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 G_q/11 and G_12/13 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 (F_q/11) lacking both Gα_qand Gα₁₁. EDNRB can activate SRF in NIH3T3 cells via G_q/11, although it can only activate SRF through G_12/13 in F_q/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 G_q/11 and G_12/13 but are not specific for either one. However, in the first inner loop, a substitution of three Ala residues for Met¹²⁸-Arg¹²⁹-Asn¹³⁰abolished the ability to activate SRF only in F_q/11 cells, suggesting that this mutation might specifically disrupt the coupling to G_12/13 rather than to G_q/11. Further characterization of this first inner loop mutant revealed that exogenous expression of Gα₁₂ or Gα_q could restore SRF activation, whereas the expression of Gα₁₃did 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.