Regions of Melanocortin 2 (MC2) Receptor Accessory Protein Necessary for Dual Topology and MC2 Receptor Trafficking and Signaling

Abstract

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 ...

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

“…In the absence of MC2R accessory protein (MRAP), MC2R is known to be retained in the endoplasmatic reticulum (ER) where it is degraded. However, in the presence of MRAP, MC2R is glycosylated and translocated to the plasma membrane where it is capable ACTH binding (Sebag & Hinkle 2009). Novel research has suggested that all MCRs may employ an accessory protein.…”

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

“…Construction of mouse MRAP and MRAP2 plasmids has been described before (4). ER-tracker blue-white DPX was purchased from Invitrogen.…”

“…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).…”

“…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).…”

Structural determinants regulating cell surface targeting of melanocortin receptors

“…Chan et al (2009) demonstrated that both MRAP1 and MRAP2 enhance MC2R but inhibit MC1R, MC3R, MC4R and MC5R signalling ability. By contrast, Sebag & Hinkle (2009b) showed defects on MC2R signalling when co-expressed with MRAP2 despite its correct trafficking to the cell membrane. These contradictory results may be explained considering the ACTH concentration used in the assays, as only supraphysiological doses of ACTH used by Chan et al (2009) were capable of inducing an activation of MC2R in the presence of MRAP2 (Sebag & Hinkle 2009b).…”