RT Journal Article SR Electronic T1 Molecular Determination of Agouti-Related Protein Binding to Human Melanocortin-4 Receptor JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 94 OP 103 DO 10.1124/mol.64.1.94 VO 64 IS 1 A1 Yingkui Yang A1 Min Chen A1 Yumei Lai A1 Ira Gantz A1 Aydin Yagmurlu A1 Keith E. Georgeson A1 Carroll M. Harmon YR 2003 UL http://molpharm.aspetjournals.org/content/64/1/94.abstract AB 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.