Julien A. Sebag

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.

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.

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 NH2 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 NH2-terminal to the transmembrane domain, forced MRAP into a single Nexo/Ccyt 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 in an Nexo/Ccyt orientation. When expressed with MRAPDelta1-30, MRAPDelta10-20, or MRAPDelta21-30, MC2 receptor was localized on the plasma membrane but unable to respond to ACTH. Residues 18-21 of MRAP were critical; MC2 receptor expressed with MRAP(18-21A) localized to the plasma membrane but did not bind 125I-ACTH or increase cAMP in response to ACTH. A newly identified MRAP homolog, MRAP2, lacks amino acids 18LDYI21 of MRAP and, like MRAP(18-21A), allows MC2 receptor trafficking but not signaling. MRAP2 with an LDYI insertion functions like MRAP. These results demonstrate that MRAP not only facilitates MC2 receptor trafficking but also allows properly localized receptor to bind ACTH and consequently signal.