W276 Mutation in the Endothelin Receptor Subtype B Impairs Gq Coupling but Not Gi or Go Coupling

The mutation of W276 to cysteine within the human endothelin receptor subtype B (ETBR) is associated with Hirschsprung's disease, a congenital intestinal disease. The sequence surrounding W276 is highly conserved between the endothelin receptor subtypes A and B. We have introduced sets of mutations into W275 and W276 of the ETBR gene, and the corresponding W257 and W258 of the ETAR gene, and studied their coupling properties with Gi, Go, and Gq in reconstituted phospholipid vesicles. The prepared mutants all showed a similar affinity for endothelin-1. The W276C/ETBR and W276A/ETBR mutants had reduced activities in Gq coupling but not in Gi/Go coupling, while the W275A/ETBR displayed reduced activities in Gi/Gq coupling, with normal Go coupling. On the other hand, W257A/ETAR and W258A/ETAR exhibited wild-type activities in all examined G protein couplings. These results suggest that the defects in the Gq signaling pathway by the ETBR are connected with Hirschsprung's disease and that the two conserved tryptophans play distinct roles in signal transduction by the two receptor subtypes. In addition, W275 and W276, which are thought to be located near the extracellular side of the transmembrane helix 5, play important roles in forming the active structure of ETBR.