Molecular determination of agouti-related protein binding to human melanocortin-4 receptor.
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.
Yang
Yingkui
Y
Division of Pediatric Surgery, University of Alabama at Birmingham, 300 ACC, 1600 7th Avenue South, Birmingham, AL 35233, USA. ying-kui.yang@ccc.uab.edu
Chen
Min
M
Lai
Yumei
Y
Gantz
Ira
I
Yagmurlu
Aydin
A
Georgeson
Keith E
KE
Harmon
Carroll M
CM
D054369:Agouti-Related Protein; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D009074:Melanocyte-Stimulating Hormones / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000494:pharmacology; D044105:Receptor, Melanocortin, Type 4; D018043:Receptors, Corticotropin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection; D000521:alpha-MSH / Q000378:metabolism
C515198:AGRP protein, human; D054369:Agouti-Related Protein; D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D011506:Proteins; D044105:Receptor, Melanocortin, Type 4; D018043:Receptors, Corticotropin; D011993:Recombinant Fusion Proteins; C430443:agouti-related protein (86-132), human; D000521:alpha-MSH; D009074:Melanocyte-Stimulating Hormones; D000242:Cyclic AMP