Agonist-induced signaling, desensitization, and internalization of a phosphorylation-deficient AT(1A) angiotensin receptor

An analysis of the functional role of a diacidic motif (Asp(236)-Asp(237)) in the third intracellular loop of the AT(1A) angiotensin II (Ang II) recep tor (AT(1)-R) revealed that substitution of both amino acids with alanine ( DD-AA) or asparagine (DD-NN) residues diminished Ang II-induced receptor ph osphorylation in COS-7 cells. However, Ang II-stimulated inositol phosphate production, mitogen-activated protein kinase, and AT(1) receptor desensiti zation and internalization were not significantly impaired. Overexpression of dominant negative G protein-coupled receptor kinase 2 (GRK2)K-220M decre ased agonist-induced receptor phosphorylation by similar to 40%, but did no t further reduce the impaired phosphorylation of DD-AA and DD-NN receptors. Inhibition of protein kinase C by bisin-dolylmaleimide reduced the phospho rylation of both the wild-type and the DD mutant receptors by similar to 30 %. The inhibitory effects of GRK2(K220M) expression and protein kinase C in hibition by bisindolylmaleimide on agonist-induced phosphorylation were add itive for the wild-type AT(1)-R, but not for the DD mutant receptor. Agonis t-induced internalization of the wild-type and DD mutant receptors was simi lar and was unaltered by coexpression of GRK2(K220M). These findings demons trate that an acidic motif at position 236/237 in the third intracellular l oop of the AT(1)-R is required for optimal Ang II-induced phosphorylation o f its carboxyl-terminal tail by GRKs. Furthermore, the properties of the DD mutant receptor suggest that not only Ang II-induced signaling, but also r eceptor desensitization and internalization, are independent of agonist-ind uced GRK-mediated phosphorylation of the AT(1) receptor.