High-Resolution NMR Structure of the Chemically-Synthesized Melanocortin Receptor Binding Domain AGRP(87−132) of the Agouti-Related Protein^,

Abstract

The agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin receptors MC3R and MC4R found in the hypothalamus and exhibits potent orexigenic (appetite-stimulating) activity. The cysteine-rich C-terminal domain of this protein, corresponding to AGRP(87-132), contains five disulfide bonds and exhibits receptor binding affinity and antagonism equivalent to that of the full-length protein. The three-dimensional structure of this domain has been determined by 1 H NMR at 800 MHz. The first 34 residues of AGRP(87-132) are well-ordered and contain a three-stranded antiparallel sheet, where the last two strands form a hairpin. The relative spatial positioning of the disulfide cross-links demonstrates that the ordered region of AGRP(87-132) adopts the inhibitor cystine knot (ICK) fold previously identified for numerous invertebrate toxins. Interestingly, this may be the first example of a mammalian protein assigned to the ICK superfamily. The hairpin's turn region presents a triplet of residues (Arg-Phe-Phe) known to be essential for melanocortin receptor binding. The structure also suggests that AGRP possesses an additional melanocortin-receptor contact region within a loop formed by the first 16 residues of its C-terminal domain. This specific region shows little sequence homology to the corresponding region of the agouti protein, which is an MC1R antagonist involved in pigmentation. Consideration of these sequence differences, along with recent experiments on mutant and chimeric melanocortin receptors, allows us to postulate that this loop in the first 16 residues of its C-terminal domain confers AGRP's distinct selectivity for MC3R and MC4R.Obesity and associated disorders such as diabetes are now at epidemic levels in the United States and other developed countries (1, 2). An understanding at the molecular level of the normal processes governing energy homeostasis and weight regulation is therefore essential for developing a comprehensive understanding of these disorders and producing effective pharmaceutical treatments. Recent studies have demonstrated the key role played by brain melanocortin receptors (MCRs) in the regulation of energy homeostasis (3-6). Although MCRs are found in various tissues, these studies indicate that the specific receptors MC3R and MC4R are directly implicated in energy balance. All MCRs are G-protein-coupled receptors (GPCRs) that up-regulate the production of cAMP in the presence of small endogenous peptide agonists (7). These agonists are derived in vivo from the single pro-opiomelanocortin peptide (POMC) that is cleaved post-translationally to form the melanocortinstimulating hormones (MSHs) and the adrenocorticotropin hormone (ACTH). MCR function is also modulated by potent endogenous antagonists known as the agouti protein and the agouti-related protein (AGRP) that indeed may exert even greater control over MCR signaling than the peptide agonists (8-11). Relative to the small peptide agonists, these antagonists are larger in size and possess a five-dis...

Molecular Determination of Agouti-Related Protein Binding to Human Melanocortin-4 Receptor

“…7. The three loops are held together at the base by an apparent scaffold of five disulfide bonds; these bonds stabilize the base of the active loop resi- dues 110 to 117 that present the Arg-Phe-Phe triple on the protein surface and are exposed to the surrounding solvent (McNulty et al, 2001). The central loop AGRP 106 -119 containing Arg-Phe-Phe may be critical for AGRP function, whereas the N-and C-terminal loops of AGRP 87-132 may confer receptor subtype specificity (Bolin et al, 1999).…”

Agouti-related protein (83-132) is a competitive antagonist at the human melanocortin-4 receptor: no evidence for differential interactions with pro-opiomelanocortin-derived ligands

“…However, this seems difficult to reconcile with the fact that the structures of MSH and AGRP are substantially different and the two peptides have different receptor subtype specificities (Yang et al 2000). Mutational analyses of the MC4-R (Yang et al 1999a, HaskellLuevano et al 2001, and three dimensional structure analysis of AGRP (Tota et al 1999, McNulty et al 2001, indicate that melanocortin peptides and AGRP share binding determinants to the MC4-R, but that AGRP has additional points of contact. Unfortunately, it is impossible to discern whether the pharmacological consequences of receptor mutagenesis are due to direct or indirect disruption of ligand receptor interactions.…”

New ligands for melanocortin receptors

“…Nonetheless, although Agrp and Asip do not appear in the tree of life until after the divergence of jawless fishes from the branch leading to mammals, their structural backbone is much older, as both the characteristic disulfide bonding pattern and the three-dimensional fold are that of the inhibitor cysteine knot (ICK), an ancient protein motif that is widely distributed among invertebrate phyla and used in a variety of invertebrate toxins. [50][51][52] An analogous observation holds for b-defensins as melanocortin receptor ligands; the gene family is clearly recognizable in teleost fish (though not in jawless fishes or tunicates), but only became recruited as a melanocortin receptor ligand very recently in mammalian evolution. 22,23,26,[53][54][55] These observations underscore a central theme of melanocortin signaling: the ability to control a broad array of different physiological processes, which has been driven by a combination of receptor duplication and diversification (the latter with respect to both ligand selectivity and tissue-specific expression), together with the recruitment of previously existing small secreted peptides to serve as new types of melanocortin ligands.…”