Abstract
The human angiotensin II type 1 receptor (AT1R) is a member of the G protein-coupled receptor (GPCR) superfamily and represents an important target for cardiovascular therapeutic intervention. Agonist-activation of the AT1R induces β-arrestin-dependent endocytosis to early endosomes in which the receptor resides as a protein complex with the Rab GTPase Rab5. In the present study, we examined whether other Rab GTPases that regulate receptor trafficking through endosomal compartments also bind to the AT1R. We find that Rab4, Rab7, and Rab11 all bind to the last 10 amino acid residues of the AT1R carboxyl-terminal tail. Rab11 binds AT1R more effectively than Rab5, whereas Rab4 binds less effectively than Rab5. Alanine scanning mutagenesis reveals that proline 354 and cysteine 355 contribute to Rab protein binding, and mutation of these residues does not affect G protein coupling. We find that the Rab GTPases each compete with one another for receptor binding and that although Rab4 interacts poorly with the AT1R, it effectively displaces Rab11 from the receptor. In contrast, Rab11 overexpression does not prevent Rab4 binding to the AT1R. Overexpression of wild-type Rab4, but not Rab11, facilitates AT1R dephosphorylation, and a constitutively active Rab4-Q67L mutant reduces AT1R desensitization and promotes AT1R resensitization. Taken together, our data indicate that multiple Rab GTPases bind to a motif localized to the distal end of the AT1R tail and that increased Rab4 activity may contribute to the regulation AT1R desensitization and dephosphorylation.
Footnotes
This work was supported by the Heart and Stroke Foundation of Ontario [Grant T-5933]; an Ontario Graduate Scholarship (to J.L.E.); a Tier I Canada Research Chair in Molecular Neurobiology (to S.S.G.F.); and the Heart and Stroke Foundation of Ontario Career Investigator Award (to S.S.G.F.).
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.068379.
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ABBREVIATIONS:
- AT1R
- angiotensin II type 1 receptor
- GPCR
- G protein-coupled receptor
- β2AR
- β2-adrenergic receptor
- HEK
- human embryonic kidney
- HBSS
- HEPES-buffered saline solution
- AngII
- Angiotensin II
- PBS
- phosphate-buffered saline
- PAGE
- polyacrylamide gel electrophoresis
- GFP
- green fluorescent protein
- AT1AR
- angiotensin II type 1A receptor
- HA
- hemagglutinin
- IP
- inositol phosphate.
- Received August 24, 2010.
- Accepted October 13, 2010.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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