Heterodimerization of MC1R and MC1R-TUBB3 chimeric isoforms.

<p>(A) Co-immunoprecipitation of MC1R-001 and the MC1R-TUBB3 chimeric isoforms. HEK293T cells expressing the indicated constructs were lysed and immunoprecipitated for Flag-labelled MC1R-001, Iso1 or Iso2 using an anti-Flag monoclonal antibody. The pellets were electrophoresed and blotted for HA-labelled MC1R-001 (with a specific anti-HA monoclonal antibody) or for Flag-labelled MC1R-001, Iso1 or Iso2 (with anti-Flag monoclonal antibody) as a control for efficient immunoprecipitation. Total lysates were also electrophoresed and blotted as expression controls (n = 3, representative blots are shown). (B) Co-immunoprecipitation of V60L or R151C variant MC1R-001 and WT MC1R-TUBB3 intergenic splice isoform Iso1. The indicated constructs were expressed in HEK293T cells and immunoprecipitated for Flag-labelled MC1R-001, V60L or R151C using an anti-Flag monoclonal antibody. Immunoblots for Flag-tagged constructs and TUBB3 are shown for immunoprecipitated and total lysates. (C) Representative confocal images of MC1R-001 (green) and Iso1 or Iso2 (red) immunostaining in HBL cells transiently transfected with HA-labelled MC1R-001 and Flag-labelled MC1R-TUBB3 chimeric isoforms. Scale bar, 10 μm. Representative line scan (right panel) from multiple experimental repeats across the cell (location indicated in merged image) shows co-localization of canonical MC1R-001 and chimeric proteins. Line scan, 20 μm for MC1R-001+Iso1, and 31 μm for MC1R-001+Iso2. (D) Effect of heterodimerization on functional coupling to cAMP. Intracellular cAMP levels in HEK293T cells expressing WT, V60L or R151C MC1R-001 alone or in combination with Iso1 and Iso2 upon stimulation with 10⁻⁷ M NDP-MSH for 30 min. Results are presented as residual cAMP production relative to WT MC1R-001 (for which cAMP levels were 0.096±0.043 and 0.889±0.071 pmol/μg protein in resting and stimulated conditions respectively) (n = 3, error bars are ±SEM, two-sided one-way ANOVA was used to generate p values *p< 0.05, **p< 0.01, ***p< 0.001). (E) Specific binding of [¹²⁵I]-NDP-MSH (5x10^-11 M and 5x10⁴ cpm) to HEK293T cells expressing MC1R-001 or the MC1R-TUBB3 isoforms Iso1 and Iso2, alone or in combination (n = 3, error bars are ±SEM, two-sided one-way ANOVA was used to generate p values, ***p< 0.001). (F) Agonist internalization index in HEK293T cells co-transfected with MC1R-001 and Iso1 or Iso2 upon incubation with [¹²⁵I]-labelled NDP-MSH (5x10^-11 M and 5x10⁴ cpm) for 90 min (n = 3, error bars are ±SEM, two-sided one-way ANOVA was used to generate p values, ***p< 0.001).</p

MC1R transcripts and intergenic splice isoforms of MC1R and TUBB3.

<p>(A) Schematic panel showing the exon organization of MC1R splice variants (MC1R-001 and MC1R-002) and MC1R-TUBB3 chimeric transcripts, Iso1 and Iso2. Exons of all MC1R derived transcripts are represented in colored boxes and the number of nucleotides in the ORF is shown below. (B) Diagram representing the structural domains of MC1R-001, MC1R-002, β-tubulin III (TUBB3) and chimeric proteins Iso1 and Iso2. Structural and functional domains are depicted in colored boxes and the number of key residues in the proteins is shown. TM indicates transmembrane regions of MC1R. Dashed lines indicate residues of MC1R and TUBB3 linked in fused proteins Iso1 and Iso2. (C) MC1R-001, Iso1 and Iso2 expression in human melanoma cell lines. Data are shown as relative expression of each isoform (as indicated in each bar graph) as compared with the levels of the isoform in HBL cells. (D) Expression of Iso1 and Iso2 mRNA as a function of the levels of the canonical MC1R-001 transcript in a panel of human melanoma cell lines. Data are represented as mRNA expression of the two intergenic splicing forms relative to MC1R-001 in each cell line.</p

Effects of intergenic splicing on the functional coupling of MC1R to the cAMP and ERK1/2 pathways.

<p>(A) Agonist-induced cAMP production in HEK293T cells expressing MC1R-001, Iso1, Iso2, or the natural MC1R-001 variant allelesV60L, V92M, R151C and D294H. Cells were incubated with 10⁻⁷ M NDP-MSH for 30 min and cAMP levels were determined by an immunoassay (n = 6, error bars are ±SEM, two-sided Student´s t test was used to generate p values, *p< 0.05, *** p< 0.001). (B-C) Representative immunoblots (B) and quantification (C) of ERK1 and ERK2 phosphorylation in PC12 cells transfected to express MC1R-001, Iso1 or Iso2 and stimulated with NDP-MSH (10⁻⁷ M) for the times indicated (n = 5, error bars are ±SEM, two-sided Student´s t test was used to generate p values, *p< 0.05, ** p< 0.01).</p

Radioligand binding and intracellular trafficking properties of MC1R-TUBB3 isoforms.

<p>(A-B) Competition binding assay of HEK293T cells transfected with MC1R-001, Iso1 and Iso2. Cells were incubated with ¹²⁵I-labelled NDP-MSH (5x10^-11 M) and increasing concentrations of non-labelled competing NDP-MSH, from 10⁻¹² to 10⁻⁷ M, extensively washed and counted for radioactivity. Non-specific binding was determined with non-transfected cells or with transfected cells incubated with the radioactive tracer in the presence of excess (10⁻⁶ M) non-labelled peptide, with the same results. Values are represented as specifically bound [¹²⁵I]-NDP-MSH (A) and as percentage of residual binding (B) at the different ligand concentrations (n = 3, data are given as mean ±SEM). (C) Flow cytometric analysis of HEK293T cells expressing MC1R-001 and MC1R-TUBB3 chimeric isoforms. Non-permeabilized cells expressing the indicated proteins were incubated with an anti-Flag antibody labelled with phycoerythrin. Since the Flag epitope was fused in-frame to the extracellular N-terminus of the MC1R sequence, only cells expressing the constructs of the plasma membrane should be detected. Histograms represent cell number (counts) as a function of Flag surface staining, on a logarithmic scale. The gray filled curve refers to cells transfected with an empty pcDNA3 (n = 3, representative histograms are shown). (D) Left panel: Representative confocal images of MC1R-001 or the chimeric isoforms (red) and calnexin (green) immunostaining in HEK293T cells transiently transfected with Flag-labelled MC1R-001 and MC1R-TUBB3 constructs. Scale bar, 10 μm. Representative line scan (right panel) from multiple experimental repeats across the cell (location indicated in merged image) shows co-localization of MC1R-TUBB3 transcripts and calnexin. Line scan, 19 μm for MC1R-001, 17 μm for Iso1 and 18 μm for Iso2. (E) Radioligand internalization assay performed on HEK293T cells expressing MC1R-001, Iso1 or Iso2 incubated with ¹²⁵I-labelled NDP-MSH. The radioactive tracer was isotopically diluted to achieve a final concentration of 5x10^-11 M and 5x10⁴ counts/well. Externally bound agonist was separated by an acid wash procedure. Both the externally bound ligand present in the acid washes and the internalized ligand associated with the cell pellets were counted. The internalization index represents the percentage of ligand internalized referred to total radioligand bound (n = 3, error bars are ±SEM, two-sided one-way ANOVA was used to generate p values, *p<0.05, **p<0.01).</p

Electrophoretic analysis and intracellular stability of MC1R-TUBB3 isoforms.

<p>(A) Expression of canonical and chimeric MC1R proteins in heterologous HEK293T cells. HEK293T cells were transiently transfected to express Flag-labelled WT MC1R-001, Iso1 and Iso2. Cells were detergent-solubilized, electrophoresed and blotted. For MC1R detection, cell lysates were probed with an anti-Flag monoclonal antibody (upper blot). Membranes were also probed for TUBB3 (middle blot) and ERK2 (lower blot), as loading control (n = 5, representative blots are shown). (B) Electrophoretic pattern of MC1R-TUBB3 transcripts expressed in HBL human melanoma cells. Representative immunoblots for MC1R, TUBB3 and ERK2 are shown as in panel A (n = 5, representative blots are shown). (C) Intracellular stability of MC1R-TUBB3 chimeric fusion proteins in HEK293T cells. Flag-labelled MC1R-001, Iso1 and Iso2 were expressed in HEK293T cells. Cells were incubated with the protein synthesis inhibitor cycloheximide (Chx, 0.1 mM) for the times indicated, lysed and the levels of residual proteins in cell extracts were detected by Western blot. Representative immunoblots probed for MC1R-001, Iso1 or Iso2 with anti-Flag are shown. (D) Semi-log graph for calculation of half-lives. The intensity of receptor bands in the blots as in panel C was quantitated with ImageJ and the semi-log of residual signals was plotted against time. Half-life (t½) values correspond to the slope of the resulting lines.</p