The first inner loop of endothelin receptor type B is necessary for specific coupling to Gα13

Abstract

Endothelin (EDN) receptor type B (EDNRB) activates serum response factor (SRF) via Gq/11 and G12/13 G proteins. In this study, we investigated the involvement of intracellular loop sequences of EDNRB in coupling to these G proteins. EDNRB mutants were generated and tested for their abilities to activate SRF in NIH3T3 cells and in the mouse embryonic fibroblast cell line (Fq/11) lacking both Gαq and Gα11. EDNRB can activate SRF in NIH3T3 cells via Gq/11, although it can only activate SRF through G12/13 in Fq/11 cells. Mutants with mutations in the second and third inner loops of EDNRB functioned in the same manner in both cell lines, either able or unable to activate SRF. This finding suggests that the second and third inner loops of EDNRB either participate or not in coupling to both Gq/11 and G12/13 but are not specific for either one. However, in the first inner loop, a substitution of three Ala residues for Met128-Arg129-Asn130 abolished the ability to activate SRF only in Fq/11 cells, suggesting that this mutation might specifically disrupt the coupling to G12/13 rather than to Gq/11. Further characterization of this first inner loop mutant revealed that exogenous expression of Gα12 or Gαq could restore SRF activation, whereas the expression of Gα13 did not. Therefore, we conclude that although the three intracellular loops of EDNRB may be involved in coupling to G proteins, residues Met128-Arg129-Asn130 in the first intracellular loop are specifically required for activation of Gα13.