Phosphorylation and desensitization of human endothelin A and B receptors. Evidence for G protein-coupled receptor kinase specificity.
Although endothelin-1 can elicit prolonged physiologic responses, accumulating evidence suggests that rapid desensitization affects the primary G protein-coupled receptors mediating these responses, the endothelin A and B receptors (ETA-R and ETB-R). The mechanisms by which this desensitization proceeds remain obscure, however. Because some intracellular domain sequences of the ETA-R and ETB-R differ substantially, we tested the possibility that these receptor subtypes might be differentially regulated by G protein-coupled receptor kinases (GRKs). Homologous, or receptor-specific, desensitization occurred within 4 min both in the ETA-R-expressing A10 cells and in 293 cells transfected with either the human ETA-R or ETB-R. In 293 cells, this desensitization corresponded temporally with agonist-induced phosphorylation of each receptor, assessed by receptor immunoprecipitation from 32Pi-labeled cells. Agonist-induced receptor phosphorylation was not substantially affected by PKC inhibition but was reduced 40% (p << 0.03) by GRK inhibition, effected by a dominant negative GRK2 mutant. Inhibition of agonist-induced phosphorylation abrogated agonist-induced ETA-R desensitization. Overexpression of GRK2, -5, or -6 in 293 cells augmented agonist-induced ET-R phosphorylation approximately 2-fold (p << 0.02), but each kinase reduced receptor-promoted phosphoinositide hydrolysis differently. While GRK5 inhibited ET-R signaling by only approximately 25%, GRK2 inhibited ET-R signaling by 80% (p << 0.01). Congruent with its superior efficacy in suppressing ET-R signaling, GRK2, but not GRK5, co-immunoprecipitated with the ET-Rs in an agonist-dependent manner. We conclude that both the ETA-R and ETB-R can be regulated indistinguishably by GRK-initiated desensitization. We propose that because of its affinity for ET-Rs demonstrated by co-immunoprecipitation, GRK2 is the most likely of the GRKs to initiate ET-R desensitization.
Freedman
N J
NJ
Howard Hughes Medical Institute, Department of Medicine, Duke University Medical Center, Box 3821, Durham, North Carolina 27710, USA.
Ament
A S
AS
Oppermann
M
M
Stoffel
R H
RH
Exum
S T
ST
Lefkowitz
R J
RJ
D000818:Animals; D002417:Cattle; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D019332:Endothelin-1 / Q000378:metabolism; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008956:Models, Chemical; D008970:Molecular Weight; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000378:metabolism*; D013379:Substrate Specificity; D051552:beta-Adrenergic Receptor Kinases
D019332:Endothelin-1; D010716:Phosphatidylinositols; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; C517898:GRK5 protein, human; C517897:Grk5 protein, rat; D019204:GTP-Binding Proteins