The hypothalamic melanocortin-4 receptor (MC4R), a seven transmembrane G-protein-coupled receptor, plays an important role in the regulation of body weight. The synthetic melanocortin analog SHU9119 has been widely used to characterize the physiological role of MC4R in feeding behavior and energy homeostasis. Previous studies indicated that SHU9119 is an agonist at the melanocortin-1 receptor (MC1R) but an antagonist at the MC4R. However, the molecular basis of the interaction between hMC4R and SHU9119 has not been clearly defined. To gain insight into the molecular determinants of hMC4R in the selectivity of SHU9119 chimeras and mutants hMC1R and hMC4R were expressed in cell lines and pharmacologically analyzed. A region of receptor containing the third transmembrane of hMC4R was found to be required for selective SHU9119 antagonism. Further mutagenesis studies of this region of hMC4R demonstrated that the amino acid residue leucine 133 in the third transmembrane was critical for the selective antagonist activity of SHU9119. The single substitution of leucine 133 to methionine did not affect SHU9119 binding to hMC4R. However, this substitution did convert SHU9119 from an antagonist to an agonist. Conversely, exchange of Met 128 in hMC1R to Leu, the homologous residue 133 of hMC4R, displayed a reduction in SHU9119 binding affinity and potency. This report provides the details of the molecular recognition of SHU9119 antagonism at hMC4R and shows that amino acid Leu 133 of hMC4R plays a key role in melanocortin receptor subtype specificity. The melanocortin-4 receptor (MC4R)1 is a seven transmembrane G-protein-coupled receptor, principally expressed in the hypothalamic nucleus. This receptor plays an important role in the regulation of body weight in rodents and humans (1-3). When stimulated by its putative physiological agonist, ␣-melanocyte-stimulating hormone (␣-MSH) inhibits feeding in mice through MC4R. Furthermore, mice with MC4R deletion develop hyperphagia, hyperinsulinism, and obesity (4). Recently, mutations in the MC4R have been reported as the most common causes of monogenic human obesity (5-7). MC4R mutations may play a particularly important role in the early onset of childhood obesity, resulting in more severe obesity-related complications, such as hypertension and diabetes, when compared with late onset obesity found in adults (8,9). In light of these findings, the molecular basis of the role that MC4R plays in obesity has been the subject of intense investigation.Many new, potent, and enzyme-resistant analogs of melanocortin peptides have been developed based on the extensive studies of the melanocortin peptide, ␣-MSH (10 -12 ]amide), which have been identified as potent, non-selective agonists at human melanocortin-1, -3, -4, and -5 receptors. In addition, the analog, SHU9119, a synthetic peptide with a ␤-(2-naphthyl)-D-alanine (D-Nal) substituted in position 7 of MTII, has been found to be a potent but non-selective antagonist for the MC3 and MC4 receptors (13,14). Intracerebroventricular admini...

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.

. (2007) EMBO J. 26, 3737-3748).Here we reconstituted in vitro the export of raftassociated and raft-independent markers staged intracellularly at 19°C. Surprisingly, whereas release of the raft-associated protein influenza hemagglutinin was dependent on the addition of an ATP-regenerating system and cytosol, release of a yellow fluorescent protein (YFP)-tagged raft-independent protein (the 75-kDa neurotrophin receptor; YFP-p75) was efficient even in the absence of these constituents. Subsequent studies suggested that YFP-p75 is released from the trans-Golgi network in fragile tubules that do not withstand isolation procedures. Moreover, immunofluorescence analysis revealed that hemagglutinin and YFP-p75 segregate into distinct subdomains of the Golgi complex at 19°C. Our data suggest that raft-associated and raft-independent proteins accumulate at distinct intracellular sites upon low temperature staging, and that upon warming, they exit these compartments in transport carriers that have very different membrane characteristics and morphologies.Efficient sorting of newly synthesized proteins along the biosynthetic pathway is critical for cell function. An additional requirement for maintaining the function and integrity of polarized epithelial cells is the selective targeting of newly synthesized cargo to differentiated apical and basolateral cell surface domains. This is accomplished by the recognition of sorting information within individual proteins that confers their segregation into distinct post-Golgi transport containers (1, 2). Basolateral sorting signals generally consist of short peptide sequences in the cytoplasmic domains of these proteins that sometimes fit the consensus for binding to adaptor protein complex subunits. In contrast, apical sorting signals are more diverse. These signals can localize to the luminal, transmembrane, or cytoplasmic domains of apical proteins. Post-translational modifications, including N-and O-linked glycans (e.g. on endolyn and the 75-kDa neurotrophin receptor (p75), respectively (3)), and glycosylphosphatidylinositol anchors have been described as apical sorting signals. For some proteins, including influenza hemagglutinin (HA), 4 apical sorting is conferred by sorting information within the transmembrane domain (4). HA-and glycosylphosphatidylinositol-anchored proteins are partially insoluble in cold Triton X-100 and preferentially associate with glycolipid-enriched microdomains (also known as lipid rafts or detergent-resistant membranes), although how this association plays a role in apical targeting is not yet understood (5).The diversity in apical sorting signals suggests that proteins with distinct classes of sorting signals might be sorted and packaged into distinct transport carriers leaving the trans-Golgi network (TGN). In fact, there is growing evidence, including data from our own lab, to suggest the existence of multiple pathways from the TGN to the apical surface. These pathways have been best described in models comparing the trafficking raft-associated v...

Agouti signaling protein (ASIP), the human (h) homolog of agouti, is an endogenous melanocortin peptide antagonist. To date, characterization of this protein has been performed with recombinant protein only and without the availability of an ASIP/agouti radioligand. In this report we describe the functional characteristics of a chemically synthesized truncated ASIP variant, ASIP-[90-132 (L89Y)], and the binding characteristics of its cognate radioligand, (125)I-ASIP-[90-132 (L89Y)]. Similar to full-length recombinant ASIP/agouti, ASIP-[90-132 (L89Y)] was a potent inhibitor of alpha-melanocyte-stimulating hormone cAMP generation at the cloned human melanocortin receptor (hMCR) subtypes hMC1R and hMC4R. It also displayed a lesser degree of inhibition at the hMC3R and hMC5R. However, ASIP-[90-132 (L89Y)] was found to be less potent than full-length recombinant ASIP and, surprisingly, only exhibited weak inhibitory activity at the hMC2R. In competition binding assays with the radioligand (125)I-ASIP-[90-132 (L89Y)], ASIP-[90-132 (L89Y)] displayed a hierarchy of binding affinity that roughly paralleled its rank order of inhibitory potency at the various MCR subtypes, i.e., hMC1R approximately hMC4R > hMC3R approximately hMC5R > hMC2R. Structure-activity studies revealed that ASIP-[90-132 (L89Y)] possessed greater pharmacological potency than either the further truncated ASIP variants ASIP-(116-132) or cyclo(CRFFRSAC). Interestingly, the latter molecules were both weak agonists at the hMC1R. These studies further support the concept that ASIP/agouti inhibits melanocortin action by directly binding to target MCRs and provide additional insight into the structural requirements for maximal inhibitory potency.