Localisation of endothelin B receptor variants to plasma membrane microdomains and its effects on downstream signalling

The endothelin B (ET_B) receptor can undergo a proteolytic cleavage resulting in an unglycosylated N-terminally truncated receptor. We investigated whether ET_B receptor processing affects caveolar localisation and mitogenic signalling. Distinct subcellular localisations of ET_B receptor constructs and epidermal growth factor (EGF) receptor ligands were analysed performing detergent-free caveolae preparations and total internal reflection fluorescence microscopy. ET_B receptor-induced transactivation of the EGF receptor and its downstream signalling was investigated performing shedding assays and ERK1/2 phosphorylation analyses. In COS7 cells, the N-terminally truncated but not the full-length or glycosylation-deficient ET_B receptor localised to caveolae. In caveolae-free HEK293 cells, only ET_B receptor constructs fused to caveolin-2 localised to membrane microdomains. A caveolar accumulation of the ET_B receptor disfavoured EGF receptor ligand shedding. Nonetheless, the activation of ERK1/2 was efficient and long-lasting. In HEK293 cells, the shedding activity was also impaired by N-terminal truncation. The subsequent ERK1/2 phosphorylation was long-lasting only for the full-length ET_B receptor. We conclude that the ET_B receptor localisation might depend on the presence of caveolae within the cell investigated. The data further suggest that caveolar enrichment of ET_B receptors does not facilitate the release of EGF receptor ligands. However, independent of their localisation, ET_B receptors are able to induce an ERK1/2 phosphorylation.