrafts under the consideration of NDT was addressed by Hagemaier in [START_REF] Hagemaier | Cost benefits of nondestructive testing in aircraft maintenance[END_REF][START_REF] Hagemaier | Effective implementation of NDT into aircraft design, fabrication, and service[END_REF].

Another example is shown by Kale [START_REF] Kale | Tradeoff of structural weight and inspection cost in reliability based optimization using multiple inspection types[END_REF] who combined the weight optimization of an aluminum panel with a reliability based model of the periodic in-service inspection.

In this work, an cost/weight optimization framework is proposed that incorporates manufacturing cost, inspection cost and the weight of the component. In addition to previous studies, the NDT cost are calculated separately by means of a feature-based cost estimation model. Moreover, the laminate quality is considered as a design variable; this variable has a distinct influence on the scan parameters and therefore the scan cost, and it controls the strength prediction of the laminate.

The optimization framework herein consists of the necessary routines to calculate the objective function and the constraints. In the following these blocks are described in detail.

Optimization framework

Our problem for the combined optimization of manufacturing cost, NDT cost and weight is formulated as min DOC of a composite element subject to structural requirements (1)

x i < x i < x i , i = 1 . . . n
where x i are the design variables and x i and x i the corresponding lower and upper limits. Examples of variables are laminate thicknesses, the distance between two adjacent stiffeners, the height and width of the stiffeners or the laminate quality.

The objective function of the optimization problem is formed by a simplified version of the direct operating cost DOC. Here, the DOC is formed by the weighted sum

DOC = α 1 C man + α 2 C ndt,prod + α 3 NC ndt,serv + pW (2)
where C man is the manufacturing cost, C ndt,prod is the cost for non-destructive in-production testing, C ndt,serv is the cost of a single in-service inspection, p is a weight penalty (given as the lifetime fuel burn cost per weight) and W is the weight of the structure. The parameters α i incorporate calibration factors due to depreciation, overhead cost and other cost adjustments, and N is the number of regular inspections during the lifetime of the aircraft. In a first step this number is a constant, in future this value could be based on the location, the stress level and the exposure of the part.

In Fig. 1, the optimization routine is illustrated, including an analysis block containing an FE model, a manufacturing cost model, a weight model and an NDT model.

FE Analysis

The FE evaluation is done in ABAQUS/Standard and comprises a linear buckling analysis and the computation of a composite failure criterion. It is important to note that the results and conclusions presented here are specific to the flaw type and size included in the analysis and the failure modes assumed (buckling and maximum strain failure) and cannot be generalized before other failure modes (e.g. crippling, local buckling of a delamination, impact damage) are accounted for. Hence, the structural constraints used herein are only examples of possible criteria; the modular design of the optimization framework allows also the implementation of a more advanced analysis.

The FE model has been parameterized in terms of variables, shown as x i in Equation [START_REF] Geiger | Automated design-to-cost: Integrating costing into the design decision[END_REF]. Changes in the variables result in a new set of buckling eigenvalues or maximum strain numbers; these form the structural constraints to the optimization problem and have to be fulfilled in order to provide a feasible solution. All changes in the ABAQUS model are done by means of Python scripts.

Manufacturing Cost

During the manufacturing of composites, several operations and processes can be related to costs. Examples are the stacking of prepreg plies, debulking, consumables, curing or tooling. Each process represents a time need and the material cost. Time needs can be translated into labor costs; the sum of all cost plus an overhead represents the total cost to manufacture the panel. This kind of feature-based model (see Niazi et al. [START_REF] Niazi | Product cost estimation: Technique classification and methodology review[END_REF]) is easily parameterizable, as features can be added, removed or modified. The commercially available cost estimation software SEER-DFM1 incorporates all these capabilities and is used therefore. Similar to the approach done for the FE calculation, an initial model has to be built up in the graphical user interface of the software. This model contains all the necessary assumptions and work steps of the manufacturing process; it is exported to a text file, parameterized in terms of variables x i and prepared for running in command line mode, the so-called server mode.

Weight estimation

The estimation of the weight is done by a simple calculation of the part volume in any given iteration, based on the CAD geometry, the material properties and the actual variables x i .

Non-Destructive Testing

Unlike the estimation of the manufacturing cost, it was decided to develop an in-house feature-based tool for the evaluation of the non-destructive testing cost. It contains the estimation of the NDT cost for in-production and in-service testing, and the adjustment of the material strength.

Some preconditions that apply to the manufacturing of composite aircraft structures can be assumed. For example, all components are thoroughly tested before the assembly, which includes that the total surface of each structural member is scanned. Testing is performed on condition of series production, i.e. the testing process is cost optimal in terms of operator level and test equipment and no non-recurring costs (such as the purchase of probes or other equipment) are incorporated. No reject rate has been implemented; the development of a reliability-based cost model will be developed in a later stage.

In the case of NDT, the generic database provides the following features:

• Thin flat laminate with access from both sides

• Thin flat laminate with access from one side

• Thick flat laminate with access from both sides

• Thick flat laminate with access from one side

• Radius • Adhesive bond
Each of these features requires input data in form of the length l k , the width w k , the thickness t k and -in case of radii -the radius r k , see Fig. 2. On the other hand, the feature definition includes a scanning technique, a complexity index, the educational level of the operator and associated with the latter a cost per hour or per scanned area.

For rectangular scans of plates, flanges and adhesive bonds, the NDT cost of the k th feature is calculated as

C ndt,k = l k w k v k d k • C machine,k + C operator,k (3)
where v k is the scan speed and d k is the scan pitch (distance between two scan paths) of the feature k. The parameters C machine,k and C operator,k represent the hourly rate of the test equipment and the operator, respectively.

For linear scans of radii, the NDT cost is calculated as

C ndt,k = l k v k • C machine,k + C operator,k (4)
and the total cost of non-destructive testing is the sum of all features

C ndt = N k=1 C ndt,k N = Number of features. ( 5
)
Apart from the scanning cost per area, one has to consider a dependency of the cost on the laminate thickness. Several approaches of how to tackle this problem have been examined. Finally, it has been decided to introduce the very flexible method of adjusting

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the structural allowable due to the higher risk of porosity or flaws in thicker structures.

Hence, a relationship between testing cost and weight is found through the quality of the laminate. The following two reductions of the material strength are evaluated.

• Strength σp,k due to possible porosity; a function of the thickness and the material of the cross-section.

• Strength σf,k due to possible flaws or foreign bodies; a function of the scan pitch, the thickness, the material properties (based on coupon tests) and the NDT equipment.

Note that all strengths are calculated feature-wise and the strength that is applied in the FE calculation is the minimum of each σp,k or σf,k . Both mechanisms are described below.

Strength reduction due to porosity

In the design of composite structures, a certain level of porosity is tolerated and included in the calculation of the structural performance. Typically, a level of two volume percents is stated, a level that has been kept constant during the last two decades. With regard to new material systems, optimized manufacturing methods and more accurate NDT techniques, this porosity level is about to be challenged.

A lot of work has been done to investigate the effect of the laminate thickness and the laminate porosity on the mechanical properties, see [START_REF] Judd | Voids and their effects on the mechanical properties of composites -an appraisal[END_REF][START_REF] Uhl | Mechanical strength degradation of graphite fiber reinforced thermoset composites due to porosity[END_REF][START_REF] Rubin | Effect of porosity on elastic constants of representative aircraft laminates[END_REF][START_REF] Costa | Critical void content for polymer composite laminates[END_REF][START_REF] Lee | Thickness effect on the compressive strength of t800/924c carbon fibre-epoxy laminates[END_REF][START_REF] Lee | A study on the compressive strength of thick carbon fibre-epoxy laminates[END_REF]. It has been concluded that there exists an exponential degradation of the compressive strength σc , although this decline does not appear as distinctive as for the interlaminar shear strength τ . In the context of isotropic materials, the equation

σ = σmax e Bvp (6)
has elevated to describe this behavior; here, σmax is the strength at zero porosity, v p is the volume fraction of porosity and B is an empirical constant that depends on orientation, shape and size of the pores.

As the proposed method is a part of the design process, the laminate thickness t has to be passed into an anticipated porosity level v p for a given material and manufacturing technique. Lee and Soutis [START_REF] Lee | Thickness effect on the compressive strength of t800/924c carbon fibre-epoxy laminates[END_REF] performed a study on the void content with respect to the thickness of the specimen and concluded that this value is highly dependent on the stacking sequence of the laminate.

Strength reduction due to foreign bodies

All manufactured composite parts have to be tested for the inclusion of foreign bodies (e.g. parts of the release paper of the prepreg) or other delaminations. Nowadays, the composite manufacturer defines a minimum flaw size that has to be detected, typically being around 6mm in diameter.

The probability of detection never reaches 100%. Instead, a stochastic value based on the material, the NDT method and the thickness of the material is assumed; quoted levels are 90% or 95%, see [START_REF] Georgiou | Pod curves, their derivation, applications and limitations[END_REF]. According to Huang et al., typical stochastic models used are the Gamma distribution or the two-parameter Weibull distribution, see [START_REF] Huang | A method for reliability assessment of aircraft structures subject to accidental damage[END_REF].

For the Weibull distribution, the cumulative distribution function of the probability of detection (POD) is given as

POD = 1 -e -(f /λ) k ( 7
)
where f is the flaw size, and λ and k are the two shape parameters. A typical curve plot for different settings of λ is shown in Fig. 3.

As can be seen in this figure, the POD is higher for thinner laminates (the actual value depends on the material, the stacking sequence and the NDT method) and for larger The variable quality level can be explained as follows: Let us assume that an allowed flaw diameter f of less than 6mm is applied to a laminate. All flaws that are bigger than f will be detected, thus causing the part to be rejected. In this case, the (cost) optimum is found as the tradeoff between the higher NDT cost on one side and the lower structural weight and manufacturing cost due to higher strength on the other side. On the other hand, a flaw diameter f of more than 6mm would lead to lower NDT cost, a higher structural weight and higher manufacturing cost due to the additional plies needed.

The resulting algorithm is shown in Fig. 4. It can be seen that the thickness t and the maximum allowed flaw size f form the inputs to the NDT module. In the first step, the scan pitch d k is adapted until a desired probability of detection is met. The direct cost for non-destructive testing C ndt is a function of the scanned area (length l and width w)

and the scan pitch d k . Simultaneously, the strength reduction is calculated and fed into the FE module where the constraints for the optimization are computed.

Solver

Based on the results of the analysis, the objective function and the optimization constraints are evaluated. Then the solver calculates adjusted variables and triggers the execution of the next iteration. For that purpose, a solver was sought that would incorporate the analysis blocks in the form of input-output models, not be too sensitive to disturbances in the form of non-smooth objective and constraint functions, and thus lead to a good convergence rate. In order to minimize the number of FE evaluations, the method of moving asymptotes (MMA) has been chosen, see Svanberg [START_REF] Svanberg | Method of moving asymptotes -a new method for structural optimization[END_REF][START_REF] Svanberg | A globally convergent version of MMA without linesearch[END_REF]. One can see in the case study below that the parametrization of the design variables as continuous variables is a rough simplification which accommodates the use of this gradient-based method.

In reality, the thickness is not a continuous variable and it is possible that a gradient-based method will miss some designs that may be more efficient that the ones obtained from a gradient-based optimization. Hence, the results from the present study are viewed as representative of the trends and relative differences of alternate designs but may not point to the "best" solution.

Case Study

In a case study, a generic skin/stringer element has been optimized in order to show the influence of the NDT cost and the quality level on the overall design. The panel (seen in For the sake of simplicity, it has been chosen to neglect any depreciation factors and cost adjustments (i.e. α 1 = α 2 = α 3 = 1) and apply the same equipment and operator rates for in-production and in-service inspection. The prescribed probability of detection has been set to 95%.

Here, five regular inspections during the lifetime of the aircraft have been assumed.

Therefore, the objective function results in

DOC = C man + C ndt,prod + 5C ndt,serv + pW ( 8
)
The attention is drawn to the quantification of the weight penalty. One can imagine that the cost/weight tradeoff is dependent on factors such as accumulated flight distance during aircraft life, engine specific fuel burn, thrust level during flights, environmental impact penalties or fuel price. Note that an objective determination of p is difficult, as it may vary from customer to customer, the application (civil, cargo, military version of the same plane), whether it is optimized for additional payload, fuel saving or additional passengers and the way the customer evaluates his product. The literature proposes a weight penalty between €45/kg and €380/kg. Own research based on the fuel consumption of an airliner in the A330 class and today's fuel price, however, reveals a different picture. In this case, the weight penalty p = lifetime fuel burn cost/kg aircraft mass would then result in about €1840/kg. The mass reduction of one kilogram might involve a reduction of the gross weight that is significantly higher, as the weight of engines and systems can be lowered. And when taking the revenue of an additional passenger into account, this value could be one order of magnitude higher. Nevertheless, it has been decided to apply a value of €1500/kg to the case study in order to accommodate both values from the literature and our own calculations.

The FE model has been built up in ABAQUS/CAE according to the drawing in Fig. 5.

It is constrained by periodical boundaries along the sides and meshed with shell elements of the type S4R. The introduction of the compressive load has been realized by two rigid bodies, simulating the adjacent frame structure. The adhesive bonds have been approximated by tie constraints, thus preventing any separation of skin and stringers.

Two failure criteria were checked during the FE analysis. First, the maximum strain values (MSTRN) were sought in all integration points of the skin and stringer nodes, and second, the eigenvalues of a linear perturbation analysis is calculated. Both are fed back to the optimization solver. Rather high loads have been applied to this structure (2.5 kN per mm width) in order to obtain comparable failure loads for buckling and strain; as can be seen below, the model will consist of rather short and stubby stiffeners.

The maximum strain criterion has been set to 0.005, -0.004 and 0.008 for tension, compression and shear, respectively.

Referring to the description of the NDT model above, the structure is divided into features, such as the skin laminate, further two stringers with two flange laminates, one web laminate and four radii each; the two adhesive bonds between skin and stringer are two other features.

The adjustment of the allowables due to porosity is estimated according to Equation [START_REF] Marx | A knowledge-based system integrated with numerical analysis tools for aircraft life-cycle design[END_REF]. The value of B = -3.14 has been adopted from Uhl et al. [START_REF] Uhl | Mechanical strength degradation of graphite fiber reinforced thermoset composites due to porosity[END_REF] who performed a number of test series for a 16 ply quasi-isotropic carbon/epoxy laminate. The void content v p as a function of the thickness is required as an input for the equation above.

The approximate function v p = 0.0025t has been extracted from the figures presented by Lee and Soutis [START_REF] Lee | Thickness effect on the compressive strength of t800/924c carbon fibre-epoxy laminates[END_REF]; in their work they studied the void content with respect to the thickness of the specimen. In this first attempt, the strength due to flaws of a given size f is based on the work done by Soutis et al. [START_REF] Soutis | Predicting the compressive engineering performance of carbon fibre-reinforced plastics[END_REF], from which the value of σf (f ) = σ0 (-12.4 444 (f -6) + 1) has been extracted. Here, σ0 refers to the strength of a coupon test with 6mm hole size.

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NDT tool presented in this paper.

Finally, the behavior of each configuration is investigated when varying the quality level and laminate thicknesses while the geometry is kept constant. Further the influence of the probability of detection POD on the direct operating cost is examined.

Results and Discussion

The first part of this section refers to a parameter study done without the activation of the NDT model. Hence, the results are obtained by optimizating a skin/stringer element with an objective function given as DOC = C man + pW and the weight penalty p varying between 0 and infinity. The results are shown in Fig. 6.

A distinctive change in configuration can be seen at a weight penalty of €1000-10'000/kg. To the left of this change, a low-cost solution is preferable. There, the skin/stringer panel consists of a thick skin and bulky stiffeners with maximum pitch -as fewer stringers are used in the low-cost solution, manufacturing costs can be saved in spite of the higher cost per stiffener. Further, the buckling constraint but not the maximum strain constraint is active. Hence, the structure is not performing optimally, and some more cost saving could be possible by enlarging the design space. To the right, the low-weight configuration comprises a densely stiffened, thin skin with rather fine stringers and both the structural constraints are active. Three design solutions have now been chosen as the starting point for the subsequent investigation, see Table 1.

Properties marked with asterisks are kept constant in the subsequent investigation.

Quality optimization of the low-cost panel (p =€0/kg) First, the cost optimized panel with a stringer pitch of 300mm has been investigated.

This panel has been optimized for a series of flaw sizes, varying from 2 to 20mm. The results of this study are compiled in Table 2, where a flaw size of 6mm represents the baseline.

As can be seen, the variation of the laminate quality has a considerable impact on the objective function DOC. At a flaw size of 2mm, the laminates have to be scanned much tighter in order to guarantee a probability of detection of 95%. The result is an NDT cost that is 2.74 times the baseline and a DOC that is 46% higher than the baseline. It also can be seen that only the buckling constraint is active for small flaw sizes. As the buckling load is independent of the strength of the material, it is evident that no weight reduction is possible in this case.

The cost for inspection C ndt decreases when the flaw size increases, as the scan time is reduced. The optimum configuration can be found at a flaw size of 16.3mm. There, the active constraint switches from buckling to maximum strain and the objective function DOC is 14% lower than the one of the baseline. Any further increase of the flaw size is disadvantageous, as the impact of the manufacturing cost due to additional material (to resist material failure) is too high.

Quality optimization of the intermediate panel (p =€1500/kg)

Second, an intermediate panel design with €1500/kg has been investigated. Similar to the preceding example, the cost of this panel was recalculated adding the cost for in-production and in-service inspection with a standard flaw size of 6mm. Then the panel has been optimized for a series of flaw sizes, see Table 3.

As seen before, the objective function is the highest for a very small flaw size of 2mm due to the high NDT cost. As the panel thicknesses are constraint by the active buckling condition, no weight or cost saving is possible for this laminate quality.

As long as buckling governs the design, an increased flaw size mainly results in lower As anticipated, the optimum laminate quality is found at very small flaw sizes, and both structural constraints are active.

Variation of the POD

Non-destructive testing theoretically never reaches a probability of detection of 100%. In practice, an artificial limit is therefore set to this value, representing a measure of trustworthiness for the testing method. The effect of the POD limit on the results is now investigated.

The results shown in Table 5 apply for the intermediate panel with a weight penalty of p =€1500/kg. As expected, the quality of laminate is not influenced by the POD, and the optimal design solution is found independently. On the other hand, an increase in NDT cost and thus an increase in the objective function can be seen for higher probabilities. This is the cost one has to account for when a higher NDT reliability is desired.

Conclusion

A framework for the cost/weight optimization of composite structures under consideration of a variable laminate quality has been proposed. A generic skin/stringer element has been adopted as a case study; the main focus has been brought to a parameter study in which the allowed flaw size was varied.

According to the definition of direct operating cost, an objective function has been selected that included manufacturing cost, in-production and in-service inspection and a weight penalty. Starting from a standard flaw size, the effects of the laminate quality on the objective function have been investigated. The optimal flaw size depends on the structure, the material data, the load case and the weight penalty; hence, it has been shown that cost savings are possible by revising the laminate quality already in the design phase. Nevertheless, the conclusions drawn here suggesting that the flaw size does not have to be 6 mm should be viewed as specific to the assumptions and analysis made and they may not be valid when, for example, fatigue is taken into account.

Larger flaw sizes may shorten the fatigue life. Further work is needed in this area before the conclusions can be generalized.

This optimization framework may be used in applications outside the aerospace industry; as an example, one could consider its implementation for the production of rotor blades for windmills.

Future Work

The proposed optimization framework represents a first approach to include quality management aspects into the design process. More work, however, has to be done in order to improve the NDT cost and the NDT strength reduction model. First, the difference between in-production and in-service testing has to be more elaborate by applying different scanning techniques and overhead adjustments. Second, the strength reduction should be applied as a function of the stacking sequence, the material properties and the manufacturing technique. Third, it has to be investigated whether a stiffness reduction due to porosity should be included in the structural model. Fourth, the inspection interval should be adapted to the stress level and the structural function of each concerning member. And finally, a probabilistic damage model should be included to capture the failure and repair of a structural member. This could include a more detailed ultrasonic analysis, which in turn causes a cost increase.

  flaw diameters. Today's 6mm flaw diameter would require a scan pitch d k of approximately 2mm, the latter having a direct influence on the NDT costs. By varying the maximum flaw size, the quality of the laminate becomes a design variable and the material strength experiences an according modification. Different ways to relate the strength to the flaw size exist. Here, open hole compression is proposed, as it represents the most conservative strength reduction for a flaw of a certain size. For examples on the strength reduction of notched laminates, see Soutis et al. [30]. Other options could include a separate strength prediction model in FE (e.g. the simulation of a CAI coupon test) which takes into account the actual stacking sequence and the flaw size of the section, or the implementation of local delamination buckling as a function of the flaw size and the through-the-thickness location of the flaw.

Fig. 5 )

 5 Fig.5) consists of a laminated composite skin and two I-shaped stiffeners that are adhesively bonded to the skin. As can be seen the skin/stringer element is parameterized in terms continuous variables, such as the stringer pitch, the panel thickness, the profile thickness, the web height and the flange width. The discrete stacking stacking sequence [0 m /90 n / ± 45 p ] s has been simplified to a [0/90/ ± 45] s layup with continuous thickness variables. Note that the laminate generally consists of a 70% fibers in the direction of 0 degree, 20% in ±45 degrees and 10% in 90 degrees. This allows the application of one single variable while maintaining the design guidelines that require at least 10% fibers in each ply direction. Here, it is again emphasized on the fact that the chosen test case is representative for a typical problem in aircraft design, and that the geometry, the load case, the material and the failure criteria could easily be exchanged. Moreover, further optimization is possible by allowing the stacking sequence to change. This can lead to more efficient load distribution among skin and stiffeners and result in lower weight and cost configurations.

First, a panel

  has been optimized for a series of weight penalties, thus resulting in a parameter study covering the whole range from pure cost optimization (p = 0) to pure weight optimization (p = ∞). Therefrom, three configurations have been chosen: A low-cost panel (a), a cost/weight optimized panel with a fuel penalty of €1500/kg (b), and a low-weight panel (c). All three baselines have been obtained without the novel

  NDT while the weight and the manufacturing cost remain the same. At a flaw size of 12.4mm, buckling and maximum strain are active, and the DOC achieves its minimum at 96%. Any increase of flaw size results in increased weight and manufacturing cost, thus forcing up the objective function. The stringer cross-sections are very thick at flaw sizes of 14mm and 20mm due to the reduced strength. As the cross-sections are extraordinarily thickened, more scanning is necessary in order to guarantee a probability of detection of 95%. The result is an increase of NDT cost, despite the existence of a lower laminate quality.Quality optimization of the low-weight panel (p =€∞/kg) Third, a weight optimized panel has been exposed to the inclusion of NDT cost. In Table4, these results are shown. As the manufacturing and non-destructive testing costs are a very small part of this objective function, the DOC mainly depends on the weight.

Fig. 1 .Fig. 2 .FEFig. 4 .Fig. 5 .Fig. 6 .

 12456 Fig.1. The optimization loop; the module within the dashed line is subject to further explanation below.

Table 2

 2 Quality optimization of the low-cost panel (p =€0/kg)

	flaw size 2mm 6mm 10mm 12.4mm 14mm 20mm
	DOC	1.16 1.00	0.97	0.96	1.00	1.37
	weight	1.00 1.00	1.00	1.00	1.05	1.46
	C ndt	2.38 1.00	0.72	0.63	0.65	0.68
	C man	1.00 1.00	1.00	1.00	1.05	1.46
	buckling 1.00 1.00	1.00	1.00	0.95	0.50
	MSTRN 0.78 0.77	0.90	1.00	1.00	1.00

Table 3

 3 Quality optimization of the intermediate panel (p =€1500/kg)

	flaw size 2mm 6mm 10mm 14mm 20mm
	DOC	0.99 1.00	1.13	1.36	1.93
	weight	0.99 1.00	1.13	1.36	1.93
	C ndt	2.07 1.00	0.85	0.75	0.74
	C man	1.00 1.00	1.15	1.24	1.36
	buckling 1.00 1.00	0.82	0.48	0.22
	MSTRN 1.00 0.98	1.00	1.00	1.00

Table 4

 4 Quality optimization of the low-weight panel (p =€∞/kg)

	POD	80% 85% 90% 95% 97.5% 99%
	DOC	0.97 0.98 0.99 1.00	1.01 1.03
	weight	1.00 1.00 1.00 1.00	1.00 1.00
	C ndt	0.76 0.81 0.89 1.00	1.11 1.24
	C man	1.00 1.00 1.00 1.00	1.00 1.00
	buckling 1.00 1.00 1.00 1.00	1.00 1.00
	MSTRN 0.78 0.78 0.78 0.78	0.78 0.78

Table 5

 5 Optimization with variable POD for the intermediate panel (p =€1500/kg)

Preprint submitted to Elsevier

see Galorath Inc. at http://www.galorath.com

Acknowledgments

This work is part of the European Framework Program 6, project ALCAS, AIP4-CT-2003-516092. Special thanks go to Alfgam AB for the use of Xopt and to Galorath International for the use of the cost estimation package SEER-DFM.