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Regulation of Melanocortin-4 Receptor Signaling: Agonist-Mediated Desensitization and Internalization

Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Jeffrey S. Flier, M.D., Beth Israel Deaconess Medical Center, Finard 204, 330 Brookline Avenue, Boston, Massachusetts 02215. E-mail: jflier{at}caregroup.harvard.edu.

Disruption of the hypothalamic melanocortin-4 receptor (MC4R) pathway results in obesity both in humans and rodents, demonstrating a crucial role for hypothalamic MC4Rs in the regulation of energy homeostasis. Because even haploinsufficiency of the MC4R gene can cause obesity in humans and mice, subtle changes in receptor numbers or signaling are likely to impact upon the regulation of food intake and energy expenditure. Little is known about the intracellular regulation of MC4R signaling. Using GT1–7 cells, we show for the first time that the MC4R undergoes ligand-mediated desensitization. We then addressed the possible mechanisms underlying the desensitization using HEK293 and COS-1 cells transfected with hemagglutinin-tagged human MC4R. Preexposure of GT1–7 cells that express endogenous MC4R to the agonist for MC4R, -melanocyte-stimulating hormone, resulted in impaired cAMP formation to a second challenge of -melanocyte-stimulating hormone. The desensitization of MC4R was accompanied by time-dependent internalization of the receptor in HEK293 cells, which was partly inhibited by pretreatment with a specific protein kinase A (PKA) inhibitor, H89. In COS-1 cells, overexpression of dominant-negative G protein-coupled receptor kinase (GRK) 2-K220R partly inhibited the agonist-mediated internalization of MC4R, whereas it did not in HEK293 cells. Overexpression of dominant-negative mutants of ß-arrestin1-V53D and dynamin I-K44A prevented agonist-mediated internalization of MC4R. Mutagenesis studies revealed that Thr312 and Ser329/330 in the C-terminal tail are potential sites for PKA and GRK phosphorylation and may play an essential role in the recruitment of ß-arrestin to the activated receptor. Our data demonstrate that, through PKA-, GRK-, ß-arrestin-, and dynamin-dependent processes, MC4R undergoes internalization in response to agonist, thereby providing novel insights into the regulation of MC4R signaling.

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