Pharmacological modulation of two melanocortin-5 receptors by MRAP2 proteins in zebrafish

in Journal of Molecular Endocrinology
Correspondence should be addressed to C Zhang: zhangchao@tongji.edu.cn

*(M Zhu and M Wang contributed equally to this work)

Restricted access

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.

Downloadable materials

  • 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 &#x00B1; SEM, ***, p < 0.001 compared with control group.

 

An official journal of

Society for Endocrinology

Sections

Figures

  • View in gallery

    Phylogenetic and synteny analysis of zebrafish MC5R receptors. (A) Phylogenetic tree of MC5Rs constructed by NJ method with Mega 6.0 software. Jones–Taylor–Thornton (JTT) model was used. The strength of branch relationships was assessed by bootstrap replication (N = 1000 replicates). Asterisk (*) indicates zebrafish mc5ra and mc5rb. (B) Synteny analysis of MC5Rs. Genes in solid black square are conserved among zebrafish, spotted agar, human, mouse, and rat.

  • View in gallery

    MRAP2s interact and regulate surface expressions of MC5Rs. Co-immunoprecipitation assay exhibits the interactions between zMC5Ra and zMRAP2a (A), zMRAP2b (B), also between zMC5Rb and zMRAP2a (C), zMRAP2b (D). *IgG heavy chain. Surface expression of zMC5Ra and zMC5Rb measured by whole-cell ELISA in 293T cells transfected with zMC5Ra or zMC5Rb and different amounts of zMRAP2a or zMRAP2b (E, F, G and H). Data are shown as mean ± s.e.m. and analyzed by two-tailed t test. **P < 0.01; ***P < 0.001. Surface expression of mammalian MC5R measured by whole-cell ELISA in 293T cells transfected with mammalian MC5R and different amounts of mammalian MRAP2 (I and J).

  • View in gallery

    Pharmacological modulation of MC5Rs by MRAP2s. Dose response curves of α-MSH induced cAMP production in 293T cells upon transfection with zMC5Ra (A and B) or zMC5Rb (C and D) in presence of different amount of zMRAP2a or zMRAP2b. Dose response curves of SHU9119 induced cAMP production in 293T cells transfected with zMC5Ra (E and F) or zMC5Rb (G and H) in presence of different amount of zMRAP2a or zMRAP2b. Dose response curves of α-MSH (I) and SHU9119 (J) induced cAMP production in 293T cells transfected with mouse MC5R and the indicated amount of mouse MRAP2. Binding competition of agonist (α-MSH) and antagonist (AgRP) of MC5Rs modulated by MRAP2s (K, L, M and N).

  • View in gallery

    Zebrafish MC5R forms homo- and heterodimers. YFP fluorescence (green) of zMC5Ra homodimers (A), zMC5Rb homodimers (B) and zMC5Ra zMC5Rb heterodimers (C) (scale bar = 10 µm). Effect of zMRAP2a and zMRAP2b on zMC5Rs dimerization. CHO cells co-expressing zMC5Ra homodimers (D and E), zMC5Rb homodimers (F and G) or zMC5Rs heterodimers (H and I) with zMRAP2a (D, F and H) or zMRAP2b (E, G and I). Surface expression of zMRAP2a and zMRAP2b is shown in red, detected by anti-V5 antibody and secondary anti-mouse Alexa594 (abcam). MRAP2 suppressed the dimer formation of MC5R proteins.

  • View in gallery

    Fasting induced expression alternation of MC5R in zebrafish brain. Adult zebrafish (TU) were fasted 0, 2, 10, 15 days and the mc5ra and mc5rb expression in the brain were analyzed by qRT-PCR. Data are analyzed by two-tailed t test compared with Day 0, graphs are shown as mean ± s.e.m. **, P < 0.01; ***, P < 0.001; ns, not significant.

Index Card

PubMed

Google Scholar

Related Articles

Altmetrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 27 27 27
Full Text Views 50 50 50
PDF Downloads 17 17 17