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Regulation of melanocortin-4 receptor signaling: agonist-mediated desensitization and internalization
Hiroshi Shinyama1, Hiroaki Masuzaki, Hui Fang
1Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.
Abstract:
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.
Insights
The melanocortin-4 receptor (MC4R) pathway is key for energy balance. This study reveals MC4R desensitization and internalization mechanisms involving PKA, GRK, beta-arrestin, and dynamin, offering new insights into MC4R signaling regulation.
Area of Science:
- Neuroscience
- Molecular Biology
- Endocrinology
Background:
- The melanocortin-4 receptor (MC4R) pathway is critical for regulating energy homeostasis, with disruptions leading to obesity.
- Even minor alterations in MC4R function can significantly impact food intake and energy expenditure.
- Intracellular mechanisms regulating MC4R signaling remain largely unexplored.
Purpose of the Study:
- To investigate the intracellular regulation of MC4R signaling.
- To characterize the phenomenon of ligand-mediated desensitization of MC4R.
- To elucidate the molecular mechanisms underlying MC4R internalization.
Main Methods:
- Utilized GT1-7, HEK293, and COS-1 cell lines.
- Employed alpha-melanocyte-stimulating hormone as the MC4R agonist.
- Investigated receptor desensitization via cAMP assays.
- Analyzed receptor internalization using dominant-negative mutants of PKA, GRK, beta-arrestin, and dynamin.
- Performed mutagenesis studies on MC4R C-terminal tail residues.
Main Results:
- Demonstrated ligand-mediated desensitization of MC4R in GT1-7 cells.
- Observed time-dependent internalization of MC4R in HEK293 cells, partly inhibited by a PKA inhibitor.
- Showed that GRK2, beta-arrestin1, and dynamin play roles in MC4R internalization.
- Identified Thr312 and Ser329/330 as potential phosphorylation sites crucial for beta-arrestin recruitment.
Conclusions:
- MC4R signaling is regulated by ligand-induced desensitization and internalization.
- These processes involve complex interactions between PKA, GRK, beta-arrestin, and dynamin.
- The findings provide novel insights into the intracellular regulation of MC4R, crucial for understanding energy balance and obesity.