hMRAPα, but Not hMRAP2, Enhances hMC4R Constitutive Activity in HEK293 Cells and This Is Not Dependent on hMRAPα Induced Changes in hMC4R Complex N-linked Glycosylation

MRAP1 but not MRAP2, is essential for melanocortin receptor 2 functional expression. Human MRAP1 splice variant (hMRAPα) and human MRAP2 (hMRAP2) also interact with the other melanocortin receptor subtypes in vitro, although the physiological significance of these interactions is unknown. Previously we showed that HA-hMC4R co-expression with hMRAPα, but not hMRAP2, specifically alters HA-hMC4R complex N-linked glycosylation. hMRAPα-FLAG also enhances hMC4R constitutive activity in vitro. Here we directly compare hMRAPα and hMRAP2 effects on hMC4R constitutive activity in HEK293 cells. In contrast to hMRAPα, co-expression with hMRAP2 had no effect on HA-hMC4R or untagged hMC4R constitutive coupling to adenylyl cyclase. We used fixed and live cell imaging of HA-hMC4R and hMC4R-eGFP respectively, to further characterise effects of hMRAPα on hMC4R subcellular trafficking. hMRAPα-FLAG co-expression did not alter the partitioning of either HA-hMC4R or hMC4R-eGFP into either the ER or the Golgi apparatus, therefore the hMRAPα effect on hMC4R complex N-linked glycosylation is probably not due to hMC4R retention in the ER. We also observed that unlike HA-hMC4R, hMC4R-eGFP lacks complex glycosylation both in the presence and absence of hMRAPα, although both HA-hMC4R and hMC4R-eGFP exhibited increased constitutive coupling to adenylyl cyclase following co-expression with hMRAPα. We conclude that hMRAPα and not hMRAP2 modulates hMC4R constitutive activity. Furthermore, hMRAPα does not increase hMC4R constitutive activity by altering hMC4R complex N-linked glycosylation. Instead we hypothesise that hMRAPα alters hMC4R conformational states leading to increased hMC4R constitutive activity.