The melanocortin-4 receptor (MC4R) is essential for control of energy homeostasis in vertebrates. MC4R interacts with melanocortin receptor accessory protein 2 (MRAP2) in vitro, but its functions in vivo are unknown. We found that MRAP2a, a larval form, stimulates growth of zebrafish by specifically blocking the action of MC4R. In cell culture, this protein binds MC4R and reduces the ability of the receptor to bind its ligand, α–melanocyte-stimulating hormone (α-MSH). A paralog, MRAP2b, expressed later in development, also binds MC4R but increases ligand sensitivity. Thus, MRAP2 proteins allow for developmental control of MC4R activity, with MRAP2a blocking its function and stimulating growth during larval development, whereas MRAP2b enhances responsiveness to α-MSH once the zebrafish begins feeding, thus increasing the capacity for regulated feeding and growth.

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, ␤2-adrenergic, or TSH-releasing hormone receptors. MRAP is the first eukaryotic membrane protein identified with an antiparallel homodimeric structure.ACTH ͉ G protein-coupled receptor ͉ membrane ͉ orientation T he G protein-coupled receptor (GPCR) family is the largest group of membrane proteins in the human genome. GPCRs respond to a wide array of signals and regulate numerous intracellular signaling pathways. They are characterized by an extracellular amino terminus that is usually glycosylated, seven transmembrane domains, and a cytoplasmic carboxyl terminus. After synthesis in the endoplasmic reticulum, GPCRs must move to the plasma membrane before they can signal in response to extracellular ligands.In the adrenal gland, the melanocortin-2 (MC2) receptor, also referred to as the corticotropin (ACTH) receptor, is activated by the pituitary hormone ACTH and promotes glucocorticoid biosynthesis. The MC2 receptor is expressed primarily in the adrenal cortex and is positively coupled to adenylyl cyclase. The MC2 receptor is a member of the class A rhodopsin-like family and the smallest known GPCR. Individuals harboring inactivating mutations in the MC2 receptor suffer from familial glucocorticoid deficiency, or hereditary unresponsiveness to ACTH (1). Recently, it was discovered that familial glucocorticoid deficiency can also arise from mutations in an accessory protein required for ACTH signaling, the MC2 receptor accessory protein (MRAP) (2). Lack of functional MRAP, like the lack of an MC2 receptor, causes ACTH re...

The regulated release of anorexigenic α-MSH and orexigenic Agouti-related protein (AgRP) from discrete hypothalamic arcuate neurons onto common target sites in the CNS plays a fundamental role in the regulation of energy homeostasis. Both peptides bind with high affinity to the melanocortin-4 receptor (MC4R); existing data showα-MSH is an agonist that couples the receptor to the Gαs signaling pathway1, while AgRP binds competitively to block α-MSH binding2, and block the constitutive activity mediated by the ligand-mimetic amino terminal domain of the receptor3. Here, we show that regulation of firing activity of hypothalamic PVN neurons by α-MSH and AgRP can be mediated independently of Gαs signaling by ligand-induced coupling of MC4R to closure of inwardly rectifying potassium channel, Kir7.1. Further, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding. Consequently, Kir7.1 signaling appears central to melanocortin-mediated regulation of energy homeostasis within the PVN. Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signaling, including the gene dosage effect of MC4R4, and the sustained effects of AgRP on food intake5.

MRAP, melanocortin 2 (MC2) receptor accessory protein, is required for trafficking by the MC2 (ACTH) receptor. MRAP is a single transmembrane protein that forms highly unusual antiparallel homodimers. We used molecular complementation to ask where MRAP achieves dual topology. Fragments of yellow fluorescent protein (YFP) were fused to the NH 2 or COOH terminus of MRAP such that YFP fluorescence could occur only in antiparallel homodimers; fluorescence was present in the endoplasmic reticulum. MRAP retained dual topology after deletion of most of the amino terminus. In contrast, deletion of residues 31-37, just NH 2 -terminal to the transmembrane domain, forced MRAP into a single N exo /C cyt orientation and blocked its ability to promote MC2 receptor trafficking and homodimerize. When the transmembrane domain of MRAP was replaced with the corresponding region from RAMP3, dual topology was retained but MRAP was inactive. Insertion of MRAP residues 29 -37 conferred dual topology to RAMP3, normally Pituitary adrenocorticotropic hormone (ACTH)2 activates melanocortin 2 (MC2) receptors in the adrenal cortex, stimulating the biosynthesis of glucocorticoids. In familial glucocorticoid deficiency, patients are resistant to ACTH and unable to make sufficient glucocorticoids. Unless adrenal corticosteroids are replaced, the failure to respond to ACTH can lead to hypoglycemia, infection, and death. Some individuals with familial glucocorticoid deficiency (type 1) have inactivating mutations in the MC2 receptor (1-3). As shown by Metherell et al. (4), another group of patients with familial glucocorticoid deficiency (type 2) has mutations in a protein needed for MC2 receptor function, termed MRAP (melanocortin 2 receptor accessory protein) (1, 2).MRAP is required for MC2 receptor maturation and trafficking to the plasma membrane (4 -6). It is a small protein containing a single transmembrane domain with no signal peptide. In the absence of MRAP, MC2 receptor is retained in the endoplasmic reticulum (ER), lacks mature carbohydrate, and is rapidly degraded. In the presence of MRAP, MC2 receptor is glycosylated and localized on the plasma membrane, where it binds ACTH and activates adenylyl cyclase (6).The NH 2 -terminal and transmembrane segments of MRAP are strongly conserved, whereas the COOH-terminal domains are highly divergent and apparently nonessential. Two splice variants of human MRAP that differ completely in the region COOH-terminal to the transmembrane region (5) and a truncated mouse MRAP lacking the entire COOH terminus (6) promote surface expression and signal transduction by the MC2 receptor. MRAP forms a stable, immunoprecipitable complex with the MC2 receptor (4, 6, 7). The MC2 receptor is one of five melanocortin receptors, class A G protein-coupled receptors (GPCRs) that are coupled to G proteins and cause an increase in cAMP when activated (8). ACTH is the natural agonist for the MC2 receptor, whereas the various melanocyte-stimulating hormone peptides bind with high affinity to all other melanocortin ...

Ghrelin is the only known circulating orexigenic hormone. It is primarily secreted by the stomach and acts at its receptor, the growth hormone secretagogue receptor 1a (GHSR1a), in the hypothalamus to signal hunger and promote food intake. The melanocortin receptor accessory protein 2 (MRAP2) was previously shown to regulate energy homeostasis through the modulation of the activity of the melanocortin-4 receptor and prokineticin receptors. In this study we identify MRAP2 as a partner of ghrelin-GHSR1a signaling. We show that MRAP2 interacts with GHSR1a and potentiates ghrelin-stimulated signaling both in vitro and in vivo. We demonstrate that in the absence of MRAP2, fasting fails to activate agouti-related protein neurons. In addition, we show that the orexigenic effect of ghrelin is lost in mice lacking MRAP2. Our results suggest that MRAP2 is an important modulator of the energy homeostasis machinery that operates through the regulation of multiple GPCRs throughout the hypothalamus.