Identification and Characterisation of Melanocortin Receptor Accessory Protein Splice Variants in Human Tissues

Abstract

Human melanocortin receptor accessory protein (hMRAP) is essential for the human melanocortin 2 receptor (hMC2R) to functionally express in vivo and in vitro. hMRAP also interacts with the other four hMCR subtypes in vitro. The hMRAP gene gives rise to hMRAPα and hMRAPβ through alternative exon splicing after nucleotide 206 which aligns with the Cterminus. These splice variants share identical N-terminal and TMDs while the C-terminal domains are divergent. The Genbank contains three hMRAPα sequences that differ by one or two amino acids after nucleotide 206. Previous work in the Mountjoy laboratory identified a hMRAPβ splice variant with a 32bp deletion after nucleotide 206. hMRAP2 is a paralogue of the hMRAP gene and there are no known hMRAP2 splice variants. It is hypothesised that functional hMRAP and hMRAP2 splice variants exist in human tissues. To test this, hMRAPα, hMRAPβ and hMRAP2 were amplified from seven human RNA samples using RT-PCR and the amplicons were sequenced. Full characterisation of these splice variants, including hMRAPα, hMRAPβ and hMRAP2, requires detection using epitope tags incorporated into the proteins. We hypothesise that epitope tagging of these splice variants could interfere with their function. To test this, full length coding sequences for each identified splice variant with or without N- or C-terminal epitope tags were subcloned into a mammalian expression vector and the recombinant DNA transiently co-transfected together with a hMCR subtype into HEK293 cells. The cells were stimulated with melanocortin peptides and adenylyl cyclase activity was measured. Two novel splice variants, hMRAPα-RW and hMRAP2-Short, were identified with divergent Cterminals. hMRAPα-RW was functional and preliminary data suggests that it differentially modulates hMC2R and hMC4R compared to hMRAPα-WT. Preliminary data suggests that hMRAP2-Short is functional and influences hMCR function compared to hMRAP2-WT. N-terminal HA and C-terminal FLAG epitope tags on hMRAPα and either N- or C-terminal FLAG epitope tags on hMRAPβ had minimal effects on hMRAPα-WT and hMRAPβ-WT function, respectively. We conclude that functional novel hMRAP and hMRAP2 splice variants with divergent Cterminal domains exist in human tissues and they may have important physiological roles in the modulation of hMCR signalling in vivo.