Regulation of G Protein–Coupled Receptor Signaling: Specific Dominant-Negative Effects of Melanocortin 2 Receptor Accessory Protein 2

Abstract

Heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs), which constitute the largest family of membrane proteins, mediate responses to diverse physiological stimuli. The presence of melanocortin 2 receptors (MC2Rs) on the plasma membrane requires the presence of either MC2R accessory protein (MRAP) or MRAP2, which are homologous accessory proteins. Here, we show that, whereas MRAP was essential for activation of MC2R signaling, MRAP2 was an endogenous inhibitor that competed with MRAP for binding to MC2R and decreased the potency of adrenocorticotropic hormone (ACTH), the endogenous agonist for MC2Rs, in stimulating the production of adenosine 3′,5′-monophosphate (cAMP). ACTH bound with high affinity to MC2Rs in the presence of MRAP, but not MRAP2. The ability of MRAP and MRAP2 to influence ligand-binding affinity was specific to MC2R, because these proteins had little effect on the binding of NDP-α-melanocyte–stimulating hormone to MC4R or on its stimulation of cAMP responses. These results demonstrate that the balance of stimulatory and inhibitory accessory proteins can control the sensitivity of a GPCR to its natural agonist.

Adrenocorticotropic Hormone (ACTH) Responses Require Actions of the Melanocortin-2 Receptor Accessory Protein on the Extracellular Surface of the Plasma Membrane

“…Although only the MC2 receptor requires an accessory protein, MRAP and MRAP2 both co-precipitate with all five MC receptors, and MRAP2 regulates responses of several of them, most notably the MC4 receptor involved in food intake and energy expenditure (6, 13-15, 31, 32). MRAP and MRAP2 heterodimerize readily, and MRAP2 can act in a dominant negative fashion to antagonize the effects of MRAP on the MC2 receptor (8). The results shown here raise the possibility that multiple dimers of MRAP and MRAP2 can interact with a given receptor, adding a new dimension to an already complex signaling system.…”

“…Substantial evidence indicates that MRAP forms an antiparallel homodimer (4,6,8,9). This structure is highly unusual and possibly unique among single pass membrane proteins.…”

Melanocortin receptors and their accessory proteins

“…MRAP and MRAP2 are able to form heterodimeric complexes in vitro, however, whether such a complex is physiologically relevant is not known (Chan et al, 2009b;Sebag and Hinkle, 2010). In one study MC2R was expressed alongside both MRAP and MRAP2, the resulting dose response curve appeared sensitive to the ratio of MRAP to MRAP2, with increasing proportions of MRAP2 shifting the curve further to the right (Sebag and Hinkle, 2010). Importantly, this effect has not been observed in other studies (Chan et al, 2009b;Agulleiro et al, 2010).…”

“…It was suggested that this difference was due to lack of the leucine, aspartic acid, tyrosine, isoleucine (LDYI) motif in MRAP2, which was present in MRAP, and insertion of the LDYI residues enabled the MRAP2/MC2R complex to respond to lower concentrations of ACTH (Sebag and Hinkle, 2009b). MRAP and MRAP2 are able to form heterodimeric complexes in vitro, however, whether such a complex is physiologically relevant is not known (Chan et al, 2009b;Sebag and Hinkle, 2010). In one study MC2R was expressed alongside both MRAP and MRAP2, the resulting dose response curve appeared sensitive to the ratio of MRAP to MRAP2, with increasing proportions of MRAP2 shifting the curve further to the right (Sebag and Hinkle, 2010).…”

“…Signaling in these experiments was assessed by measuring cAMP generation using a competitive protein binding assay in response to treatment with a single concentration of agonist (Chan et al, 2009b). When the signaling properties of MC4R in the presence of MRAP were studied by measuring cAMP generation with a luciferase assay across a range of agonist concentrations using mouse constructs, co-expression of MRAP was not found to influence MC4R signaling (Sebag and Hinkle, 2010). Further examination of this interaction is needed to definitively understand whether MRAP interacts with MC4R in vivo and to determine the impact of this interaction on MC4R signaling.…”