Adina Ciomaga

Pascal Monasse

Jean Michel Morel

LEVEL LINES SHORTENING YIELDS AN IMAGE CURVATURE MICROSCOPE

Keywords: Partial differential equations, scientific visualization, image shape analysis, image reconstruction curvature scale space

This paper presents an image processing algorithm simulating a sub-pixel evolution of an image by mean curvature motion or by affine curvature motion. The sub-pixel algorithm computes the image curvature directly on the smoothed level lines, and yields a microscopic visualization of the curvature map revealing many image details, and getting rid of aliasing effects. This "curvature microscope" showing curvatures in false colors runs on line on any image proposed by users at http://www.ipol.im/pub/algo/cmmm image curvature microscope/.

INTRODUCTION

Attneave's founding 1954 paper [START_REF] Attneave | Some informational aspects of visual perception[END_REF] on image perception anticipated the numerical analysis of digital pictures by stating that in images: "information is concentrated along contours (i.e., regions where color changes abruptly), and is further concentrated at those points on a contour at which its direction changes most rapidly (i.e., at angles or peaks of curvature)". Yet, a direct computation of curvature on a raw image is impossible. We show in this paper how curvatures can be accurately estimated by a direct computation on level lines after their independent smoothing, as illustrated in Fig. 1. Asada and Brady [START_REF] Asada | The curvature primal sketch[END_REF] introduced the concept of "multiscale curvature". In their paper the contours are approximated by splines and smoothed by a 1D heat equation. Their explicit goal was to implement Attneave's idea that shapes must be represented by curvature extrema. This paper led to increasingly sophisticated attempts to represent planar shapes from their curvatures, and to compute curvature correctly. One of the difficulties is the extraction of the contours on which the curvature should be computed.

(a)

The subject was clarified by several mathematical contributions. Grayson [START_REF] Grayson | The heat equation shrinks embedded plane curves to round points[END_REF] proved that the intrinsic heat equation shrinks and smooths Jordan curves and preserves their topology. The Osher-Sethian level set method [START_REF] Osher | Fronts propagating with curvaturedependent speed: Algorithms based on the Hamilton-Jacobi formulation[END_REF] implemented the motion by mean curvature of a manifold by applying a new PDE (the scalar mean curvature motion) to the distance function to the manifold.

In parallel Mackworth and Moktharian [START_REF] Mackworth | A theory of multiscale, curvature-based shape representation for planar curves[END_REF] proposed a fast numerical scheme for curve shortening. But their shape extraction algorithm was unconvincing. Caselles et al. realized the potential of using directly level lines instead of edges and proposed to perform image analysis directly on the topographic map [START_REF] Caselles | A Kanizsa program[END_REF]. A fast algorithm computing the topographic map was proposed by Monasse and Guichard [START_REF] Monasse | Fast computation of a contrast invariant image representation[END_REF]. Sapiro and Tannenbaum [START_REF] Sapiro | Affine invariant scale space[END_REF] discovered the affine curve shortening and Alvarez et al. [START_REF] Alvarez | Axioms and fundamental equations of image processing[END_REF] the affine invariant and contrast invariant image smoothing. A remarkably fast and simple geometric algorithm for affine shortening was published by [START_REF] Moisan | Affine plane curve evolution: A fully consistent scheme[END_REF]. It has been used for shape identification algorithms in the works of Cao et al. [START_REF] Cao | A Theory of Shape Identification[END_REF].

The present paper builds on the above mentioned contributions and describes a complete image processing numerical chain starting from a digital image and ending with an accurate computation and visualization tool of its curvatures. This chain, which had been outlined in [START_REF] Koepfler | Geometric multiscale representation of numerical images[END_REF], is now completed with a subpixel image reconstruction from an arbitrary tree of level lines. It, hopefully, advances Attneave's program and yields anyway what we shall term an image curvature microscope. The algorithm can be tested on line and many examples of all kinds are provided. 1

LEVEL LINES SHORTENING

Curvature evolutions

Let u(x) be the continuous image, which is assumed to be at least C 2 in a neighborhood of a point x 0 ∈ R 2 , and assume that its gradient is not null, Du(x 0 ) = 0. The scalar curvature of u at x 0 , denoted by curv(u)(x 0 ), is defined by

curv(u)(x 0 ) = u xx u 2 y -2u xy u x u y + u yy u 2 x (u 2 x + u 2 y ) 3/2 (x 0 ). ( 1
)
This scalar curvature at x 0 is linked to the curvature κ(x 0 ) of the level line passing by. Recall that the curvature of a C 2 curve x(s) parameterized by a length parameter s is defined by κ(x) := x (s). The link between the curvature of an image level line κ(x) and the scalar curvature curv(u)(x) at nonsingular points is given in the next formula. Denote by x = x(s) the level line of u passing by x 0 . Then

κ(x 0 ) = -curv(u)(x 0 ) Du |Du| (x 0 ). (2)
This relation already suggests that the curvature can be computed in two quite different ways: either as the curvature of a level line extracted from the image and parameterized by length, or as a 2D differential operator. In both cases, a previous smoothing is necessary. This introduces a new parameter, the smoothing scale, denoted hereafter by t. Hence the notion of curvature scale space which is associated with curve or image evolutions. The smoothing can be therefore alternatively performed by curve shortening,

∂x ∂t = κ(x) (CS) (3)
or by mean curvature motion

∂u ∂t = |Du|curv(u). (M CM ) (4)
As shown by Grayson [START_REF] Grayson | The heat equation shrinks embedded plane curves to round points[END_REF], this (nonlinear) evolution can be applied to any piecewise C 1 curve. The curve instantly becomes smooth and shrinks asymptotically to a circle. Based on this principle, the level lines shortening (LLS) algorithm gives a subpixel evolution by curvature driven flows of both level lines and images. It starts by extracting all level lines of a digital image, with a number of levels sufficient to grant an exact reconstruction of the initial image. Then it simulates a subpixel evolution of the image by moving independently all of its level lines by curve shortening (CS) (resp. affine curve shortening (AS)). The evolved image is eventually reconstructed from its evolved level lines. Thus the algorithm realizes the commutative diagram in Fig. 2. This setting is based on a topological structure, the inclusion tree structure of the level lines as a full and non-redundant representation of an image (see [START_REF] Caselles | Geometric Description of Images as Topographic Maps[END_REF]), and on a topological property, namely the Fig. 2. Level Lines Shortening scheme. monotonicity with respect to inclusion of the curve shortening evolution. The inclusion of the level lines is maintained while performing the smoothing. Thus, the reconstruction can start with the largest level line, namely the frame of the image, and continue by filling from top to bottom in this inclusion tree each region surrounded by each level line. At each step the interior of the current level line is filled in with its own level, and these levels are updated when passing to its descendants.

Denote by BL(Ω) the space of bilinear interpolations of digital images defined on a grid of Ω. We prove in a companion paper the next theorem which justifies the algorithm: Theorem 2.1 Let u 0 ∈ BL(Ω). Then its Level Lines Shortening evolution u(x, t) = LLS(t)u 0 (x) is a (viscosity) solution for the curvature PDE

∂u ∂t = curv(u)|Du|, in R 2 × [0, ∞) u(•, 0) = u 0 , on R 2 . (5)
(A similar result holds for ∂u ∂t = curv(u)

1 3 |Du|, the affine curvature motion (ACM).)

Image Reconstruction from a set of level lines

The algorithm described here performs an accurate image reconstruction from a topographic map, i.e. from an arbitrary family of Jordan curves organized in a tree structure with respect to geometrical inclusion. It starts with a topographic map, namely a family of discrete level lines (typically obtained after (affine) curve shortening) {Σ λ,i } i∈F λ ,λ∈Λ organized in an inclusion tree structure. This tree is walked down in preorder (parent before children) and the interior of the current level line filled in with its level λ. Using that order, each level line is painted before its descendants, ensuring that its private pixels (that is, pixels of its interior and not in the interior of any child) are at the correct level while non-private pixels get painted over by the children.

Each closed curve Σ is stored as a polygon defined by its ordered vertices {P k (x k , y k )} 1≤k≤N . N depends on Σ, and x k and y k are floating point coordinates. We need to fill in all pixels with integral coordinates (j, i) inside the polygon. The filling in of each curve is performed by a fast ray casting algorithm described below.

Polygon intersections with the grid. The goal of this algorithm, which is a preliminary to the filling algorithm, is to find the intersections of a polygonal level line Σ with all horizontal lines y = i. For any given i, these are given by the intersections x i l , l = 1 . . . p of the segments [P k P k+1 ] with the line y = i. These intersections are ordered by their abscissae so that

x i 1 ≤ x i 2 ≤ • • • ≤ x i p .
Translating if necessary Σ by a tiny amount ε vertically, the algorithm secures that no y k is an integer. Therefore p is even, because Σ is a Jordan curve. This gives a simple and fast decision rule: a pixel (j, i) is surrounded by the polygon if and only if j is within an odd interval Filling the interior. Line by line all odd intervals are enumerated and pixels with ordinate i whose abscissa is inside such an interval are filled in with level λ. Due to the inclusion principle it is possible to go from the 2D topology of the level lines to the 1D topology on a dual edge and conversely. As shown in Fig. 3, suppose that two or more level lines belonging to different gray levels intersect a dual edge, leaving the same data points outside and inside: denote them P in and P out . Then the restored gray value at P out is the gray value associated to the largest shape ordered by inclusion which leaves the pixel outside, whereas P in belongs to the smallest shape that includes the pixel. If curves with different orientation cross the same dual edge it is enough to update the gray value at P in . This conforms to our choice of filling the interiors of the lines in the order given by the tree of inclusions.

[x i 2l+1 , x i 2l+2 ].

IMAGE CURVATURE MICROSCOPE

Whenever we talk about curvatures in a digital image, we actually refer to the curvatures of the level lines associated to the image. Most curvature computation algorithms are based on finite difference schemes (FDS) for formula [START_REF] Attneave | Some informational aspects of visual perception[END_REF]. In this case, the curvature depends on the gray values of the neighbor pixels and consequently, high oscillations along transverse level lines appear.

For the sake of precision, we suggest that curvatures should be computed directly on level lines and not on a discrete grid. For a given digital image, one can extract the level lines at given quantization levels. Each level line is stored as a set of ordered points Σ = {P i (x i , y i )} i=0..n , with P 0 = P n . A polygonal line approximation followed by uniform and fine sampling allows to compute realiable curvatures, but only after level line smoothing. The reason for smooth-ing is that bilinear level lines present oscillations due to the grid, which is an interpolation artefact and thus curvatures wouldn't correspond to our visual perception. In this setting, we give meaning to the curvature of a polygonal line, in terms of pointwise behavior at each vertex. The simplest way is to consider at each point P i a discrete curvature k i , by taking the triple (P i-1 , P i , P i+1 ) and compute k i as the inverse of the circumscribed radius R i of this triangle. Set ----→ P i-1 P i := -→ u i = (u 1 i , u 2 i ) and its length

u i = | ----→ P i-1 P i |, re- spectively - → v i = ------→ P i-1 P i+1 = (v 1 i , v 2 i ), with v i = | ------→ P i-1 P i+1 |.
Lemma 3.1 The curvature at vertex P i is given by

k i = 2 u 1 i u 2 i+1 -u 2 i u 1 i+1 u i u i+1 v i . ( 6
)
The computed curvature is then attributed to the pixel surrounding P i . A curvature image is created by associating to each dual pixel (a pixel of vertices at integer coordinates) an average of all curvatures computed in it.

To summarize, the curvature map is computed as follows: 1. extract the tree of level lines at given quantization levels; 2. perform uniform, fine sampling of the level lines; 3. short time curve shortening to each level line; 4. compute discrete curvatures; 5. register at each dual pixel the average of all discrete curvatures computed in and create thus the curvature image.

NUMERICAL RESULTS

A simple binary shape has its curvatures computed in two different ways: by a FDS for formula [START_REF] Attneave | Some informational aspects of visual perception[END_REF] and by smoothing level lines and displaying with high resolution the corresponding curvature map. In the first case the curvature presents oscillations, whereas the second result is coherent with our perception of curvature. It is zero on straight edges, constant on arcs of circles and high at bent parts, such as corners of shapes. An example is presented in Fig. 4, where zero curvatures are displayed in gray, positive ones shade from gray to white and negatives from gray to black. As illustrated in Fig. 5, LLS can be used for a graphic quality improvement of contours. Performing a scaled zoom on the image one can expect to have one level line passing through each dual pixel, and thus to observe more and more exactly the level lines and curvatures at microscopic scale. We display in Fig. 6 an original image, its level lines and the corresponding curvature map before level lines filtering, respectively its LLS evolution, smoothed level lines and the curvature map. Observe that LLS removes JPEG artefacts and performs a nice geometric restoration.

CONCLUSION

The first outcome of Level lines Shortening algorithm is the evolved image, which presents some sort of denoising, simplification, and desaliasing. But the main outcome is an accurate curvature estimate on all level lines. As a visualization tool, the fact that all level lines are polygons with real coordinates allows to zoom in the image at an arbitrary resolution. This is necessary to explore visually the intricacy of the local image structure. Hence the name of curvature microscope given to the final visualization.

  Attneave's cat (b). Curvature map.

Fig. 1 .

 1 Fig. 1. Attneave's figure illustrating the prominent role of curvature peaks in image perception and its corresponding curvature map computed by Level Lines Shortening.

Fig. 3 .

 3 Fig. 3. The 1D topology of level lines corresponding to different gray values on a dual edge.

Fig. 4 .

 4 Fig. 4. Curvatures computed with a FDS and by LLS.

Fig. 5 .

 5 Fig. 5. Scaled zoom on a texture. Curvatures in absolute value are displayed along the level lines.

Fig. 6 .

 6 Fig.6. Piece of a map, its corresponding level lines and curvatures before (top) and after (botom) LLS filtering.

Fig. 7 .

 7 Fig. 7. LLS tears apart ridges and emphasizes crossovers.

http://www.ipol.im/pub/algo/cmmm image curvature microscope/


Dr Andreas Breit
email: andreas.breit@lrz.uni-muenchen.de

Thomas R H Büch

Ingrid Boekhoff

Hans Jürgen Solinski

Ellen Damm

Thomas Gudermann

Alternative G protein-coupling and biased agonism: new insights into melanocortin-4 receptor signalling

Keywords: melanocortins, MC4R, AGRP, obesity, alternative G protein coupling, biased agonism ACTH, adrenocorticotropic hormone, cAMP, cyclic 3',5'-adenosine monophosphate, AGRP, agouti-related protein, CTX, cholera toxin, ER, endoplasmic reticulum

The melanocortin-4 receptor (MC4R) is a prototypical G protein-coupled receptor (GPCR) that plays a considerable role in controlling appetite and energy homeostasis. Signalling initiated by MC4R is orchestrated by multiple agonists, inverse agonism and by interactions with accessory proteins. The exact molecular events translating MC4R signalling into its physiological role, however, are not fully understood. This review is an attempt to summarize new aspects of MC4R signalling in the context of its recently discovered alternative G protein coupling, and to give a perspective on how future research could improve our knowledge about the intertwining molecular mechanisms that are responsible for the regulation of energy homeostasis by the melanocortin system.

The melanocortin system 1.Agonists, receptors and physiological role

The melanocortin precursor molecule proopiomelanocortin (POMC) gives rise to at least 4 different melanocortins: the melanocyte-stimulating hormones (MSH) which are named α-, β-or γ-MSH and the adrenocorticotropic hormone (ACTH) (Pritchard et al., 2003;Pritchard et al., 2002). Melanocortins are small peptide hormones that bind with different affinities to 5 distinct melanocortin receptors (MCR), which belong to the super family of G protein-coupled receptors (GPCR) (Tab. 1). Melanocortins regulate pigmentation, adrenal hormone secretion, immune functions, lipid metabolism and feeding behaviours (Brzoska et al., 2008). The MC1R is expressed in many different tissues such as skeletal muscle, brain and lung, but its most established scene of action is the skin, where MC1R signalling is responsible for pigmentation (Suzuki et al., 1996). The MC2R is the receptor for ACTH and, thus, a strong stimulator of glucocorticoid production in vertebrates (Chida et al., 2007). MC3R and MC4R are abundantly found in the brain, where they A c c e p t e d M a n u s c r i p t 3 exert catabolic effects by decreasing food intake and increasing energy expenditure [START_REF] Cone | The Central Melanocortin System and Energy Homeostasis[END_REF]. Both MCR subtypes bind αand β-MSH with similar affinity (Biebermann et al., 2006;[START_REF] Huszar | Targeted disruption of the melanocortin-4 receptor results in obesity in mice[END_REF][START_REF] Lee | A POMC variant implicates betamelanocyte-stimulating hormone in the control of human energy balance[END_REF], whereas, γ-MSH binds to the MC4R with a considerably lower affinity (by two orders of magnitude) compared to α-MSH (Voisey et al., 2003). Given the higher affinity of γ-MSH to the MC3R, it is assumed that this peptide induces its physiological effects mainly through the latter subtype. Interestingly, a newly discovered GPCR family, termed "mas-related GPCR" (Mrg), binds γ-MSH and its derivatives with similar high affinity, suggesting, that this peptide interacts with members of two unrelated GPCR families. The focus of γ-MSH research might turn to this novel aspect of melanocortin signalling in upcoming years [START_REF] Han | Orphan G protein-coupled receptors MrgA1 and MrgC11 are distinctively activated by RFamide-related peptides through the Galpha q/11 pathway[END_REF][START_REF] Lembo | Proenkephalin A gene products activate a new family of sensory neuron--specific GPCRs[END_REF].

The MC5R shows a broad expression pattern and targeted disruption of this gene results in widespread dysfunction of exocrine glands including a marked decrease in the production of sebum (Thiboutot et al., 2000). Since the complexity of signalling initiated by all 5 receptor subtypes and the melanocortins in different tissues is way beyond the scope of one review, in the following, we will concentrate on the MC4R.

The MC4R has been shown to play a pivotal role in controlling meal size and energy homeostasis. Adipose tissue-derived hormones such as leptin increase POMC expression in α-MSH-releasing neurons located in the arcuate nucleus of the hypothalamus (Shimizu et al., 2007). Secreted α-MSH activates MC4R expressing neurons of various hypothalamic nuclei, which, in turn, enhance the release of anorexigenic stimuli (e.g. thyrotropinand corticotropin-releasing hormone) or inhibit the liberation of orexigenic peptides (e.g. orexins and melanin-concentrating hormone) [START_REF] Ellacott | The central melanocortin system and the integration of short-and long-term regulators of energy homeostasis[END_REF].

The importance of MC4R signalling in the regulation of human metabolism has been highlighted by the finding that mutations in the MC4R gene are the most frequent monogenic cause of severe obesity (Biebermann et al., 2003;[START_REF] Govaerts | Obesity-associated mutations in the melanocortin 4 receptor provide novel insights into its function[END_REF][START_REF] Hinney | Prevalence, spectrum, and functional characterization of melanocortin-4 receptor gene mutations in a representative population-based sample and obese adults from Germany[END_REF][START_REF] Hinney | Melanocortin-4 receptor gene: case-control study and transmission disequilibrium test confirm that functionally relevant mutations are compatible with a major gene effect for extreme obesity[END_REF]Tao and Segaloff, 2003;Vaisse et al., 1998;Yeo et al., 2003). Accordingly, targeted disruption of the MC4R or the POMC gene in mice causes an obesitydiabetes syndrome characterized by hyperphagia, hyperinsulinemia and hyperglycemia (Balthasar et al., 2005;[START_REF] Huszar | Targeted disruption of the melanocortin-4 receptor results in obesity in mice[END_REF].

Besides its effects on the regulation of energy homeostasis, MC4R has also been shown to sensitise sexual sensation of males and females in humans [START_REF] Hadley | Discovery that a melanocortin regulates sexual functions in male and female humans[END_REF], to exhibit anti-inflammatory effects (Brzoska et al., 2008), to increase systolic blood pressure [START_REF] Greenfield | Modulation of blood pressure by central melanocortinergic pathways[END_REF], to stimulate neurite elongation in DRG neurons after axonal injury (Tanabe et al., 2007) and to directly enhance allodynia (Starowicz et al., 2002).

Melanocortin-induced signalling pathways of the MC4R

Since the cloning of the MC4R by [START_REF] Gantz | Molecular cloning, expression, and gene localization of a fourth melanocortin receptor[END_REF], it has been established that this receptor subtype regulates intracellular concentrations of cyclic 3',5'-adenosine monophosphate (cAMP) by increasing adenylyl cyclase (AC) activity through G s proteins and afterwards enhances activity of protein kinase A (PKA) [START_REF] Gantz | Molecular cloning, expression, and gene localization of a fourth melanocortin receptor[END_REF]. Further, it has been reported that ligand-induced activation of the MC4R modifies the activity of extracellularregulated kinases (ERK-1/2) (Chai et al., 2006;Patten et al., 2007;Vongs et al., 2004), AMP-activated kinase (Minokoshi et al., 2004), c-jun kinase (Chai et al., 2009), phosphatidylinositol-3-kinase (PI3K) (Vongs et al., 2004) and protein kinase C (PKC) (Chai et al., 2006). Down-stream of these kinases, it has been shown that MC4R signalling regulates ion channel activity [START_REF] Fu | Agoutirelated peptide and MC3/4 receptor agonists both inhibit excitatory hypothalamic ventromedial nucleus neurons[END_REF], gene expression [START_REF] Lee | Differential regulation of cAMPmediated gene transcription and ligand selectivity by MC3R and MC4R melanocortin receptors[END_REF] and enhances insulin signalling by decreasing insulin receptor

Alternative G protein coupling and signalling of the MC4R

Numerous studies clearly indicated that the MC4R interacts with G s proteins in endogenous or recombinant expression systems. The importance of the G s /PKA pathway has been shown by targeted disruption of the RIIβ-subunit of PKA in mice [START_REF] Czyzyk | Disruption of the RIIbeta subunit of PKA reverses the obesity syndrome of Agouti lethal yellow mice[END_REF]. This work demonstrates that disruption of PKA activity alters adiposity and locomotor activity downstream of the MC4R in the hypothalamus.

Interestingly, when recombinantly expressed, adipositasassociated MC4R mutants exerted inconsistent effects on G s signalling. Some mutants showed a full loss-of-function phenotype, whereas others showed no functional alterations on G s signalling. Even more surprisingly, some mutants constitutively increased adenylyl cyclase activity [START_REF] Hinney | Melanocortin-4 receptor gene: case-control study and transmission disequilibrium test confirm that functionally relevant mutations are compatible with a major gene effect for extreme obesity[END_REF]. Therefore, no clear correlation could be drawn between the cellular phenotype resulting from these mutations and obesity observed in vivo. Thus, one might suggest that signalling pathways other than G s might contribute to the physiological effects of the MC4R. In addition to the established G s coupling, observations have, indeed, been made either suggesting direct coupling of the MC4R to other members of the G protein family or the activation of signalling pathways that are insensitive to specific inhibitors of the G s /PKA pathway. Newman et al., reported that NDP-MSH increases intracellular calcium concentrations in murine, immortalized hypothalamic neurons (GT1-1 cells) endogenously expressing MC4R (Newman et al., 2006). This calcium response was blocked by the MC4R specific antagonist SHU-9119 and sensitive to a specific phospholipase C (PLC) inhibitor. Thus, as suggested by the authors, in GT1-1 cells MC4R might be able to activate the G q /PLC/calcium pathway. However, in closely related GT1-7 cells, no melanocortin-induced activation of this pathway was observed (Buch et al., 2009). Mountjoy et al., (Mountjoy et al., 2001) and Nickolls et al., (Simon et al., 2009) reported calcium transients in HEK293 cells recombinantly overexpressing the MC4R, thus confirming the results obtained in GT1-1 cells. However, in these cells melanocortin-induced calcium signals were blocked by the Gα s specific inhibitor, cholera toxin (CTX), and most probably not due to the activation of G q proteins. These data suggest that MC4R-mediated calcium signalling strongly depends on the cellular context.

Considering melanocortin-induced activation of ERK-1/2, a similar picture arises. In HEK293 cells melanocortinmediated activation of ERK-1/2 was sensitive to the Gα i/o specific inhibitor pertussis toxin (PTX) (Chai et al., 2006), whereas in GT1-1 or GT1-7 cells ERK-1/2 activation induced by melanocortins was resistant to PTX (Buch et al., 2009;Chai et al., 2006). These observations indicate engagement of the MC4R with PTXsensitive G proteins. However, it seems that the propensity of this receptor to activate PTX-sensitive G proteins depends on the cell line used. Melanocortin-induced activation of ERK-1/2 is of particular interest, since Sutton et al., reported that a specific ERK-1/2-inhibitor completely blocked anorexigenic effects of the MC4Rspecific agonist MTII in rats (Sutton et al., 2005). Given the importance of PKA and ERK-1/2 signalling for the anorexigenic effects of melanocortins, one might assume that the G s /PKA pathway directly increases ERK-1/2 activity, as shown for other GPCRs (Vossler et al., 1997;Wan and Huang, 1998). Indeed, in the above mentioned study of Sutton et al, MTIIinduced activation of ERK-1/2 was abolished by the cAMP inhibitor Rp-cAMPs. However, other studies reported that in GT1-1 cells melanocortin-induced activation of ERK-1/2 does not require PKA but PKC activity and the increase of intracellular calcium concentrations (Chai et al., 2006). Similarly, in MC4R

A c c e p t e d M a n u s c r i p t overexpressing CHO-KI cells, melanocortin-induced ERK-1/2 activation was insensitive to PKA inhibitors but sensitive to PI3K blockade and thus most probably conveyed through inositol trisphosphate (Vongs et al., 2004). Thus, it remains unclear whether PKA and ERK-1/2 belong to the same or diverging pathways and which signalling components associate the MC4R with these kinases.

Considering recently discovered signalling pathways of GPCR that are not mediated by G proteins but initiated by adapter proteins like arrestins, this scenario becomes even more complex: initially, arrestins have been shown to terminate G protein-dependent signalling after agonist binding to the receptor. However, recent work from many outstanding laboratories has been shown that arrestins also activate cellular effectors like ERK-1/2 in a G protein-independent manner (Shenoy and Lefkowitz, 2005). Thus, MC4R-mediated ERK-1/2 activation could also be explained by this new aspect of GPCR signalling. This is of particular interest, since data from our laboratory suggest that α-MSH and AGRP induce interactions between the MC4R and arrestins in HEK293 or Cos-1 cells (Breit et al., 2006). Thus, arrestinmediated ERK-1/2 activation by the MC4R could also contribute to melanocortin signalling.

Differences observed in the sensitivity of melanocortin-induced ERK-1/2 signalling to PTX in GT1-1 and GT1-7 cells on the one hand and HEK293 cells on otherhand, suggest that the MC4R couples to members of the G i/o family only when overexpressed in HEK293 cells. However, again the situation appears much more complex, since recently it has been reported that although α-MSH-induced activation of ERK-1/2 signalling in GT1-7 cells is not sensitive to PTX, the toxin affects α-MSHinduced accumulation of cAMP and incorporation of GTPγS 35 (Buch et al., 2009). Thus, similar to over expressing HEK293 cells, the MC4R has the potential to functionally interact with G i/o proteins, when endogenously expressed in GT1-7 cells.

The D90N mutation of the MC4R has been associated with severe early-onset obesity. This MC4R variant binds melanocortins with unchanged high affinity, but agonist binding does not initiate G s signalling. Thus, in the context of alternative G protein coupling the D90N variant of the MC4R represents an excellent tool to analyze putative G s -independent signalling pathways and to identify structures of the MC4R that are responsible for the coupling to distinct G proteins. Indeed, one study from our laboratory revealed that although this mutant is deficient in any detectable G s signalling, it induced incorporation of GTPγS 35 in PTXsensitive G proteins and decreases basal concentrations of cAMP, indicating that the MC4R-D90N selectively activates G i/o (Buch et al., 2009). This finding strengthens the idea of dual coupling of the MC4R to G s and G i/o proteins and provides new insights into the molecular mechanism of MC4R-mediated G protein activation.

It has been proposed that receptors exist in an inactive, G protein-uncoupled (R) and an active, G protein-coupled (R*) conformation (Samama et al., 1993). Over the last decade, it has been debated whether GPCR form only one or more R*s [START_REF] Galandrin | The evasive nature of drug efficacy: implications for drug discovery[END_REF][START_REF] Kenakin | Functional selectivity through protean and biased agonism: who steers the ship?[END_REF]. Data obtained for the D90N mutant indicate that a single point mutation in the coding sequence of the MC4R allows for specific activation of G i/o but not of G s . Hence, one may postulate that the MC4R exists in R*s specific for G s or G i/o proteins, indicating that MC4R-propagated signalling is best described in a model consistent with multiple R* conformations. A multi-statemodel of receptor activation raises the intriguing possibility that distinct ligands might be able to differentially stabilize distinct R*s and therefore selectively activate a particular signalling pathway (ligand-directed signalling). Such so-called biased agonists have recently been identified for members of other GPCR families [START_REF] Galandrin | Conformational rearrangements and signaling cascades involved in ligand-biased mitogen-activated protein kinase signaling through the beta1-adrenergic receptor[END_REF][START_REF] Leduc | Functional selectivity of natural and synthetic prostaglandin EP4 receptor ligands[END_REF] , 2009;Michel and Alewijnse, 2007;Ramachandran et al., 2009;Reversi et al., 2005;Schonbrunn, 2008) and have also been suggested for the MC4R by Nickolls et al.. This work reported that when considering cAMP levels, calcium signals and, in addition, agonist-dependent receptor endocytosis induced by non-peptide versus peptide MC4R agonists, agonist-specific receptor conformations exist that translate agonist binding into different efficacies to activate a given pathway (Nickolls et al., 2005).

In sum, it appears that the MC4R is able to functionally engage with other G proteins than G s , most probably G i/o and/or G q (see Tab. 2), and that different active receptor conformations might be responsible for this alternative G protein coupling. However, the ability of the MC4R protein to create these distinct conformations might depend on the cellular context and/or the expression level of the receptor.

AGRP-induced signalling pathways of the MC4R

In contrast to all other hormone systems known so far, the melanocortin system is the only one that is not just regulated by agonistic peptides but also by two endogenously occurring neuropeptides that block ligand-induced MCR signalling: agouti and "agouti-related protein" (AGRP). Agouti binds to almost all MCR with high affinity, whereas AGRP exhibits rather selective binding towards the MC3R and MC4R (Ollmann et al., 1997) (Tab. 1).

Since binding of AGRP prevents or displaces the binding of melanocortins to the MC3R or MC4R, it has been defined as a classic competitive antagonist (Ollmann et al., 1997). In line with these inhibitory effects on melanocortin signalling, AGRP has been shown to oppose the biological activity of melanocortins in vivo and therefore represents an orexigenic stimulus (Bewick et al., 2005;[START_REF] Dhillo | Paraventricular nucleus administration of calcitonin gene-related peptide inhibits food intake and stimulates the hypothalamopituitary-adrenal axis[END_REF][START_REF] Graham | Overexpression of Agrt leads to obesity in transgenic mice[END_REF][START_REF] Luquet | NPY/AgRP neurons are essential for feeding in adult mice but can be ablated in neonates[END_REF][START_REF] Graham | Overexpression of Agrt leads to obesity in transgenic mice[END_REF]. This model is based, for example, on the observation that AGRPdeficiency leads to a lean phenotype of mice and an extended lifespan, when the animals received a high fat diet (Redmann and Argyropoulos, 2006;Wortley et al., 2005). Furthermore, AGRP polymorphisms are associated with resistance to adipositas and development of type-2 diabetes in humans (Argyropoulos et al., 2003;[START_REF] Dubern | Mutational analysis of melanocortin-4 receptor, agouti-related protein, and alphamelanocyte-stimulating hormone genes in severely obese children[END_REF]Vink et al., 2001).

However, the molecular events responsible for AGRP-mediated physiological effects are not completely understood. Based on the common model of competitive antagonism, effects of AGRP on appetite control depend strictly on the action of melanocortins. Contrasting this assumption, recent studies revealed that the effects of AGRP on appetite control are independent of melanocortin signalling. For example, one study reported that intracerebroventricular injection of AGRP in POMC-and thus melanocortin-deficient mice induces long-lasting increased food uptake (Tolle and Low, 2008). Similarly, another study reported that ablation of AGRP-expressing neurons in the arcuate nucleus leads to starvation not only in wildtype but also in mice that have chronic blockade of melanocortin signalling (Wu et al., 2008). Absence of melanocortin signalling did not even ameliorate the extent of starvation. Both studies provide in vivo data indicating that AGRP impairs appetite control in a melanocortinindependent manner, suggesting that mechanisms other than competitive antagonism of MC4R signalling have also to be considered when discussing the physiological effects of AGRP on the molecular level.

One forward explanation for AGRPmediated actions in the absence of melanocortins is that, in addition to its antagonistic effects, AGRP decreases melanocortin-independent MC4R signalling and thus basal intracellular cAMP concentrations in MC4R expressing cells (Breit et al., 2006;[START_REF] Khong | Expression of functional melanocortin-4 receptor in the hypothalamic GT1-1 cell line[END_REF]Nijenhuis et al., 2001;Yang et al., 1999).

These actions of AGRP on basal cAMP

A c c e p t e d M a n u s c r i p t 7 production define this neuropeptide as an inverse agonist of the MC4R and could account for the melanocortin-independent effects described above. However, since the importance of basal, ligand-independent MC4R activity in the regulation of energy homeostasis is not clear, it is hard to predict whether or not blocking basal MC4R activity due to the inverse agonistic actions of AGRP can sufficiently account for the melanocortin-independent effects of AGRP observed in vivo.

In light of the above mentioned possibility that MC4R are functional engaged with G i/o proteins, it is tempting to speculate that members of this G protein family might also be involved in the effects of AGRP in the absence of melanocortins. Indeed, a recent publication of our laboratory provided data indicating that AGRP is able to induce the incorporation of GTPγS 35 in PTX-sensitive G proteins in membranes derived from MC4R overexpressing HEK293 cells (Buch et al., 2009). However, in the same cells PTX was not able to block AGRP-mediated reduction of forskolin-induced cAMP accumulation. This confusing observation might be best explained by the activation of G z proteins that have been described as the only member of the G protein family that decreases cAMP accumulation in a PTXinsensitive manner, but so far proof of an additional coupling of the MC4R to G z proteins was not successful.

Together, with the discrepancy observed between HEK293 and GT1-7 cells in melanocortin-induced ERK-1/2 activation, we suggest that MC4R signalling in overexpressing HEK293 cells differs dramatically from the signalling observed in GT1-1 or GT1-7 cells (Tab. 3), and that AGRP-mediated signalling in HEK293 cells cannot fully be described by currently known signalling pathways. However, in GT1-7 cells the picture of AGRP-induced signalling appears to be a bit clearer. AGRP-induced incorporation of GTPγS 35 in PTX-sensitive G proteins was also detectable in this cell line (Buch et al., 2009), indicating that AGRP has the potential to activate members of the G i/o family via MC4R in endogenous expression systems. In striking contrast to HEK293 cells, AGRP did not decrease but increased forskolin-induced cAMP production in GT1-7 cells (Buch et al., 2009). At first glance, this behavior of AGRP appears to be paradox. However, it should be taken into account that AC isoforms can be grouped into 3 subfamilies. Members of group 2 have been shown to be less sensitive to Gα i/o -mediated inhibition, but are sensitized by βγ-subunits released from Gα i/o . Indeed, transcripts of AC2 and AC4, which belong to group 2, were detectable in GT1-7 cells (Buch et al., 2009) and, thus, AGRP-mediated activation of G i/osignalling might increase forskolin-induced cAMP production by sensitization of group 2 AC isoforms. This hypothesis was firstly supported by the blocking effects of PTX on AGRP-mediated enhancement on forskolin-induced cAMP accumulation and secondly by the inhibitory effects of the recently described cell-permeable β 1 γ 2subunit inhibitor gallein on AGRP-induced forskolin-promoted cAMP accumulation (Buch et al., 2009). Thus, it appears that AGRP activates PTX-sensitive G proteins to release βγ-subunits that sensitize AC in GT1-7 cells.

In conclusion, besides its antagonistic and inverse agonistic actions, AGRP has the propensity to activate PTX-sensitive G proteins in GT1-7 cells and, thus, to behave like a biased agonist of the MC4R that is able to selectively activate G i/o and to simultaneously block G s signalling. We believe that in overexpressing systems (like HEK293 cells) this biased agonism affects distinct down-stream effectors of MC4R differently when compared to GT1-7 cells (see Tab. 3). Together with our results obtained for the MC4R-D90N mutant we suggest a multi-state-model of MC4R activation that rests on the extended version of the allosteric ternary complex model of receptor activation (Samama et al., 1993). This model implies at least one additional active receptor state (R**) that binds G i/o proteins with higher affinity and MSH with A c c e p t e d M a n u s c r i p t lower affinity when compared to R* (for details see Fig. 2). Biased agonistic actions of AGRP could have strong implications on MC4R-induced regulation of energy homeostasis. However, in vivo data considering this aspect are not established yet. Noteworthy, a recent publication described that melanocortins modulate the excitability of hypothalamic neurons by alterations of resting potassium conductance [START_REF] Fu | Agoutirelated peptide and MC3/4 receptor agonists both inhibit excitatory hypothalamic ventromedial nucleus neurons[END_REF]. Interestingly, inward-rectifier potassium channels (K IR 3.x) are regulated by βγsubunits released from Gα i/o [START_REF] Leaney | Contribution of Kir3.1, Kir3.2A and Kir3.2C subunits to native G protein-gated inwardly rectifying potassium currents in cultured hippocampal neurons[END_REF]Milovic et al., 2004). In agreement with biased agonistic actions of AGRP, the authors observed inhibition of these hypothalamic neurons by AGRP in a PTXsensitive manner. Due to the exclusive G s coupling of the MC4R postulated so far, it has been contended by the authors that PTX-sensitive signalling of AGRP is most probably mediated by another, yet unidentified GPCR. Based on our new model, it will be enlightening to clarify whether AGRP-mediated PTX-sensitive inhibition of hypothalamic neurons is blocked by MC4R selective antagonists, indicating that this effect is mediated by MC4R-dependent activation of G i/o proteins.

Fine-tuning of MC4R signalling

Although the importance of MC4R signalling in the regulation of energy homeostasis is well established, surprisingly little is known about the molecular events responsible for this physiological role. Furthermore, some studies obtained contradictory results (often in different cell models) and alternative G protein coupling and inverse or biased agonism additionally complicate the picture. Thus, in the following, we will discuss aspects of receptor signalling that are known to fine-tune GPCR signalling and whose considerations might contribute to the understanding of MC4R-induced signalling.

GPCR activity-modifying accessory proteins

For a long time signalling of GPCR has been described as a unidirectional process simply regulated by the affinity of the receptor to its cognate ligand and G protein.

In recent years fundamental studies delineated a pluridimensional picture of GPCR signalling, since influence of receptor multimerisation and accessory proteins has been revealed. Interactions among GPCR might appear as homo-or heteromultimerisation. The latter affects many basic functions of GPCR signalling such as ligand binding and G protein coupling (Angers et al., 2002;Milligan et al., 2006). For the MC4R heteromultimerisation with the MC3R and the G protein-coupled receptor-7 (GPR7), which are both expressed in similar hypothalamic regions compared to the MC4R, has been observed (Biebermann et al., 2003;[START_REF] Elsner | MC4R oligomerizes independently of extracellular cysteine residues[END_REF]Rediger et al., 2009). The impact of these interactions on melanocortin signalling is still unsettled, therefore, further analysis of MC4R containing receptor complexes might contribute to a better understanding of melanocortin and AGRP signalling.

The concept of accessory proteins with single transmembrane domains that regulate GPCR function has been put forward by the observation that the expression of the 3 different RAMPs (receptor-activity modifying protein) proteins (RAMP1-3) dramatically affects cell surface expression and pharmacological properties of two distinct receptors (Sexton et al., 2001). RAMPs are membrane-spanning proteins with a single transmembrane domain that exhibit no ligand-binding or signalling properties by themselves. However, coexpression of RAMP-1 or -3 with the calcitonin receptor creates the receptor for amylin, a hormone whose blood level increases similarly to that of insulin after meal uptake (Armour et al., 1999;Muff et al., 1999;Tilakaratne et al., 2000;Zumpe et al., 2000). The calcitonin-receptor-like receptor expressed without any RAMPs [START_REF] Cone | The Central Melanocortin System and Energy Homeostasis[END_REF][START_REF] Christopoulos | Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product[END_REF]Ono et al., 2000).

In the case of the MC4R three families of different accessory proteins might be of interest: syndecans, mahoganoid and MRAPs (melanocortin receptor accessory proteins).

Syndecans

Syndecans are a group (syndecan-1 to -4) of heparan-sulfate proteoglycans that exist in a membrane-bound and in a soluble, shedded form. Membrane-bound syndecans act as co-receptors and are involved in several physiological processes (Reizes et al., 2006).

For example, mice that transgenetically overexpress syndecan-1 in the hypothalamus have severe maturityonset obesity and diabetes mellitus type 2. Targeted disruption of the syndecan-3 gene leads to resistance of the mice to dietinduced obesity (Reizes et al., 2001). A positive association of obesity and syndecan-3 polymorphisms was also found in humans [START_REF] Ha | Positive association of obesity with single nucleotide polymorphisms of syndecan 3 in the Korean population[END_REF]. Interestingly, it has been shown in various mouse models that syndecans enhance AGRP-induced phenotypes and suggested that syndecans enhance the blocking effects of AGRP on MC4R-signalling (Reizes et al., 2003). Although the exact mechanisms regulating the interaction between AGRP and syndecans are not clear yet, it has been put forward that the membrane-bound form of syndecans co-localizes with the MC4R and binds AGRP. This syndecan/AGRP complex could decrease the affinity of melanocortins to the MC4R and thus inhibit melanocortin signalling (Reizes et al., 2006). This model has been supported by the observation that co-expression of MC4R and syndecan-3 in HEK293 cells enhances the inhibitory effects of AGRP on melanocortin-induced cAMP accumulation (Reizes et al., 2001). However, another study noticed that the domains of AGRP responsible for the binding to syndecan-3 are cleaved from the peptide before it leaves the cell, suggesting that the influence of syndecan-3 on food intake is independent from its interactions with AGRP and/or the MC4R [START_REF] Creemers | Agouti-related protein is posttranslationally cleaved by proprotein convertase 1 to generate agouti-related protein (AGRP)83-132: interaction between AGRP83-132 and melanocortin receptors cannot be influenced by syndecan-3[END_REF].

Mahoganoid

The mouse mahogany protein is the orthologue of the human attractin and represents a receptor involved in suppression of obesity and skin pigmentation [START_REF] Gunn | The mouse mahogany locus encodes a transmembrane form of human attractin[END_REF]Nagle et al., 1999). Since the effects of mahogany observed in various mouse models are mainly mediated by agouti and not by AGRP, they are not in the focus of this review. However, the mouse mahoganoid, also called mahogunin ring finger-1 (MGRN-1) protein, and its human orthologue (KIAA0544) are cytosolic ring domain-containing ubiquitin ligases that effect, similarly to mahogany, energy balance and pigmentation (Phan et al., 2002). Mouse models suggest that the MC4R as well as AGRP might contribute to these effects. On the molecular level three models have been suggested to describe the biological effects of mahoganoid 1) mahoganoid increases expression or activity of AGRP; 2) influences the physical proximity of AGRP to the MC4R or 3) decreases the availability of melanocortins for the MC4R (Phan et al., 2002). A more recent study analyzed the effects of mahoganoid on MC4R-induced cAMP production in HEK293 cells (Perez-Oliva et al., 2009). These experiments revealed that mahoganoid decreased MC4R signalling without affecting other G scoupled receptors. The inhibitory effects of mahoganoid were independent of receptor surface expression, ubiquitination, internalization or protein stability and occurred upstream of G s coupling and AC activation. Therefore, a competition of mahoganoid and G s proteins for the MC4R A c c e p t e d M a n u s c r i p t 10 might be a straightforward explanation for these effects, as suggested by the authors.

Melanocortin receptor accessory proteins (MRAPs)

Like RAMPs, MRAPs are proteins with one transmembrane domain that have recently been described to modify MCR signalling (reviewed by [START_REF] Hinkle | Structure and function of the melanocortin2 receptor accessory protein (MRAP)[END_REF]Webb and Clark, 2010)). In most cell types heterologous expression of the MC2R does not yield a functional receptor (Noon et al., 2002). Direct interactions of MRAPs with the MC2R are required for the receptor to traffic from the ER to the cell surface (Metherell et al., 2005). Recently, it was described that MRAP and MRAP-2 also interact with the four remaining MCR (Chan et al., 2009). In contrast to the benefit the MC2R experiences from its interaction with MRAPs, all other MCR suffer from the co-expression with MRAPs (Sebag and [START_REF] Hinkle | Structure and function of the melanocortin2 receptor accessory protein (MRAP)[END_REF]). The MC5R for example, which is well translocated from the ER to the cell surface when expressed alone, is trapped inside when co-expressed with MRAPs. Similarly but maybe to a lesser extent, MRAPs decrease the translocation of the MC4R to the plasma membrane and inhibit its efficacy to stimulate cAMP accumulation. However, to date it is not clear if the effects of MRAPs on MC4R-mediated cAMP accumulation solely result from reduced cell surface expression or if MRAPs are also able to travel with the MC4R to the cell surface and influence there its coupling to G proteins and, thus, MC4R signalling.

Organization of MC4R signalling in specialized membrane microdomains (lipid rafts)

Lipid rafts are cholesterol-and sphingolipid-rich microdomains in the plasma membrane [START_REF] Helms | Lipids as targeting signals: lipid rafts and intracellular trafficking[END_REF]Simons and Toomre, 2000). The main aspect of lipid rafts is to regulate the activity of a given pathway by their propensity to include or exclude signalling components in the signalling cascade. Organization of GPCR signalling by lipid rafts has been reported for the cAMP and calcium pathway numerous times [START_REF] Helms | Lipids as targeting signals: lipid rafts and intracellular trafficking[END_REF]Simons and Toomre, 2000).

Considering in particular cAMP signalling, it has been shown that chemically induced cholesterol depletion of the plasma membrane (resulting in the disruption of lipid rafts) increases cAMP levels, because lipid rafts separate various components of the G s /AC-pathway from each other and, thus, exert a tonic inhibition on cAMP production (Ostrom et al., 2001;Ostrom et al., 2000;Pontier et al., 2008). Most AC isoforms are found in lipid rafts [START_REF] Liu | Adenylyl cyclase type 6 overexpression selectively enhances betaadrenergic and prostacyclin receptormediated inhibition of cardiac fibroblast function because of colocalization in lipid rafts[END_REF]Thangavel et al., 2009) while Gα s and Gα o subunits are mostly found in non-lipid rafts areas (Abankwa and Vogel, 2007;Ostrom et al., 2001;Pontier et al., 2008). Interestingly, for the Gα o protein which is highly expressed in neurons, it has been reported that in cerebellar neurons the inactive form colocalizes with βγ-subunits in non-lipid rafts, but after activation, Gα o leaves its βγsubunits behind and translocates into lipid rafts, most probably to regulate AC activity (Yuyama et al., 2007). Interestingly, accumulation of AC subtypes in lipid rafts has mainly been reported for AC3, AC5, AC6 and AC8 (which are all Gα i/o -sensitive but insensitive to βγ-induced sensitization, see Tab. 4) but not for others (Schwencke et al., 1999). Thus, there might be the possibility that Gα i/o -sensitive ACs accumulate in lipid rafts while ACs that are sensitized by βγ-subunits enrich in nonlipid raft areas.

To our knowledge, so far, no data are available about the localization of the MC4R in lipid rafts; however, one study suggests that the closely related MC3R subtype is located in lipid rafts (Wachira et al., 2007). The distribution of lipid rafts over the cell surface, their lipid composition and/or inventory of scaffolding proteins has been shown to strongly depend on the cell type and morphogenesis (Helms and A c c e p t e d M a n u s c r i p t 11 [START_REF] Helms | Lipids as targeting signals: lipid rafts and intracellular trafficking[END_REF]Pontier et al., 2008;Simons and Toomre, 2000). In such a scenario, the number and/or composition of lipid rafts in different cells could affect the regulation of AC activity by Gα i/o -and βγ-subunits. Given the contrasting effects of AGRP on cAMP accumulation in HEK293 and GT1-7 cells, suggested to be mediated by βγsubunits in the latter, altered organization of these signal components in microdomains depending on the cell type might explain the observed differences. Similarly, different organization of components of the calcium signalling pathway in lipid rafts of membranes from GT1-1 and GT1-7 cells, could contribute to the differences observed in melanocortin-induced calcium transients. For future perspectives, it might be advantageous to analyze the microdomain localization of the MC4R and its downstream effectors in different cell lines and then to compare melanocortin-and/or AGRP-induced signalling in these cells.

Conclusions

Although the important role of the MC4R in decreasing food intake and increasing energy expenditure is widely accepted, pharmacological intervention in MC4R signalling plays only a minor role in the therapy of obesity or anorexia. Thus, screening for drugs that significantly modulate MC4R activity in vivo is ongoing work. Besides others, one drawback in this progress is that signalling pathways of the MC4R build up a multi-layered, entangled network: various endogenous ligands activate multiple G proteins with different efficacies; accessory proteins such as syndecans, mahoganoid and MRAPs contribute to the fine-tuning of MC4R function and organisation of signalling components in microdomains and even receptor heteromultimerisation might also be involved. Thus, disentangling the network of MC4R-induced signalling in the particular model system used might ease and improve both drug discovery and academic research.

  IRS-1) phosphorylation(Chai et al., 2009) (Fig.1).

  et

  ER, but co-expression with RAMP-2 or -3 gives rise to the adrenomedullin receptor and with RAMP-1 to the calcitonin gene-related receptor on the cell surface (Buhlmann

  MacNeil DJ, Howard AD, Guan X, Fong TM, Nargund RP, Bednarek MA, Goulet MT, Weinberg DH, Strack AM, Marsh DJ, Chen HY, Shen CP, Chen AS, Rosenblum CI, MacNeil T, Tota M, MacIntyre ED and Van der Ploeg LH (2002) The role of melanocortins in body weight regulation: opportunities for the treatment of obesity. Metherell LA, Chapple JP, Cooray S, David A, Becker C, Ruschendorf F, Naville D, Begeot M, Khoo B, Nurnberg P, Huebner A, Cheetham ME and Clark AJ (2005) Mutations in MRAP, encoding a new interacting partner of the ACTH receptor, cause familial glucocorticoid deficiency type 2. Nature genetics 37(2):166-170. Michel MC and Alewijnse AE (2007) Liganddirected signaling: 50 ways to find a lover. Molecular pharmacology 72(5):1097-1099. Milligan G, Canals M, Pediani JD, Ellis J and Lopez-Gimenez JF (2006) The role of GPCR dimerisationWeigl LG (2004) The sensitivity of G protein-activated K+ channels toward halothane is essentially determined by the C terminus. The Journal of biological chemistry 279(33):34240-34249. Minokoshi Y, Alquier T, Furukawa N, Kim YB, Lee A, Xue B, Mu J, Foufelle F, Ferre P, Birnbaum MJ, Stuck BJ and Kahn BB (2004) AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature 428(6982):569-574. Mountjoy KG, Kong PL, Taylor JA, Willard DH and Wilkison WO (2001) Melanocortin receptor-mediated mobilization of intracellular free calcium in HEK293 cells. Physiological genomics 5(1):11-19. Muff R, Buhlmann N, Fischer JA and Born W (1999) An amylin receptor is revealed following co-transfection of a calcitonin receptor with receptor activity modifying proteins-1 or -3. Endocrinology 140(6):2924-2927. Nagle DL, McGrail SH, Vitale J, Woolf EA, Dussault BJ, Jr., DiRocco L, Holmgren L, Montagno J, Bork P, Huszar D, Fairchild-Huntress V, Ge P, Keilty J, Ebeling C, Baldini L, Gilchrist J, Burn P, Carlson GA and Moore KJ (1999) The mahogany protein is a receptor involved in suppression of obesity. Nature 398(6723):148-152. Newman EA, Chai BX, Zhang W, Li JY, Ammori JB and Mulholland MW (2006) Activation of the melanocortin-4 receptor mobilizes intracellular free calcium in immortalized hypothalamic neurons. The Journal of surgical research 132peptide and nonpeptide agonists: evidence for ligandspecific conformational states. The Journal of pharmacology and experimental therapeutics 313(3):1281-1288. Nijenhuis WA, Oosterom J and Adan RA (2001) AgRP(83-132) acts as an inverse agonist on the human-melanocortin-4 receptor. Molecular endocrinology (Baltimore, Md 15(1):164-171. Noon LA, Franklin JM, King PJ, Goulding NJ, Hunyady L and Clark AJ (2002) Failed export of the adrenocorticotrophin receptor from the endoplasmic reticulum in nonadrenal cells: evidence in support of a requirement for a specific adrenal accessory factor. The Journal of endocrinology 174(1):17-25. Ollmann MM, Wilson BD, Yang YK, Kerns JA, Chen Y, Gantz I and Barsh GS (1997) Antagonism of central melanocortin receptors in vitro and in vivo by agoutirelated protein. Science (New York, NY 278(5335):135-138. Ono Y, Okano I, Kojima M, Okada K and Kangawa K (2000) Decreased gene expression of adrenomedullin receptor in mouse lungs during sepsis. Biochemical and biophysical research communications 271(1):197-202. Ostrom RS, Gregorian C, Drenan RM, Xiang Y, Regan JW and Insel PA (2001) Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase. The Journal of biological chemistry 276(45):42063-42069. Ostrom RS, Violin JD, Coleman S and Insel PA (2000) Selective enhancement of betaadrenergic receptor signaling by overexpression of adenylyl cyclase type 6: colocalization of receptor and adenylyl cyclase in caveolae of cardiac myocytes. Molecular pharmacology 57(5):1075-1079.

  Agouti-related protein-deficient mice display an age-related lean phenotype. Cell metabolism 2(6):421-427. Wu Q, Howell MP, Cowley MA and Palmiter RD (2008) Starvation after AgRP neuron ablation is independent of melanocortin signaling. Proceedings of the National Academy of Sciences of the United States of America 105(7):2687-2692. Yang YK, Thompson DA, Dickinson CJ, Wilken J, Barsh GS, Kent SB and Gantz I (1999) Characterization of Agouti-related protein binding to melanocortin receptors. Molecular endocrinology (Baltimore, Md 13(1):148-155. Yeo GS, Lank EJ, Farooqi IS, Keogh J, Challis BG and O'Rahilly S (2003) Mutations in the human melanocortin-4 receptor gene associated with severe familial obesity disrupts receptor function through multiple molecular mechanisms. Human molecular genetics 12(5):561-574. Yuyama K, Sekino-Suzuki N, Sanai Y and Kasahara K (2007) Translocation of activated heterotrimeric G protein Galpha(o) to ganglioside-enriched detergent-resistant membrane rafts in developing cerebellum. The Journal of biological chemistry 282(36):26392-26400. Zumpe ET, Tilakaratne N, Fraser NJ, Christopoulos G, Foord SM and Sexton PM (2000) Multiple ramp domains are required for generation of amylin receptor phenotype from the calcitonin receptor gene product. Biochemical and biophysical research communications 267(1):368-372.

Fig. 1 :

 1 Fig.1: Regulation of MC4R signalling by accessory proteins and alternative G protein coupling leads to the activation of ki li d i t t th difi ti f i d t ff t i l di t i ti f t

Fig. 1

 1 Fig. 1

Figure

  Figure

Table 2 Alternative Signalling of Melanocortins

 2

	effector	ligand	pathway	cell system	reference
	calcium release	α-MSH	CTX but not PTX	HEK293 cells	Mountjoj et
			sensitive: Gs		al.,
	calcium release	various, incl.	-	HEK293 cells	Nickolls et
		NDP-and α-			al.,
		MSH
	calcium release	NDP-MSH	sensitive to PLC	GT1-1 cells	Newmann et
			inhibition: Gq		al.,
	no calcium release	α-MSH or	-	GT1-7 cells	Buch et al.,
		NDP-MSH
	ERK activation	NDP-MSH	sensitive to PKC and	GT1-1 cells	Chai et la.,
			calcium inhibition
	ERK activation	NDP-MSH	PTX sensitive: Gi/o	HEK293 cells	Chai et la.,
	ERK activation	NDP-MSH	not PTX sensitive	GT1-1 cells	Chai et la.,
	ERK activation	α-MSH	not PTX sensitive	GT1-7 cells	Buch et al.,
	ERK activation	NDP-MSH	insensitive to PKA but	CHO-K1 cells Vongs et al.,
			sensitive to PI3K
			inhibition
	ERK activation	NDP-MSH	insensitive to PKA	HEK293 cells	Chai et la.,
			inhibition
	ERK activation	MTII	sensitive to PKA	in vivo	Sutton et al.,
			inhibition
	GTPγS 35	α-MSH	PTX sensitive: Gi/o	HEK293 and	Buch et al.,
	incorporation and			GT1-7 cells
	cAMP production
	signalling by Gs	α-MSH	PTX sensitive: Gi/o	HEK293 cells	Buch et al.,
	deficient mutant

Table 3 Alternative Signalling of AGRP

 3

	effector	pathway	cell system	reference
	GTPγS 35	PTX sensitive	HEK293 cells	Buch et al.,
	incorporation
	GTPγS 35	PTX sensitive	GT1-7 cells	Buch et al.,
	incorporation
	decrease of FSK-	PTX insensitive	HEK293 cells	Buch et al.,
	elevated cAMP
	decrease of FSK-	inhibition of G s	B16/G4F cells	Nijenhuis et al.,
	elevated cAMP
	increase of FSK-	PTX and gallein	GT1-7 cells	Buch et al.,
	elevated cAMP	sensitive
	ERK activation	PTX sensitive	HEK293 cells	unpublished data
	receptor endocytosis	-	HEK293 cells	Breit et al.,
	no receptor	-	HEK293 cells	Nickolls et al.,
	endocytosis
	receptor endocytosis	-	GT1-7 cells	Breit et al.,
	potassium	PTX sensitive	hypothalamic neurons	Fu et al.,
	conductance

Both events contribute to the orexigenic effects of AGRP. Of note, the efficacy of AGRP to decrease basal G s activation strongly depends on the isomerisation constant J which reflects the ratio of [R*] over [R] and might be different in various cell models. Lower panel: within a multi-state-model AGRP stabilizes an active receptor conformation (R**) that differs from R* stabilized by MSH R** Lower panel: within a multi-state-model AGRP stabilizes an active receptor conformation (R ) that differs from R stabilized by MSH. R binds G i/o proteins with higher affinity and MSH with lower affinity than R*, thus binding of AGRP to R** has three consequences: 1) displacement of MSH from R* (competitive antagonism), 2) uncoupling of G s from R* even in the absence of MSH (inverse agonism) and 3) activation of G i/o proteins (biased agonism). The exchange of D to N at position 90 of the MC4R wild-type selectively prevents the mutated protein from builting up R* (J`m utant > J`w ild-type ), thus MSH binds preferentially to R** and activates more G i/o -than G s proteins.