Functional domains of the C-terminus of the rat angiotensin AT1A receptor.
Previous work has shown that truncating the carboxyl terminus (C-terminus) of the rat angiotensin AT1A receptor to 309 amino acids abolished G-protein coupling and receptor internalization. This suggests that domains responsible for these functions lie beyond amino acid 309 of the C-terminus. The objective of this study was to determine the effect on angiotensin AT1A receptor function and regulation of deleting 41 amino acids from the C-terminus, which include the putative protein kinase C phosphorylation sites. Using site directed mutagenesis, the codon for Tyr319 was converted to a stop codon and the resulting truncated receptor permanently expressed in cultured human kidney cells. The properties of the truncated receptor were compared to those of the full length receptor. Expression of the truncated receptor was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of photolabelled membrane preparations. Angiotensin II activation of both full length and truncated receptors resulted in mobilization of inositol phosphates. However, whereas this was associated with rapid internalization of the full length receptor, the truncated receptor failed to internalize. Furthermore, pretreatment of cells with phorbol 12-myristate 13-acetate, a direct activator of protein kinase C, markedly attenuated the full length, but no the truncated receptor's ability to mobilise inositol phosphates. Thus, we conclude that the domain between amino acids 309 & 318 is important for G-protein coupling; that amino acids beyond 318 regulate internalization and one or more of the putative protein kinase C phosphorylation sites, present in the C-terminus of the angiotensin At1A receptor, actively regulate the receptor.
Balmforth
A J
AJ
Institute for Cardiovascular Research, University of Leeds, UK.
Lee
A J
AJ
Bajaj
B P
BP
Dickinson
C J
CJ
Warburton
P
P
Ball
S G
SG
D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007668:Kidney / Q000187:drug effects*; D008969:Molecular Sequence Data; D011869:Radioligand Assay; D051381:Rats; D011945:Receptors, Angiotensin / Q000187:drug effects* / Q000502:physiology*; D013997:Time Factors
D011945:Receptors, Angiotensin; D000804:Angiotensin II