Pertussis Toxin-sensitive Signaling of Melanocortin-4 Receptors in Hypothalamic GT1-7 Cells Defines Agouti-related Protein as a Biased Agonist

Abstract

Melanocortin-4 receptor (MC4R)-induced anorexigenic signaling in the hypothalamus controls body weight and energy homeostasis. So far, MC4R-induced signaling has been exclusively attributed to its coupling to G s proteins. In line with this monogamous G protein coupling profile, most MC4R mutants isolated from obese individuals showed a reduced ability to activate G s . However, some mutants displayed enhanced G s coupling, suggesting that signaling pathways independent of G s may be involved in MC4R-mediated anorexigenic signaling. Here we report that the G s signaling-deficient MC4R-D90N mutant activates G proteins in a pertussis toxin-sensitive manner, indicating that this mutant is able to selectively interact with G i/o proteins. Analyzing a hypothalamic cell line (GT1-7 cells), we observed activation of pertussis toxin-sensitive G proteins by the wild-type MC4R as well, reflecting multiple coupling of the MC4R to G s and G i/o proteins in an endogenous cell system. Surprisingly, the agouti-related protein, which has been classified as a MC4R antagonist, selectively activates G i/o signaling in GT1-7 cells. Thus, the agouti-related protein antagonizes melanocortin-dependent G s activation not only by competitive antagonism but additionally by initiating G i/o protein-induced signaling as a biased agonist.The melanocortin system has been shown to play a pivotal role in food intake and energy homeostasis. Therefore, dysfunction of the melanocortin system inevitably leads to an obese phenotype in mammals. Accordingly, targeted disruption of the melanocortin-4 receptor (MC4R) 2 gene in mice causes an obesity-diabetes syndrome characterized by hyperphagia, hyperinsulinemia, and hyperglycemia (1). The importance of MC4R signaling in the regulation of human metabolism has been highlighted by the finding that mutations in the MC4R gene are the most frequent monogenic cause of severe obesity (2-7).Signaling pathways involved in MC4R-mediated regulation of energy homeostasis have been attributed to its coupling to G s proteins and the resulting activation of the protein kinase A pathway (8, 9). Agouti and agouti-related protein (AGRP) are the only known endogenously occurring neuropeptides that block GPCR activity and are, therefore, classified as MCR antagonists. AGRP has been shown to block melanocortin signaling at MC3R and MC4R subtypes (10). In addition, it has been proposed that AGRP decreases basal as well as forskolinpromoted adenylyl cyclase activity, thus also acting as an inverse agonist on basal MCR activity (11). However, recent studies revealed that the effects of AGRP on appetite control are independent of melanocortin signaling (12, 13). For example, in mice deficient of the melanocortin precursor proopiomelanocortin starvation after AGRP neuron ablation is independent of melanocortin signaling (13). Thus, the orexigenic effects induced by AGRP appear to be mediated in a melanocortin-independent manner by a so far unknown mechanism.Interestingly, the aforementioned MC4R mutants isolated from obese pat...

Cell Adhesion Receptor GPR133 Couples to Gs Protein

“…2). Note that MC4R-transfected cells alone produce some IP response via G i coupling (44), which is, however, much lower when compared with MC4R/ G␣ qs4 -co-transfecetd cells. Transfection of either GPR133 or G␣ qs4 alone did not increase basal IP levels when compared with mock-transfected COS-7 cells.…”

Constitutive Cholesterol-dependent Endocytosis of Melanocortin-4 Receptor (MC4R) Is Essential to Maintain Receptor Responsiveness to α-Melanocyte-stimulating Hormone (α-MSH)

“…GT1-7 cells are immortalized hypothalamic neurons that have endogenous MC4R (14,17,(51)(52)(53). To compare the effect of cholesterol depletion on MC4R and ␤ 2 AR endocytosis, we carried out antibody feeding experiments.…”

Characterization of murine melanocortin receptors mediating adipocyte lipolysis and examination of signalling pathways involved

“…Besides the expression in 3T3-L1 cells, MC5R has also been identified in human adipose tissue and associated with obesity phenotypes (Chagnon et al 1997). The G s transduction pathway is established to be a validated path of signalling for all MCRs (Barrett et al 1994;Mountjoy et al 1992) all though other signalling pathways have been suggested (Buch et al 2009;Konda et al 1994;Daniels et al 2003;Rodrigues et al 2009). The natural occurring melanocortin peptide family consists of -, -and -melanocyte stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH).…”