Abstract
Agouti-related protein (AGRP) is an endogenous antagonist of melanocortin action that functions in the hypothalamic control of feeding behavior. Although previous studies have shown that AGRP binds three of the five known subtypes of melanocortin receptor, the receptor domains participating in binding and the molecular interactions involved are presently unknown. The present studies were designed to examine the contribution of extracytoplasmic domains of the melanocortin-4 receptor (MC4R) to AGRP binding by making chimerical receptor constructs of the human melanocortin-1 receptor (MC1R; a receptor that is not inhibited by AGRP) and the human MC4R (a receptor that is potently inhibited by AGRP). Substitutions of the extracytoplasmic NH2 terminus and the first extracytoplasmic loop (exoloop) of the MC4R with homologous domains of the MC1R had no effect on AGRP (87-132) binding affinity or inhibitory activity (the ability to inhibit melanocortin-stimulated cAMP generation). In contrast, cassette substitutions of exoloops 2 and 3 of the MC4R with the homologous exoloops of the MC1R resulted in a substantial loss of AGRP binding affinity and inhibitory activity. Conversely, the exchange of exoloops 2 and 3 of the MC1R with the homologous exoloops of the MC4R was found to confer AGRP binding and inhibitory activity to the basic structure of the MC1R. Importantly, these substitutions did not affect the ability of the alpha-melanocyte stimulating hormone analogue [Nle4,D-Phe7] melanocyte stimulating hormone to bind or activate the chimeric receptors. These data indicate that exoloops 2 and 3 of the melanocortin receptors are important for AGRP binding.
Highlights
The melanocortin peptides, ␣, , and ␥-melanocyte stimulating hormone (MSH)1 and adrenocorticotropic hormone, are a group of peptides derived from the pro-opiomelanocortin prohormone that share a common message sequence (His-D-PheArg-Trp)
AGRP is transcribed as 132 amino acids in man (131 amino acids in mouse), and it is not presently known whether it is post-translationally processed in mammals, we have recently shown that a 46-amino acid COOH-terminal AGRP variant has the same ability to selectively bind MCR subtypes and functionally inhibit melanocortins as the fulllength molecule (17)
Characterization of MC4R/MC1R Chimeras with 125I-NDPMSH and 125I-AGRP (87–132) Binding—Fig. 2A demonstrates that cassette substitutions of the NH2 terminus, first, second, and third exoloop of the MC4R with homologous regions of the MC1R did not alter 125I-NDP-MSH
Summary
Vol 274, No 20, Issue of May 14, pp. 14100 –14106, 1999 Printed in U.S.A. Contribution of Melanocortin Receptor Exoloops to Agouti-related Protein Binding*. AGRP is transcribed as 132 amino acids in man (131 amino acids in mouse), and it is not presently known whether it is post-translationally processed in mammals, we have recently shown that a 46-amino acid COOH-terminal AGRP variant has the same ability to selectively bind MCR subtypes and functionally inhibit melanocortins as the fulllength molecule (17). Pharmacological studies using this chemically synthesized truncated AGRP variant, AGRP (87–132), indicate that AGRP is a competitive antagonist of ␣-MSH at MCR subtypes [3, 4], and 5. Based on assumptions about its structure and presently held concepts about the way larger peptides bind seven transmembrane Gprotein coupled receptors, we hypothesized that one potential binding determinant for AGRP might be the extracytoplasmic domains of the MCRs
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