The E92K Melanocortin 1 Receptor Mutant Induces cAMP Production and Arrestin Recruitment but Not ERK Activity Indicating Biased Constitutive Signaling

Rosenkilde MM (2011) The E92K Melanocortin 1 Receptor Mutant Induces cAMP Production and Arrestin Recruitment but Not ERK Activity Indicating Biased Constitutive Signaling. PLoS ONE 6(9): e24644. doi:10.1371/journal.pone.0024644 The E92K Melanocortin 1 Receptor Mutant Induces cAMP Production and Arrestin Recruitment but Not ERK Activity Indicating Biased Constitutive Signaling Tau Benned-Jensen 0 Jacek Mokrosinski 0 Mette M. Rosenkilde 0 Wolfgang Meyerhof, German Institute for Human Nutrition, Germany 0 Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, University of Copenhagen , Copenhagen , Denmark Background: The melanocortin 1 receptor (MC1R) constitutes a key regulator of melanism. Consequently, many naturallyoccurring MC1R mutations are associated with a change in color. An example is the Glu-to-Lys substitution found at position II:20/2.60 in the top of transmembrane helix II which has been identified in melanic mice and several other species. This mutation induces a pronounced increase in MC1R constitutive activity suggesting a link between constitutive activity and melanism which is corroborated by the attenuation of a-melanocyte stimulating hormone (aMSH) induced activation. However, the mechanism by which the mutation induces constitutive activity is currently not known. Methodology/Principal Findings: Here we characterize the constitutive activity, cell surface expression and internalization of the mouse mutant, Mc1r E92K. As previously reported, only positively charged residues at position II:20/2.60 induced an increase in constitutive activity as measured by cAMP accumulation and CREB activation. Furthermore, the mutation induced a constitutive recruitment of b-arrestin. This phenomenon is only observed in MC1R, however, as the equivalent mutations in MC2-5R had no effect on receptor signaling. Interestingly, the mutation did not induce constitutive ERK1/2 phosphorylation or increase the internalization rate indicating the constitutive activity to be biased. Finally, to identify regions of importance for the increased constitutive activity of Mc1r E92K, we employed a chimeric approach and identified G102 and L110 in the extracellular loop 1 to be selectively important for the constitutive activity as this, but not aMSHmediated activation, was abolished upon Ala substitution. Conclusions/Significance: It is concluded that the E92K mutation induces an active conformation distinct from that induced by aMSH and that the extracellular loop 1 is involved in maintaining this conformational state. In turn, the results suggest that in MC1R, which lacks an extracellular loop 2, the first extracellular loop may play a more prominent role during receptor activation than in general. - Funding: The study was supported by the Danish Medical Research Council, the European Communitys Sixth Framework Program (INNOCHEM: LSHB-CT-2005518167), the NovoNordisk Foundation, the Lundbeck Foundation, AP-Moller Foundation and the Aase and Einer Danielsen Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. In mammals, melanism is regulated by the levels and distribution of the eumelanin (black/brown) and pheomelanin (red/yellow) pigments. The two major genetic loci involved in the regulation of this process are extension and agouti encoding the melanocortin 1 receptor (MC1R) and the agouti signaling protein, respectively. MC1R is a constitutively active Gas-coupled seven transmembrane (7TM) receptor which is expressed primarily in epidermal melanocytes and is activated by several proopoimelanocortin derived peptides, most potently by a melanocytestimulating hormone (aMSH). However, MC1R is unique among 7TM receptors in that it is also targeted by an endogenously expressed inverse agonist, namely the agouti signaling protein [1]. Accordingly, both constitutive and ligand-induced activity of MC1R can be inhibited by this peptide [24]. Upon MC1R activation, accumulation of cAMP activates the rate-limiting melanogenic enzyme tyrosinase leading to elevated and reduced levels of eumelanin and pheomelanin, respectively. Thus, ultimately, receptor activation results in a darker phenotype, whereas inhibition of receptor activity results in a lighter phenotype [1]. Structurally, MC1R and the four other melanocortin receptors (MC2-5R) are interesting as they possess three unique features not found in rhodopsin-like 7TM receptors in general. First, they lack the highly conserved cysteine residue in the top of TM-III (CysIII:01, 90% conserved [5]) that normally forms a functionally important disulfide bridge with a cysteine in the extracellular loop (ECL) 2. Absence of this structural hallmark is only found in few other receptors including the cannabinoid and sphingosine receptors. Secondly, the ECL2 of the melanocortin receptors (MCRs) is very short consisting of only 3 polar amino acids keeping TM-IV and -V in close proximity of one another. This loop is on average the longest of the three ECLs [5] and in the currently available 7TM receptor crystal structures ECL2 contains prominent secondary structures such as a b-sheet (rhodopsin [6,7]), an a-helix (b-adrenergic receptors [8,9]) or an inter-loop disulfide bridge (adenosine A2A receptor [10]). Finally, the ECL3 of the MCRs is unusually rich in conserved Cys and Pro residues suggesting that it is detained in a specific fold likely by an intra-loop disulfide bridge [11] (Figure 1A). MC1R as well as MC3-5R have been shown to exhibit a high level of constitutive activity i.e. signaling in the absence of an agonist [2,11,12] which likely is physiological important. For instance, in morbidly obese individuals several mutations in MC4R have been identified that selectively abolish constitutive but not ligand-induced MC4R activity [13] and still others affecting both [14]. Given that activation of MC4R has an anorexigenic effect, it is possible that the constitutive activity of MC4R provides a satiety signal. In turn, when absent this could lead to an increase in food intake and ultimately obesity. However, even stronger evidence in favor of constitutive activity being physiologically relevant comes from naturally-occurring mutations in the MC1R. Thus, loss-of-function mutations in the MC1R frequently lead to a lighter phenotype as e.g. seen in individuals with red hair harboring the high penetrance R151C, R160W or D294H mutations [15]. On the other hand, a range of MC1R mutations has been identified in different species that constitutively activate the receptor and are associated with a darker phenotype [1618]. Interestingly, among the latter, one specific mutation is found in several phylogenetically distantly related species, namely the charge-reversing Glu-to-Lys substitution at position II:20/2.60 in the top o (...truncated)