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 (ET A -R and ET B -R). The mechanisms by which this desensitization proceeds remain obscure, however. Because some intracellular domain sequences of the ET A -R and ET B -R differ substantially, we tested the possibility that these receptor subtypes might be

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... ially regulated by G protein-coupled receptor kinases (GRKs). Homologous, or receptor-spe- cific, desensitization occurred within 4 min both in the ET A -R-expressing A10 cells and in 293 cells transfected with either the human ET A -R or ET B -R. In 293 cells, this desensitization corresponded temporally with agonistinduced phosphorylation of each receptor, assessed by receptor immunoprecipitation from 32 P i -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 ET A -R desensitization. Overexpression of GRK2, -5, or -6 in 293 cells augmented agonist-induced ET-R phosphorylation ϳ2-fold (p < 0.02), but each kinase reduced receptorpromoted phosphoinositide hydrolysis differently. While GRK5 inhibited ET-R signaling by only ϳ25%, 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 ET A -R and ET B -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. ¶ Present address: Bristol Myers Squibb Pharmaceutical Research, 5 Research Pkwy., Wallingford, CT 06492-7660. 1 The abbreviations used are: G protein, guanine nucleotide-binding regulatory protein; ET-R, endothelin receptor; ET A -R, endothelin receptor, subtype A; ET B -R, endothelin receptor, subtype B; AT 1A -R, rat type 1A angiotensin II receptor; ET-1, endothelin-1; GRK, G protein-coupled receptor kinase; PKC, protein kinase C; SFLLRN, single-letter amino acid code for the human thrombin receptor agonist peptide; t-ET A -R and t-ET B -R, epitope-tagged ET A -R and ET B -R, respectively; TPA, 12-Otetradecanoylphorbol-13-acetate; 293 cells, human embryonic kidney cells; PCR, polymerase chain reaction.