Agouti-Related Protein Segments Outside of the Receptor Binding Core are Required for Enhanced Short and Long Term Feeding Stimulation

摘要

The Agouti-Related Protein (AgRP) plays a central role in energy balance by reducing signaling through the hypothalamic melanocortin receptors (McRs) 3 and 4, in turn stimulating feeding and decreasing energy expenditure. Mature AgRP(83-132), produced by endoproteolytic processing, contains a central region that folds as an inhibitor cystine knot (ICK) stabilized by a network of disulfide bonds; this domain alone carries the molecular features for high affinity McR binding and inverse agonism. Outside of the ICK domain are two polypeptide segments – an N-terminal extension and a C-terminal loop – both completely conserved but of unknown function. Here we examine the physiological roles of these non-ICK segments by developing a panel of modified AgRPs that were administered to rats through intracerebroventricular (ICV) injection. Analysis of food consumption demonstrates that basic (positively charged) residues are essential for potent short and long term AgRP stimulated feeding. Moreover, we demonstrate an approximate linear relationship between protein charge density and 24 hr food intake. Next, we developed artificial AgRP(83-132) analogs with increased positive charge and found that these species were substantially more potent than wild type. A single dose of one protein, designated AgRP-4K, results in enhanced feeding for well over a week and weight gain that is nearly double that of AgRP(83-132). These studies suggest new strategies for the development of potent orexigenic species, and may serve as leads for the development of therapeutics for treating wasting conditions such as cachexia.