Shinyama Hiroshi, Masuzaki Hiroaki, Fang Hui, Flier Jeffrey S
Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.
Endocrinology. 2003 Apr;144(4):1301-14. doi: 10.1210/en.2002-220931.
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, alpha-melanocyte-stimulating hormone, resulted in impaired cAMP formation to a second challenge of alpha-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 beta-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 beta-arrestin to the activated receptor. Our data demonstrate that, through PKA-, GRK-, beta-arrestin-, and dynamin-dependent processes, MC4R undergoes internalization in response to agonist, thereby providing novel insights into the regulation of MC4R signaling.
下丘脑黑皮质素-4受体（MC4R）通路的破坏会导致人类和啮齿动物肥胖，这表明下丘脑MC4R在能量稳态调节中起关键作用。由于MC4R基因即使单倍体不足也会导致人类和小鼠肥胖，因此受体数量或信号传导的细微变化可能会影响食物摄入和能量消耗的调节。关于MC4R信号传导的细胞内调节知之甚少。利用GT1-7细胞，我们首次表明MC4R会发生配体介导的脱敏。然后，我们使用转染了血凝素标记的人MC4R的HEK293和COS-1细胞，探讨了脱敏背后的可能机制。将表达内源性MC4R的GT1-7细胞预先暴露于MC4R激动剂α-黑素细胞刺激素，会导致对α-黑素细胞刺激素的第二次刺激时cAMP形成受损。MC4R的脱敏伴随着HEK293细胞中受体的时间依赖性内化，用特异性蛋白激酶A（PKA）抑制剂H89预处理可部分抑制这种内化。在COS-1细胞中，显性负性G蛋白偶联受体激酶（GRK）2-K220R的过表达部分抑制了激动剂介导的MC4R内化，而在HEK293细胞中则没有。β-抑制蛋白1-V53D和发动蛋白I-K44A显性负性突变体的过表达阻止了激动剂介导的MC4R内化。诱变研究表明，C末端尾巴中的Thr312和Ser329/330是PKA和GRK磷酸化的潜在位点，可能在β-抑制蛋白募集到活化受体中起重要作用。我们的数据表明，通过PKA、GRK、β-抑制蛋白和发动蛋白依赖性过程，MC4R会因激动剂而发生内化，从而为MC4R信号传导的调节提供了新的见解。
