No abstract

, did not show any specific binding to the radioligand, but were found to be constitutively active in the cAMP assay. The E and E R alleles are associated with black feather colour in chicken while the e b allele gives rise to brownish pigmentation. The three constitutively active receptors share a mutation of Glu to Lys in position 92. This mutation was previously found in darkly pigmented sombre mice, but constitutively active MC receptors have not previously been shown in any nonmammalian species. We also inserted the Glu to Lys mutation in the human MC1 and MC4 receptors. In contrast with the chicken clones, the hMC1-E94K receptor bound to the ligand, but was still constitutively active independently of ligand concentration. The hMC4-E100K receptor did not bind to the MSH ligand and was not constitutively active. The results indicate that the structural requirements that allow the receptor to adapt an active conformation without binding to a ligand, as a consequence of this E/K mutation, are not conserved within the MC receptors. The results are discussed in relationship to feather colour in chicken, molecular receptor structures and evolution. We suggest that properties for the ÔE92K switchÕ mechanism may have evolved in an ancestor common to chicken and mammals and were maintained over long time periods through evolutionary pressure, probably on closely linked structural features.Keywords: G-protein coupled; MSH; melanocortin receptor; polymorphism.Spontaneous or constitutive G-protein coupled receptor (GPCR) activity was first convincingly described for the d-opioid receptor [1], and was further established by the demonstration of constitutive activity in chimeras of the a 1B and a 2 receptors (summarized in [2]). Later it was shown that mutations in the human rhodopsin gene can constitutively activate transducin in the absence of retinal and light [3]. It is now known that naturally occurring constitutively active GPCRs are found to be responsible for a diverse array of inherited as well as somatic genetic disorders [4,5].The melanocortins (a-MSH/ACTH), secreted from a frog pituitary, were in 1912 found to cause pigmentation.In higher vertebrates including aves and mammals, these peptides are expressed throughout the body, and are involved in a variety of physiological regulatory functions [6][7][8]. In the skin, the melanocortins are synthesized locally (for birds [9], for mammals [10]), and act through a GPCR named melanocortin (MC) 1 receptor to regulate melanogenesis. Keratinocytes probably serve as the main physiological source of melanocortins acting on melanocytes in the epidermis and hair follicle. The MC1 receptor couples through G proteins to adenylate cyclase to stimulate tyrosinase, the rate-limiting enzyme in the synthesis of both classes of melanin pigments, eumelanin and phaeomelanin. A low level of tyrosinase expression leads to increased phaeomelanin synthesis, while elevated levels of tyrosinase, that can result from a-MSH stimulation of melanocytes, divert the intermediates primarily a...

We created a molecular model of the human melanocortin 4 receptor (MC4R) and introduced a series of His residues into the receptor protein to form metal ion binding sites. We were able to insert micromolar affinity binding sites for zinc between transmembrane region (TM) 2 and TM3 where the metal ion alone was able to activate this peptide binding G-protein-coupled receptor. The exact conformation of the metal ion interactions allowed us to predict the orientation of the helices, and remodeling of the receptor protein indicated that Glu 100 and Ile 104 in TM2 and Asp 122 and Ile 125 in TM3 are directed toward a putative area of activation of the receptor. The molecular model suggests that a rotation of TM3 may be important for activation of the MC4R. Previous models of G-protein-coupled receptors have suggested that unlocking of a stabilizing interaction between the DRY motif, in the cytosolic part of TM3, and TM6 is important for the activation process. We suggest that this unlocking process may be facilitated through creation of a new interaction between TM3 and TM2 in the MC4R.The G-protein-coupled receptors (GPCRs) 1 require a membrane to maintain their functionality and structural integrity. This makes crystallization of these receptors difficult, hampering structural determination. Crystallization of the first mammalian GPCR, the bovine rhodopsin, was an important breakthrough (1). Earlier three-dimensional models of GPCRs were mainly based on cryoelectron microscopy data generated from bacteriorhodopsin (2), assuming a common fold in the transmembrane (TM) regions. Such assumptions had clear limitations as bacteriorhodopsin is not a GPCR and has no sequence homology to human GPCRs. Even though the bovine rhodopsin model is detailed, it is not clear how applicable it is for different GPCRs, considering the large variety of these receptors in the human genome. Today it is still an unrealistic task to crystallize the hundreds of GPCRs found in the human genome mainly due to the difficulties in obtaining material suitable for solubilization and crystallization studies. Site-directed mutagenesis has played an important role in determining the putative interaction of a ligand to a single amino acid within a GPCR. Such interactions, however, may not always be informative about the orientation of the helix bundles, which is crucial information for building structural models. Moreover, the results of alanine replacement studies in most cases cannot discriminate between specific ligand-receptor interactions and changes that cause unspecific conformational alterations that perturb the binding. This is particularly evident when the ligand is a flexible molecule like a peptide or a protein. One alternative approach for studying the three-dimensional structure of GPCRs is construction of a high affinity zinc-binding site between the helices, using two His residues facing each other (3). Such artificial intrahelical and interhelical binding sites have been used effectively to determine the orientation and exact distance...

1 Melanocortin (MC) receptors are widely distributed throughout the body of chicken, like in mammals, and participate in a wide range of physiological functions. 2 To clarify the pharmacological impact of ligands acting in the MC system, we expressed the chicken MC1, MC2, MC3, MC4 and MC5 (cMC1-5) receptors in eukaryotic cells and performed comprehensive pharmacological characterization of the potency of endogenous and synthetic melanocortin peptides. 3 Remarkably, the cMC receptors displayed high affinity for ACTH-derived peptides and in general low affinity for a-MSH. It is evident that not only the cMC2 receptor but also the other cMC receptors interact with ACTH-derived peptide through an epitope beyond the sequence of a-MSH. 4 The synthetic ligand MTII was found to be a potent agonist whereas HS024 was a potent antagonist at the cMC4 receptor, indicating that these ligands are suitable for physiological studies in chicken. 5 We also show the presence of prohormone convertase 1 (PC1) and PC2 genes in chicken, and that these peptides are coexpressed with proopiomelanocortin (POMC) in various tissues.

The melanocortin 5 receptor (MC5R) is activated by melanocyte-stimulating hormones (MSHs) and has a widespread tissue distribution, while its detailed central expression pattern and brain functions are fairly unknown. We report cloning, pharmacological characterization, tissue distribution and detailed brain mapping of melanocortin 5 receptor in goldfish (gMC5R). The goldfish orthologue protein is 69% identical to human MC5R and is conserved in important functional domains. The gMC5R showed similar potency to a-, b-and c-MSH peptides in radioligand binding as the mammalian orthologues, while MTII and HS024 were both agonists at this receptor. The gMC5R-mRNA was found in the peripheral tissues including kidney, spleen, skin and retina, with low expression levels in the intestine, fat, muscle, gill, pituitary and ovary. In situ hybridization studies demonstrated that gMC5R transcripts are widely distributed in the goldfish brain. The gMC5R expression was found in ventral telencephalon, preoptic area, dorsal and ventral thalamus, infundibular hypothalamus, posterior tuberculum, tectum and tegmentum mesencephali, reticular formation, vagal and facial lobes and spinal cord. The cloning and characterization of this receptor provides an important tool to elucidate its participation in neuroendocrine and behavioural control.