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, ␤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...

Opposite Effects of the Melanocortin-2 (MC2) Receptor Accessory Protein MRAP on MC2 and MC5 Receptor Dimerization and Trafficking

“…Surface and Total Epitope Detection by Fixed Cell ELISA-To measure epitopes on the extracellular side of the plasma membrane, cells in 12-or 24-well plates were washed with phosphate-buffered saline, fixed for 10 min with 2% paraformaldehyde, washed, blocked in 5% nonfat dry milk in phosphatebuffered saline, incubated with 1:5000 mouse monoclonal anti-HA or anti-V5 antibody in blocking buffer for 2 h at room temperature, washed three times for 5 min in phosphate-buffered saline, and incubated with secondary antibody and processed for ELISA as described (3). The same protocol was performed in permeabilized cells to measure total expression of proteins of interest.…”

“…MRAP forms antiparallel homodimers, which are exceedingly rare structures, and it is these dimers that co-precipitate with the MC2 receptor (3). Mutagenic analysis has revealed that the carboxyl-terminal region of MRAP is not essential, but the amino-terminal and transmembrane regions are necessary for function (3)(4)(5).…”

“…Deletion or alanine substitution of a critical four-amino acid segment, LDYI, at residues 18 -21 of the mouse sequence, results in an MRAP molecule that facilitates expression of the MC2 receptor on the plasma membrane but does not allow the receptor to bind agonist or signal (4). MRAP is not required for cell surface expression of other melanocortin receptors and can inhibit expression or signaling in some cases (3,6).…”

“…MRAP forms antiparallel homodimers, which are exceedingly rare structures, and it is these dimers that co-precipitate with the MC2 receptor (3). Mutagenic analysis has revealed that the carboxyl-terminal region of MRAP is not essential, but the amino-terminal and transmembrane regions are necessary for function (3)(4)(5). Deletion or alanine substitution of a critical four-amino acid segment, LDYI, at residues 18 -21 of the mouse sequence, results in an MRAP molecule that facilitates expression of the MC2 receptor on the plasma membrane but does not allow the receptor to bind agonist or signal (4).…”

“…The MC2 receptor is also unusual in its requirement for an accessory protein, the MC2 receptor accessory protein (MRAP) (2). MRAP must be expressed with the MC2 receptor in order for the receptor to undergo glycosylation, traffic to the plasma membrane, bind ACTH, and stimulate adenylyl cyclase (2)(3)(4). Individuals with inactivating mutations of either the MC2 receptor or MRAP suffer from ACTH resistance and severe glucocorticoid deficiency (2).…”