Interactions of endothelin receptor subtypes A and B with G(i), G(o), and G(q) in reconstituted phospholipid vesicles

To understand the biochemical basis for the functional divergence of the hu man endothelin receptor subtypes A (ETAR) and B (ETBR), they were expressed , purified from insect Sf9 cells, and reconstituted into phospholipid vesic les with the G(o), G(q), and G(i) proteins. For each G protein, a unique pa ttern of reactivity was observed with the different receptor subtypes. Both ETAR and ETBR activated G(o) to a similar maximal extent, and both subtype s activated G(q) with similar EC50 values; however, the ETAR displayed a 2- 3-fold higher maximal extent of activation. In contrast, both subtypes acti vated G(i) to a similar maximal extent, but the ETAR displayed a 4-fold hig her EC50 value as compared to the ETBR. To test whether these coupling spec ificities are influenced by C-terminal palmitoylation of the receptor, we m utated a cluster of-cysteine residues near the end of the seventh transmemb rane helix in both receptors. While the cysteine mutations in the ETBR resu lted in a partially palmitoylated receptor, the replacement of these cystei ne residues in the ETAR yielded a mostly palmitoyl-deficient receptor and h ad no effect on G(o) activation, but caused a reduction in the extents of G (i) and G(q) stimulation. Together, these studies provide important insight s into the specificity of G protein coupling in the endothelin receptors. T he ability to discriminate between the different G proteins under various p hysiological conditions may be a key element in the selection of distinct s ignal transduction pathways by the two receptor subtypes.