Dataset Information

G-Protein binding domains of the angiotensin II AT1A receptors mapped with synthetic peptides selected from the receptor sequence.

ABSTRACT: The vascular angiotensin II type 1 receptor (AT1AR) is a member of the G-protein-coupled receptor superfamily. We mapped the G-protein binding domains of the AT1AR using synthetic peptides selected from the receptor sequence, which interfere with AT1AR-G-protein coupling. Membrane GTPase activity was used as a measure of the functional coupling in rat vascular smooth muscle cells. Peptides corresponding to the N-terminal region of the second intracellular loop (residues 125-137), the N-terminal region of the third intracellular loop (217-227) and the juxtamembranous region of the C-terminal tail (304-316) inhibited angiotensin II-induced GTPase activation by 30%, 30%, and 70%, respectively. The latter two domains (217-227 and 304-316) are predicted to form amphiphilic alpha-helices. Only the peptide representing residues 217-227 stimulated basal activity (45%). No synthetic peptide had a significant effect on either the number or the affinity of the AT1AR binding. These observations indicate that domains of the second and third regions and the cytoplasmic tail of the AT1AR interact with G-proteins, and that multiple contacts with these receptor domains may be important for binding and activation of the G-proteins.

SUBMITTER: Kai H

PROVIDER: S-EPMC1219541 | biostudies-other | 1998 Jun

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Protein kinase C (PKC) phosphorylates the C-terminus of the type 1 angiotensin II receptor (AT(1)), although the exact site(s) of phosphorylation are unidentified. In the present study, we examined the phosphorylation of epitope-tagged wild-type AT(1A) receptors, transiently expressed in Chinese hamster ovary K1 cells, in response to angiotensin II (AngII) and following selective activation and inhibition of PKC. This phosphorylation was compared with mutant receptors where C-terminal serine residues (Ser(331), Ser(338) and Ser(348)) within three putative PKC consensus sites were replaced with alanine, either individually or in combination. Stimulation by AngII or the phorbol ester PMA to activate PKC induced an increase in phosphorylation of the wild-type AT(1A) receptor, which was prevented by truncation of the receptor C-terminus to remove the last 34 amino acids, including Ser(331), Ser(338) and Ser(348). Whereas single alanine mutation (Ser(331)Ala, Ser(338)Ala and Ser(348)Ala) resulted in decreased receptor phosphorylation, no single mutant completely inhibited either AngII- or PMA-induced phosphorylation. Combined mutation of the three PKC consensus sites caused an approximately 70% reduction in PMA-mediated phosphorylation. The approximately 60% reduction in AngII (1 microM)-induced phosphorylation of this triple mutant and the partial inhibition of wild-type receptor phosphorylation by bisindolylmaleimide, a specific PKC inhibitor, suggest a significant contribution of PKC to agonist-stimulated regulation. The ratio of PKC to total receptor phosphorylation was greatest at low doses of AngII (1 nM), consistent with the idea that PKC phosphorylates and regulates receptor function at low levels of stimulation, whereas phosphorylation by other kinases is more prevalent at high levels of agonist stimulation. To determine if a single PKC site is favoured when the contribution of PKC varies, the phosphorylation of wild-type and mutant receptors was examined over a range of AngII concentrations (0, 1, 10 and 100 nM). At all AngII concentrations, single mutation of Ser(331), Ser(338) or Ser(348) was incapable of completely preventing receptor phosphorylation, suggesting no clear preference for PKC consensus-site utilization. Together, these results indicate a redundancy in PKC phosphorylation of the AT(1A) receptor, whereby all three consensus sites are utilized to some degree following homologous (AngII) and heterologous (PMA) stimulation. The contribution of PKC phosphorylation to receptor regulation is unclear, but multiple PKC phosphorylation of the AT(1A) receptor may allow independent and/or complementary events to occur at the three separate sites of the C-terminus.

Dataset Information

G-Protein binding domains of the angiotensin II AT1A receptors mapped with synthetic peptides selected from the receptor sequence.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets