CDB15:0000042 AGRP — MC4R

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 1H NMR at 800 MHz. The first 34 residues of AGRP(87-132) are well-ordered and contain a three-stranded antiparallel beta sheet, where the last two strands form a beta 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.

ABSTRACT: Agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin-4 receptor (MC4R) that functions in the hypothalamic control of feeding behavior. Our previous studies have suggested that in addition to exoloops 2 and 3, several transmembrane domains of MC4R may be important for AGRP binding. However, the detailed molecular basis of MC4R domains in AGRP binding is presently unclear. The present studies were designed to determine the specific contribution of MC4R exoloops and transmembrane domains to AGRP binding by using chimeric receptor constructs of the human melanocortin-1 receptor (hMC1R), a receptor that is not inhibited by AGRP, and the human MC4R (hMC4R), a receptor that is potently inhibited by AGRP. Our results indicate that substitutions of the second and third extracellular loops of the MC4R with homologous domains of the MC1R dramatically decreased AGRP 87-132 binding affinity, but did not affect AGRP 110-117 binding affinity. In contrast, cassette substitutions of the third or fourth transmembrane domain of the MC4R with the homologous domain of the MC1R resulted in a substantial decrease of AGRP 87-132 binding affinity and loss of AGRP 110-117 binding affinity. These data suggest that the AGRP fragment 110-117 has no binding sites at exoloops of hMC4R and that transmembrane domains of MC4R may play an important role in AGRP 110-117 binding and function, whereas the exoloops do not. The second and third extracellular loops of MC4R are important for AGRP 87-132 N-terminal binding, whereas the third and fourth transmembrane domains of hMC4R are crucial for AGRP 110-117 binding.

ABSTRACT: The mRNA encoding an agouti related protein (ART) of unknown biochemical function was previously reported to be up-regulated in the hypothalamus of two genetically obese mouse strains. We have expressed human ART as a secreted protein in COS-7 cells, and show that recombinant ART is functionally active in inhibiting the binding of a radiolabeled alpha-melanocyte stimulating hormone (alpha-MSH) analog to the human melanocortin-3 (MC-3) and melanocortin-4 (MC-4) receptors, while it is not a potent inhibitor of the human melanocortin-5 (MC-5) receptor. ART is an antagonist of the human MC-3 and MC-4 receptors as determined in functional assay. ART appears to be approximately 100-fold more potent than agouti with reference to the MC-3R and MC-4R binding affinity. These data suggest that ART may be a physiological regulator of feeding behavior.

ABSTRACT: Expression of Agouti protein is normally limited to the skin where it affects pigmentation, but ubiquitous expression causes obesity. An expressed sequence tag was identified that encodes Agouti-related protein, whose RNA is normally expressed in the hypothalamus and whose levels were increased eightfold in ob/ob mice. Recombinant Agouti-related protein was a potent, selective antagonist of Mc3r and Mc4r, melanocortin receptor subtypes implicated in weight regulation. Ubiquitous expression of human AGRP complementary DNA in transgenic mice caused obesity without altering pigmentation. Thus, Agouti-related protein is a neuropeptide implicated in the normal control of body weight downstream of leptin signaling.

ABSTRACT: Agouti protein and the Agouti-related protein (AGRP) are antagonists of the melanocortin-3 receptor and melanocortin-4 receptor. Both proteins contain 10 cysteines in the C-terminal domain arranged in five disulfide bonds. One possible arrangement of the disulfide bonds predicts an octapeptide loop, and the chemical properties of four residues within this loop (residues 111-114 in human AGRP) bear striking resemblance to those of several melanocortin peptides, including alpha-MSH, MT-II, and SHU-9119. We showed that cyclic synthetic octapeptides based on the sequence of this loop from Agouti protein or human AGRP are functional antagonists of the human melanocortin-4 receptor. All peptides had a lower affinity for the melanocortin-3 receptor than for the melanocortin-4 receptor. Substitution of serines for cysteines resulted in linear peptides which had reduced binding affinities for both receptors. Mutational analysis of human AGRP indicated that its C-terminal domain is functionally equivalent to the intact human AGRP. The RFF111-113 triplet appears to be the most critical portion of AGRP in determining the binding affinity for both melanocortin-3 and melanocortin-4 receptors. These data strongly suggest that the loop defined by Cys-110 and Cys-117 is critical in determining the antagonist activity of human AGRP. Our data provide indirect evidence for the suggestion that the Cys-110 to Cys-117 octapeptide loop of human AGRP mimics the conformation of alpha-MSH, MT-II, and SHU-9119.