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Melanocortin-2 receptor accessory protein MRAP forms antiparallel homodimers.

ABSTRACT: The melanocortin-2 (MC2) receptor accessory protein (MRAP) is required for trafficking of the G protein-coupled MC2 receptor to the plasma membrane. The mechanism of action and structure of MRAP, which has a single transmembrane domain, are unknown. Here, we show that MRAP displays a previously uncharacterized topology. Epitopes on both the N- and C-terminal ends of MRAP were localized on the external face of CHO cells at comparable levels. Using antibodies raised against N- and C-terminal MRAP peptides, we demonstrated that both ends of endogenous MRAP face the outside in adrenal cells. Nearly half of MRAP was glycosylated at the single endogenous N-terminal glycosylation site, and over half was glycosylated when the natural glycosylation site was replaced by one in the C-terminal domain. A mutant MRAP with potential glycosylation sites on both sides of the membrane was singly but not doubly glycosylated, suggesting that MRAP is not monotopic. Coimmunoprecipitation of differentially tagged MRAPs established that MRAP is a dimer. By selectively immunoprecipitating cell surface MRAP in one or the other orientation, we showed that MRAP homodimers are antiparallel and form a stable complex with MC2 receptor. In the absence of MRAP, MC2 receptor was trapped in the endoplasmic reticulum, but with MRAP, the MC2 receptor was glycosylated and localized on the plasma membrane, where it signaled in response to ACTH. MRAP acted specifically, because it did not increase surface expression of other melanocortin, beta2-adrenergic, or TSH-releasing hormone receptors. MRAP is the first eukaryotic membrane protein identified with an antiparallel homodimeric structure.

SUBMITTER: Sebag JA

PROVIDER: S-EPMC2154416 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Regulation of Lipolytic Response and Energy Balance by Melanocortin 2 Receptor Accessory Protein (MRAP) in Adipocytes.

Project description:Melanocortin 2 receptor accessory protein (MRAP) is highly expressed in adrenal gland and adipose tissue. In adrenal cells, MRAP is essential for adrenocorticotropic hormone (ACTH)-induced activation of the cAMP/protein kinase A (PKA) pathway by melanocortin 2 receptor (MC2R), leading to glucocorticoid production and secretion. Although ACTH was known to stimulate PKA-dependent lipolysis, the functional involvement of MRAP in adipocyte metabolism remains incompletely defined. Herein, we found that knockdown or overexpression of MRAP in 3T3-L1 adipocytes reduced or increased ACTH-induced lipolysis, respectively. Moreover, an unbiased proteomics screen and coimmunoprecipitation analysis identified Gαs as a novel interacting partner of MRAP. An MRAP mutant disabled in Gαs association failed to augment the activation of PKA and lipolytic response to ACTH. Furthermore, compared with wild-type mice, transgenic mice (aP2-MRAP) overexpressing MRAP fat specifically exhibited increased lipolytic response to ACTH. When fed a high-fat diet (HFD), the transgenic mice displayed a significant decrease in the gain of adiposity and body weight as well as an improvement in glucose and insulin tolerance. These phenotypes were accompanied by increased adipose expression of genes for mitochondrial fatty acid oxidation and thermogenesis, and overall energy expenditure. Collectively, our data strongly suggest that MRAP plays a critical role in the regulation of ACTH-induced adipose lipolysis and whole-body energy balance.

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