Aberrant trafficking of human melanocortin 1 receptor variants associated with red hair and skin cancer: Steady-state retention of mutant forms in the proximal golgi

Abstract

The melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor (GPCR) expressed in melanocytes, is a major determinant of skin pigmentation and phototype. MC1R activation stimulates melanogenesis and increases the ratio of black, strongly photoprotective eumelanins to reddish, poorly photoprotective pheomelanins. Several MC1R alleles are associated with red hair, fair skin, increased sensitivity to ultraviolet radiation (the RHC phenotype) and increased skin cancer risk. Three highly penetrant RHC variants, R151C, R160W, and D294H are loss-of-function MC1R mutants with altered cell surface expression. In this study, we show that forward trafficking was normal for D294H. Conversely, export traffic was impaired for R151C, which accumulated in the endoplasmic reticulum (ER), and for R160W, which was enriched in the cis-Golgi. This is the first report of steady-state retention in a post-ER secretory compartment of a GPCR mutant found in the human population. Residues R151 and R160 are located in the MC1R second intracellular loop (il2). Two other mutations in il2, T157A preventing T157 phosphorylation and R162P disrupting a (160)RARR(163) motif, also caused intracellular retention. Moreover, T157 was phosphorylated in wild-type MC1R and a T157D mutation mimicking constitutive phosphorylation allowed normal traffic, and rescued the retention phenotype of R160W and R162P. Therefore, MC1R export is likely regulated by T157 phosphorylation and the (160)RARR(163) arginine-based motif functions as an ER retrieval signal. These elements are conserved in mammalian MC1Rs and in all five types of human melanocortin receptors. Thus, members of this GPCR subfamily might share common mechanisms for regulation of plasma membrane expression.

Figure 1. Structure of the human MC1R

A. Putative MC1R secondary structure, according to the two dimensional model of Ringholm et al (Ringholm et al., 2004) with minor modifications. Residues in il2 are shown in grey. Residues R151, R160 and D294 frequently mutated in red hair individuals are shown in black. Other residues discussed in this study are hatched. el, extracellular loop; il, intracellular loop. B. Sequence alignment of il2 and flanking residues in melanocortin receptors. The upper block shows the sequence of MC1R from various vertebrate species. Amino acids likely forming the il2 are shown on a grey background. The lower block shows the sequences of the remaining human melanocortin receptors. The invariant T157 is highlighted in bold and by a solid arrow. Residues R151 and R160 are highlighted by broken arrows. Basic residues in the C-terminal vicinity of T157 are underlined.

Figure 2. Expression and processing of wild type and variant MC1R in human melanoma cells

Clones of HBL melanoma cells stably expressing epitope-tagged wtMC1R or variant receptors were metabolically labelled with ³⁵S, chased for 30 min or 2 h as indicated and immunoprecipitated for MC1R. Immunoprecipitated proteins were digested with EndoH (EH), solubilized and electrophoresed. C stands for undigested control samples.

Figure 3. Retention of the R151C variant of MC1R in the ER

A. HBL melanoma cells expressing wtMC1R or the indicated variants were fixed, permeabilized, stained for MC1R and calnexin and examined in a confocal microscope. MC1R is shown in red (upper) and calnexin in green (middle). In merged images (lower) co-localization is shown in yellow. Note the higher co-localization of R151C with the ER marker (arrow). Scale bar: 20 μm B. Same as in panel A, except that HEK293 cells transiently transfected with wtMC1R or mutant MC1R were used. Co-localization of R151C with calnexin is highlighted (arrow). Scale bar: 15 μm

Figure 4. Bafilomycin A-promoted accumulation of the R151C variant in a post-ER site

HEK293 cells co-transfected with wtMC1R or R151C and the small GTPase Rab1 fused to the fluorescent protein EGFP (EGFP-Rab1) were treated with bafilomycin A (1 μM) for 4 h. Cells were stained for MC1R (red, upper row). The EGFP-Rab1 signal was detected directly (green, middle row). Merged images are shown in the lower row. The higher co-localization with the proximal Golgi marker Rab1 following bafilomycin treatment is highlighted by an arrow. Scale bar: 10 μm

Figure 5. Selective steady-state enrichment of R160W MC1R variant in the proximal Golgi

A. HBL melanoma cells stably expressing wtMC1R or variant MC1R were transfected with EGFP-Rab1, CFP-GalTr, or empty vector. Cells were stained for MC1R and, if appropriate, for GM130 or COPI, as indicated. Occasionally, nuclei were labelled with DAPI (shown in blue). Cells were examined in a confocal microscope. MC1R is shown in red, all markers in green and co-localization in yellow. Only merged images are shown. Stronger co-localization signals are shown with arrows. Scale bar: 15 μm B. Same as in A, except that HEK293 cells were analyzed. Scale bar: 15 μm

Figure 6. Disruption of the staining pattern of the R160W MC1R variant by brefeldin treatment

A. HEK293 cells transfected with wtMC1R or R160W and EGFP-Rab1 were treated with brefeldin (1 μg/ml, 30 min), permeabilized and stained for MC1R (red). Disruption of the Golgi apparatus is shown by comparison of the EGFP-Rab1 signal (green) in treated and control cells. Scale bar: 10 μm B. Same as in panel A except that cells were treated with baf A (1 μM, 4 h). Scale bar: 10 μm

Figure 7. Distribution of the T157A and T157D MC1R mutants

HEK293 cells were transiently transfected to express the T157A (panel A) or T157D (panel B) mutants, alone or in combination with EGFP-Rab1 or CFP-GalTr. Permeabilized cells were stained for MC1R (red, upper row in each panel), and for calnexin, COPI or GM130, when needed. The EGFP-Rab1 and CFP-GalTr signals were detected directly. All organelle markers are shown on green (middle row in each panel). Strong co-localization signals are highlighted by arrows. Scale bar: 15 μm

Figure 8. Functional analysis of the T157A and T157D mutants

A. Functional coupling to cAMP generation. wtMC1R, T157A or T157D were transiently expressed in HEK293 cells. Cells were challenged with 10⁻⁷ M NDP-MSH for 30 min, lysed and their cAMP content was determined by radioimmunoassay. Empty bars represent the cAMP concentration in resting controls and solid bars the cAMP levels after stimulation. B. Binding of [¹²⁵ I]NDP-MSH. Cells were incubated with 10⁻¹⁰ M [¹²⁵ I]NDP-MSH for 1 h, washed and the radioactivity associated was determined. C. Phosphorylation of T157. HEK293 cells expressing the MC1R form indicated on top of each lane were washed with PBS and solubilized in 200 μl solubilization buffer supplemented with 1% phosphatase inhibitor mix (Calbiochem). Extracts were immunoprecipitated with a resin for affinity capture of Flag-tagged proteins. Released proteins were electrophoresed and immunoblotted with an anti-phosphothreonine antibody (upper). Comparable input was checked by staining for MC1R with anti-Flag (lower). A representative blot out of three experiments with similar results is shown.

Figure 9. Distribution of the R162P mutant

HEK293 cells were transiently transfected to express the R162P mutant, alone or in combination with EGFP-Rab1 or CFP-GalTr. Permeabilized cells were stained for MC1R (red, upper row), and for calnexin, COPI or GM130, when needed. Organelle markers are shown in green (middle row). Stronger co-localization signals are highlighted by arrows. Scale bar: 15 μm

Figure 10. The T157D mutation rescued normal forward traffic of R160W and R162P

HEK293 cells were transfected to express the T157D/R151C, T157D/R160W or T157D/R162P double mutants alone or in combination with EGFP-Rab1 and CFP-GalTr. Permeabilized cells were stained for MC1R (red, upper row in each panel), and for organelle markers when needed. Markers are shown in green (middle row). Arrows point to positive co-localization signals. A. Behaviour of the T157D/R151C mutant. Note the significant co-localization with calnexin and the noticeable co-distribution with CFP-GalT. Scale bar: 15 μm B. Behaviour of T157D/R160W. Note the detectable plasma membrane expression and the lack of the strong co-localization with Rab1, COPI or GM130 observed for R160W. Scale bar: 15 μm C. Staining of T157D/R162P. For convenience only merged images are shown. Bar: 10 μm

Figure 11

Flow cytometric analysis of HEK293 cells transfected with Flag-tagged wtMC1R or variant MC1R. Histograms display cell number (counts) as a function of MC1R surface staining, on a logarithmic scale. The blank (filled curve) refers to cells transfected with a native wtMC1R construct lacking the Flag epitope. Representative histograms out of triplicates are shown.