Abstract
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.
Highlights
Two melanocortin receptor accessory protein genes known as MRAP1 and MRAP2 exist in most vertebrates
We determined that hMRAPα-FLAG co-transfection with HA-hMC4R increased HA-hMC4R constitutive coupling to adenylyl cyclase compared to baseline coupling of HA-hMC4R coexpressed with pcDNA 3.1 (~17% increase, p = 0.02) (Fig 1A, Table 1)
This is consistent with the enhanced untagged hMC4R constitutive activity induced by hMRAPα-FLAG that we previously observed [3]. human MRAP2 (hMRAP2)-FLAG co-transfection with HA-hMC4R did not increase nor decrease HA-hMC4R baseline coupling to adenylyl cyclase (Fig 1A, Table 1)
Summary
Two melanocortin receptor accessory protein genes known as MRAP1 and MRAP2 exist in most vertebrates. HMRAPα and hMRAP2 interact with the other four human melanocortin receptor (hMCR) subtypes when they are co-expressed in vitro, but the physiological significance of these interactions is poorly understood [2]. We previously showed that hMRAPα-FLAG co-expression with hMCR subtypes in HEK293 cells has differential effects on the functional expression of all five hMCR subtypes. Sebag et al showed that expression of mouse MRAP2 or the zebrafish homologue MRAP2b with the mouse or zebrafish MC4R respectively, reduced MC4R constitutive activity and increased MC4R sensitivity to α-MSH and coupling to adenylyl cyclase [5]. In this study we aimed to directly compare hMRAPα and hMRAP2 effects on hMC4R constitutive coupling to adenylyl cyclase in HEK293 cells. HMRAP2-FLAG neither increases nor decreases HAhMC4R constitutive coupling to adenylyl cyclase
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