The first inner loop of endothelin receptor type B is necessary for specific coupling to Galpha 13.
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 Galpha(q) and Galpha(11). 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 Met(128)-Arg(129)-Asn(130) abolished 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 Galpha(12) or Galpha(q) could restore SRF activation, whereas the expression of Galpha(13) did not. Therefore, we conclude that although the three intracellular loops of EDNRB may be involved in coupling to G proteins, residues Met(128)-Arg(129)-Asn(130) in the first intracellular loop are specifically required for activation of Galpha(13).
Liu
Bo
B
Department of Genetics and Developmental Biology, University of Connecticut Medical Center, Farmington 06030, USA.
Wu
Dianqing
D
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000378:metabolism; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D017930:Genes, Reporter; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000737:chemistry* / Q000502:physiology*; D026362:Serum Response Factor / Q000378:metabolism; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D004268:DNA-Binding Proteins; D008024:Ligands; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; D026362:Serum Response Factor; D008156:Luciferases; D010738:Type C Phospholipases; D044365:GTP-Binding Protein alpha Subunits, G12-G13