UC Santa Cruz

The melanocortin 4-receptor (MC4R) is a G-protein coupled receptor (GPCR) that is expressed in the hypothalamus and is essential for energy homeostasis. Mutations in MC4R are the most common monogenic cause of obesity. MC4R signaling is regulated by its agonist ???? -MSH and by its endogenous antagonist the agouti-related peptide (AgRP). AgRP’s orexigenic effects are extraordinarily long-lasting. Additionally, several accessory proteins have been implicated in the regulation of MC4R. This dissertation focuses on two accessory proteins, syndecan-3 and the melanocortin receptor accessory protein 2 (MRAP2). Syndecan-3 is a cell-surface heparan sulfate proteoglycan that is hypothesized to increase the local concentration of AgRP at MC4R neurons. AgRP’s prolonged orexigenic effects are dependent on positively charged residues outside of its inhibitor cystine knot (ICK) core. These positively charged residues are not required for MC4R binding or antagonism but are crucial for electrostatic interactions with heparan sulfate. Using AgRP peptides with varying positive charge in its non-ICK segments along with glycan arrays and feeding studies with AgRP-treated mice, we find that AgRP increases food intake and reduces energy expenditure. These actions are dependent on AgRP’s positive charges and the AgRP variant that has the most protracted feeding response also has the greatest affinity for heparan sulfate. These findings further support the role of syndecan-3 in facilitating AgRP signaling. MRAP2 increases ????-MSH-induced MC4R signaling and also modulates several other hypothalamic GPCRs that regulate metabolism. MRAP2 is a single-pass membrane protein that forms highly unusual anti-parallel dimers. Despite MRAP2’s important role in energy balance, very little is known regarding the molecular details that drive its unique structure and how this relates to its ability to modulate MC4R. Using a panel of MRAP2 variants, biochemical experiments reveal that MRAP2 dimerizes through its transmembrane domain. We also show that MRAP2 can form parallel dimers using a luciferase-based molecular complementation assay. Elucidating the functions and structures of MC4R’s accessory proteins will not only further our understanding of GPCR signaling but also aid in the development of therapeutics aimed at modulating this complex signaling system.