Agouti protein and Agouti-related protein (Agrp) are novel ligands for the melanocortin 1 receptor (Mc1r) and Mc4r, respectively, and are thought to exert their physiologic effects by inhibition of alpha-MSH binding, and by a second, as yet undefined, mechanism that is mediated through melanocortin receptors but is distinct from melanocortin displacement. Previously existing mutations of Agouti and the Mc1r have provided some of the critical tools and key insights for understanding these pathways; another coat color mutation, mahogany, has been implicated in Agouti protein and Agrp signaling because it suppresses the effects of Agouti on both pigmentation and obesity. Double mutant studies indicate that mahogany is genetically downstream of Agouti transcription but genetically upstream of melanocortin receptor signaling. To investigate how Agrp and the gene mutated in mahogany are involved in the normal regulation of body weight, we propose to 1) Identify the mahogany gene by positional cloning and determine it's pattern of RNA and protein expression; 2) construct a mahogany null allele and determine its effects on obesity induced by ectopic expression of Agrp, or by deficiency for the Mc4r; 3) use a natural cell culture model for mahogany gene action established with primary melanocytes to investigate whether mahogany affects melanocortin receptor number, ligand affinity, or coupling to downstream effectors; and 4) develop a quantitative binding assay for Agrp to determine if mahogany acts at the level of ligand processing, stabilization, or cell surface binding.
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