1 Translational Medical Center for Stem Cell Therapy and Institute for Regenerative Medicine, Shanghai East Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, China
Correspondence should be addressed to C Zhang: email@example.com
*(M Zhu and M Wang contributed equally to this work)
Melanocortin receptor accessory protein 2 (MRAP2) plays an important role in regulating melanocortin receptors. In zebrafish, MRAP2a and MRAP2b show distinct pharmacological effects on MC4R activity, but how MRAP2 protein regulates other zebrafish melanocortin receptors is barely studied. Zebrafish have two mc5r genes: mc5ra and mc5rb, it is still vague which one is the homologous isoform to the mammalian paralog. Here, we utilize synteny and phylogenetic analysis to demonstrate the evolutionary conservation of zebrafish MC5Ra and MC5Rb among different species. We also show that MRAP2a and MRAP2b could interact and regulate surface expression of two MC5R receptors. Bimolecular fluorescence complementation (BiFC) studies suggest that zebrafish MC5Rs could form homo- and heterodimers, which are suppressed by co-expression with MRAP2 proteins. In comparison with mammalian MC5R-MRAP2 system and different pharmacological effects of zMRAP2 protein on MC5Rs, zmc5ra is identified as the evolutionary homologous paralog to the mammals, and it is regulated by metabolic state in zebrafish brain region.
Supplementary figure 1: Amino acid alignment of multiple MC5R sequences. MC5Rs from multiples species were analyzed with MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/). The alignment was colored with DNAMAN software (Lynnon Biosoft). ENSEMBLE or GenBank or NCBI Reference Sequence accession numbers for MC5Rs: human Homo sapiens (ENSP00000318077), house mouse Mus musculus (ENSMUSP00000130497), cow Bos taurus (ENSBTAP00000012049), chicken Gallus gallus (ENSGALP00000049188), xenopus Xenopus tropicalis (ENSXETP00000057015), fugu Takifugu rubripes (ENSTRUP00000032203), tetraodon Tetraodon nigroviridis (AAQ55179), zebrafish Danio rerio MC5Ra (ENSDARP00000040072) and MC5Rb (ENSDARP00000071694), elephant shark Callorhinchus milii (XP_007893314) and mud shark Squalus acanthias (AAS67890). For interpretation of the references to color in this figure legend, the reader is referred to theWeb version of this article.
Supplementary figure 2: FACS analysis of YFP fluorescent cells. Percent of YFP+ cells co-expressing zMC5Ra homodimers (A), zMC5Rb homodimers (B) and zMC5Ra zMC5Rb heterodimers (C) with RAMP3 (negative control), MRAP2a and MRAP2b. Data were analyzed by two-tailed t test, graphs are shown as mean ± SEM, ***, p < 0.001 compared with control group.