The melanocortin 1 receptor (MC1R) is a key regulator of human pigmentation and is expressed on the surface of melanocytes and melanoma cells. Alpha-melanocyte stimulating hormone (aMSH), the ligand for MC1R, is synthesised from pro-opiomelanocortin (POMC) through cleavage of this precursor by pro-hormone convertase (PC)-1 and PC-2. Recent work has shown that aMSH and PC-1 and PC-2 are located within the melanosome (Peters et al, J. Invest. Dermatol. 114: 430–437, 2000). The presence of aMSH in the melanosome could imply a role for aMSH independent of MC1R in this organelle, but also raises the issue of whether MC1R is also present at the melanosome. To address this question, we have transfected B16G4F Mc1r-null melanoma cells with human MC1R tagged with enhanced green fluorescent protein (EGFP), and have investigated the sub-cellular localisation of MC1R within these cells.

B16G4F cells were stably transfected with wild type (WT) and separately Asp294His variant human MC1R tagged at the C terminus with EGFP. aPEP-13 and aPEP-1 antibodies were used to label Pmel-17 protein in early melanosomes and tyrosinase related protein-1 (TRP-1) in late melanosomes respectively. An Alexa-fluor 633-conjugated secondary antibody emitting far red fluorescence was employed for detection in order to ensure there was no crossover into the EGFP excitation / emission spectra by these markers. Images were obtained using laser scanning fluorescence confocal microscopy, and analysed with accompanying Leica software for double positive fluorescence within pixels in Z planes.

Confocal microscopy demonstrated expression of EGFP-WT MC1R and separately EGFP-Asp294His MC1R in a punctate pattern within the cell body as well as at the cell membrane. Co-localisation of WT and Asp294His EGFP-MC1R was observed with both early and late melanosome markers. Co-localisation of EGFP-MC1R was also detected with early and late melanosomes along dendrites following induction of dendricity by culturing in the presence of 10^-3M IBMX. These results suggest that MC1R is present close to or within the melanosome as well as at the cell surface, and suggests that part of the function of MC1R may be mediated through its association with the melanosome.