Agonist-Induced Phosphorylation of the Angiotensin AT1a Receptor Is Localized to a Serine/Threonine-Rich Region of Its Cytoplasmic Tail
- Roger D. Smith1,
- László Hunyady2,
- J. Alberto Olivares-Reyes1,
- Balázs Mihalik2,
- Suman Jayadev1 and
- Kevin J. Catt1
- 1Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 (R.D.S., J.A.O-R., S.J., K.J.C.), and2Department of Physiology, Semmelweis University of Medicine, H-1088, Budapest, Hungary (L.H., B.M.)
Abstract
The agonist-induced phosphorylation sites of the rat AT1aangiotensin receptor were analyzed using epitope-tagged mutant receptors expressed in Cos-7 cells. Angiotensin II-stimulated receptor phosphorylation was unaffected by truncation of the cytoplasmic tail of the receptor at Ser342 (Δ342) but was abolished by truncation at Ser325 (Δ325). Truncation at Ser335 (Δ335), or double-point mutations of Ser335 and Thr336 to alanine (ST-AA), reduced receptor phosphorylation by ∼50%, indicating that in addition to Ser335 and/or Thr336, amino acids within the Ser326-Thr332 segment are also phosphorylated. Agonist-induced phosphorylation of the ST-AA and Δ335 receptors was partially inhibited by staurosporine, suggesting that the single protein kinase C consensus site in the Ser326-Thr332 segment (Ser331) is phosphorylated. The impairment of receptor phosphorylation was broadly correlated with the attenuation of agonist-induced internalization rates (Δ325 < Δ335 < ST-AA < Δ342 < wild-type) and with the increasing rank order of magnitude of inositol phosphate production normalized to an equal number of receptors (Δ325 > Δ335 > ST-AA = Δ342 > wild-type). These results demonstrate that agonist-induced phosphorylation of the AT1a receptor is confined to an 11-amino-acid serine/threonine-rich segment of its carboxyl-terminal cytoplasmic tail and implicate this region in the mechanisms of receptor internalization and desensitization.
Footnotes
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Send reprint requests to: Dr. Kevin J. Catt, ERRB, NICHD, NIH, Bldg. 49, Room 6A-36, 49 Convent Drive, Bethesda, MD 20892-4510. E-mail: catt{at}helix.nih.gov
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R.D.S. was supported in part by an International Fellowship (FS/95018) from the British Heart Foundation. L.H. was supported in part by an International Research Scholar’s award from the Howard Hughes Medical Institute and grant FKFP-0776/1997 from the Hungarian Ministry of Culture and Education. J.A.O.-R. was supported by a Pan American Fellowship (NIH/CONACyt-979004). R.D.S. and L.H. contributed equally to this work.
- Abbreviations:
- GPCR
- G protein-coupled receptor
- Ang II
- angiotensin II
- AT1a-R
- type 1a angiotensin receptor
- DMEM
- Dulbecco’s modified Eagle’s medium
- FBS
- fetal bovine serum
- GRK
- G protein-coupled receptor kinase
- HA
- hemagglutinin
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- LB
- lysis buffer
- PAGE
- polyacrylamide gel electrophoresis
- PKC
- protein kinase C
- PNGase
- peptide N-glycosidase
- SDS
- sodium dodecyl sulfate
- TPA
- 12-O-tetradecanoylphorbol-13-acetate
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- Received July 16, 1998.
- Accepted September 10, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
