Independence of type I angiotensin II receptor endocytosis from G protein coupling and signal transduction.
The relationship between angiotensin II-induced activation of G proteins and receptor internalization was analyzed by transiently expressing mutant and wild type cDNAs for the rat AT1a receptor in COS-7 cells. Pertussis toxin-sensitive G proteins did not appear to play a role in endocytosis since the receptor showed normal internalization kinetics in pertussis toxin-treated cells. Three deletion mutants of the third cytoplasmic loop revealed that the N-terminal part of this region is important for both receptor endocytosis and intracellular signaling. Three point mutations of Asp74, which has been implicated in signal transduction by the AT1a receptor, caused impaired G protein coupling and inositol phosphate responses. However, each of these mutants (D74N, D74H, and D74Y) showed markedly different internalization kinetics. The D74Y mutant showed the greatest impairment of internalization but retained the highest degree of inositol phosphate stimulation. In contrast, the D74N mutant, which showed the most impaired G protein coupling and inositol phosphate responses, had similar internalization kinetics to the wild type receptor. The combined mutant receptor containing the D74N substitution and deletion of residues 221-226 from the third cytoplasmic loop showed no G protein coupling or inositol phosphate response but was internalized about 60% as rapidly as the wild type receptor. These data demonstrate that endocytosis of the AT1 receptor is independent of agonist-activated signal transduction and indicate that receptor internalization and activation of phospholipase C have different structural requirements.
Hunyady
L
L
Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892.
Baukal
A J
AJ
Balla
T
T
Catt
K J
KJ
D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D004705:Endocytosis; D019204:GTP-Binding Proteins / Q000502:physiology*; D007295:Inositol Phosphates / Q000378:metabolism; D009154:Mutation; D051381:Rats; D011945:Receptors, Angiotensin / Q000378:metabolism*; D015398:Signal Transduction
D007295:Inositol Phosphates; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D019204:GTP-Binding Proteins