The Melanocortin system of G-Protein coupled receptors and ligands plays a vital role in cell signaling of many important physiological processes, including pigmentation, steroidogenesis, and metabolic control. These receptors are all activated by ligands derived from the proopiomelanocortin (POMC). This prohormone is processed by tissue specific expression of various proteases. Three receptors within this system have a secondary level of regulation. Melanocortin receptors (McRs) 1, 3 and 4 also respond to endogenously expressed antagonist proteins, the agouti-signaling protein (ASIP) and agouti-related protein (AgRP). ASIP and AgRP compete with POMC ligands for receptor binding and signal for reduction in cAMP. Both antagonists are sequentially and structurally similar; yet show differing tissue expression and receptor binding specificities. AgRP is expressed in the hypothalamus and signals for an increase in appetite and lower energy expenditure through Mc3R and Mc4R. ASIP is primarily expressed in the skin and affects pigmentation through Mc1R, but can interact with Mc3R and Mc4R through some genetic mutations.

The ligand expression and binding specificity at McRs 1, 3 and 4 provide a unique set of tools for understanding structural determinants required for regulation of pigmentation and energy balance and homeostasis. Here study a novel third antagonist sequence, AgRP2, discovered in teleost fish and expressed in the light sensitive Pineal gland of the brain. This protein shares the same cysteine spacing observed for ASIP and AgRP, and pharmacological studies revealed AgRP2 is selective for the zebrafish Mc1R. Unlike the human homologue, the zebrafish Mc1R is highly expressed in the brain. Knockdown studies reveal that the AgRP2 protein signals for pigment granule aggregation and is a vital molecule in the background adaptive process. Next, NMR analysis of a β-MSH mutant sequence, identified a tyrosine residue, not involved in receptor binding contacts, that participates in forming a turn motif. Mutation of this residue to cysteine significantly reduces this turn structure and dramatically reduces the agonist binding and activity at Mc4R. This mutation predisposes carriers to early-onset obesity. Finally, we study the non-ICK regions of the AgRP protein. Isothermal titration calorimetry experiments implicate the C-loop in proteoglycan binding and motivated rat feeding studies on C-terminal constructs. Through truncation of this domain, a correlation between peptide charge and appetite stimulation was characterized and led to the design of so-called "Super-AgRP" proteins that stimulate feeding in rodents by more than twice that of wild-type AgRP. These studies have the potential to revolutionize the treatment of cachexia and other negative energy balance metabolic disorders.