The Melanocortin 2 Receptor Accessory Protein Exists as a Homodimer and Is Essential for the Function of the Melanocortin 2 Receptor in the Mouse Y1 Cell Line

Abstract

The ACTH receptor [melanocortin 2 receptor (MC2R)] gene produces a functional receptor only when transfected into cells of adrenocortical origin, implying that it may require an adrenal-specific accessory factor. Recently we showed that the MC2R accessory protein (MRAP) is essential for the cell surface expression of the MC2R in such models. Using RNA interference (RNAi) technology, we have further explored the action of MRAP in the functioning of the MC2R in Y1 mouse adrenocortical cells that endogenously express MRAP and MC2R. We created stable cell lines expressing mouse MRAP short hairpin RNA (shRNAs) by transfecting cells with an expression vector containing the MRAP small interfering RNA sequence. The knockdown of MRAP resulted in a reduction in MC2R signaling. The overexpression of a mouse MRAP-Flag construct did not restore the expression of MRAP due to its degradation by the mouse shRNAs. The introduction of human MRAP that is resistant to silencing by mouse MRAP shRNAs resulted in the rescue of the MC2R signaling. MRAP migrates on SDS-PAGE with markedly lower mobility than predicted for a 14.1-kDa protein. Coimmunoprecipitation and mass spectroscopy suggests that MRAP exists as a homodimer that is resistant to dissociation by sodium dodecyl sulfate and reducing agents.

H295R expression of melanocortin 2 receptor accessory protein results in ACTH responsiveness

“…The MRAP gene is encoding a protein of 172 residues and predicted molecular weight of 19 kDa. MRAP forms a stable antiparallel homodimer at the plasma membrane and in the ER and shows glycosylation of the protein (Sebag & Hinkle 2007, Cooray et al 2008. In the present study, both anti-V5 antibody and anti-MRAP antibody detected the bands in H295RA cells between 30 and 40 kDa on western blotting.…”

“…In fact, a previous study demonstrated that endogenously expressed Y-1 adrenal cell MRAP was immunodetected as a band of more than 30 kDa despite the predicted molecular mass of 14 kDa (Cooray et al 2008). H295RA cells showed higher basal MC2R expression levels and greater response to ACTH stimulation compared to the parental H295R cells in terms of steroidogenesis and mRNA expression of steroidogenic enzymes.…”

“…Mutations in MRAP gene cause familial glucocorticoid deficiency (FGD) type 2, which is an autosomal recessive disorder characterized by severe cortisol deficiency with high plasma ACTH levels (Metherell et al 2005). In the Y-1 mouse cell line that endogenously express both MC2R and MRAP, knockdown of MRAP by shRNA resulted in a loss of response to ACTH, which was rescued by overexpression of the shRNAinsensitive human MRAP (Cooray et al 2008). It has also been demonstrated that mRNA levels of both MC2R and MRAP are regulated by ACTH in human adrenal primary cultures (Xing et al 2010).…”

Promiscuity among the MRAPs

“…Human MRAP1 existed as one of two splice variants -MRAP1 α and β that had distinct 3′ ends encoding different C-termini. We found MRAP1 existed as a very stable dimer that was relatively resistant to dissociation by detergents and reducing agents (Cooray et al 2008). Remarkably, Sebag and Hinkle (2007) demonstrated using a number of techniques that this was an antiparallel homodimer in which one N-terminus was extracellular and the other intracellular.…”

A corticotroph pituitary adenoma as the initial presentation of familial glucocorticoid deficiency

“…The functional expression of MC2R requires the MC2R accessory protein (MRAP), a protein involved in the trafficking of MC2R (2,3). Mutations in MC2R or MRAP cause familial glucocorticoid deficiency (FGD).…”