Agonist-induced signaling, desensitization, and internalization of a phosphorylation-deficient AT1A angiotensin receptor.
An analysis of the functional role of a diacidic motif (Asp236-Asp237) in the third intracellular loop of the AT1A angiotensin II (Ang II) receptor (AT1-R) revealed that substitution of both amino acids with alanine (DD-AA) or asparagine (DD-NN) residues diminished Ang II-induced receptor phosphorylation in COS-7 cells. However, Ang II-stimulated inositol phosphate production, mitogen-activated protein kinase, and AT1 receptor desensitization and internalization were not significantly impaired. Overexpression of dominant negative G protein-coupled receptor kinase 2 (GRK2)K220M decreased agonist-induced receptor phosphorylation by approximately 40%, but did not further reduce the impaired phosphorylation of DD-AA and DD-NN receptors. Inhibition of protein kinase C by bisindolylmaleimide reduced the phosphorylation of both the wild-type and the DD mutant receptors by approximately 30%. The inhibitory effects of GRK2K220M expression and protein kinase C inhibition by bisindolylmaleimide on agonist-induced phosphorylation were additive for the wild-type AT1-R, but not for the DD mutant receptor. Agonist-induced internalization of the wild-type and DD mutant receptors was similar and was unaltered by coexpression of GRK2K220M. These findings demonstrate that an acidic motif at position 236/237 in the third intracellular loop of the AT1-R is required for optimal Ang II-induced phosphorylation of its carboxyl-terminal tail by GRKs. Furthermore, the properties of the DD mutant receptor suggest that not only Ang II-induced signaling, but also receptor desensitization and internalization, are independent of agonist-induced GRK-mediated phosphorylation of the AT1 receptor.
Olivares-Reyes
J A
JA
Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
Smith
R D
RD
Hunyady
L
L
Shah
B H
BH
Catt
K J
KJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D004705:Endocytosis; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D051381:Rats; D011945:Receptors, Angiotensin / Q000819:agonists* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
D007211:Indoles; D007295:Inositol Phosphates; D008301:Maleimides; D011945:Receptors, Angiotensin; D000803:Angiotensin I; C088060:bisindolylmaleimide