Cell-Type Specific Effects of Endocytosis Inhibitors on 5-Hydroxytryptamine2A Receptor Desensitization and Resensitization Reveal an Arrestin-, GRK2-, and GRK5-Independent Mode of Regulation in Human Embryonic Kidney 293 Cells
- John A. Gray1,
- Douglas J. Sheffler1,
- Anushree Bhatnagar1,
- Jason A. Woods1,
- Sandra J. Hufeisen1,
- Jeffrey L. Benovic4 and
- Bryan L. Roth1,2,3
- Departments of 1Biochemistry (J.A.G., D.J.S, A.B., J.A.W., S.J.H., B.L.R.), 2Psychiatry (B.L.R.), and 3Neurosciences (B.L.R.), Case Western Reserve University School of Medicine, Cleveland, Ohio and the 4Kimmel Cancer Center (J.L.B.), Thomas Jefferson University Medical School, Philadelphia, Pennsylvania
- Dr. Bryan L. Roth, Department of Biochemistry, Room W438, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4935. E-mail:roth{at}biocserver.cwru.edu
Abstract
The effect of endocytosis inhibitors on 5-hydroxytryptamine2A (5-HT2A) receptor desensitization and resensitization was examined in transiently transfected human embryonic kidney (HEK) 293 cells and in C6 glioma cells that endogenously express 5-HT2A receptors. In HEK-293 cells, 5-HT2A receptor desensitization was unaffected by cotransfection with a dominant-negative mutant of dynamin (DynK44A), a truncation mutant of arrestin-2 [Arr2(319–418)], or by two well-characterized chemical inhibitors of endocytosis: concanavalin A (conA) and phenylarsine oxide (PAO). In contrast, β2-adrenergic receptor desensitization was significantly potentiated by each of these treatments in HEK-293 cells. In C6 glioma cells, however, DynK44A, Arr2(319–418), conA, and PAO each resulted in the potentiation of 5-HT2A and β-adrenergic receptor desensitization. The cell-type-specific effect of Arr2(319–418) on 5-HT2Areceptor desensitization was not related to the level of GRK2 or GRK5 expression. Interestingly, although β2-adrenergic receptor resensitization was potently blocked by cotransfection with DynK44A, 5-HT2A receptor resensitization was enhanced, suggesting the existence of a novel cell-surface mechanism for 5-HT2Areceptor resensitization in HEK-293 cells. In addition, Arr2(319–418) had no effect on 5-HT2A receptor resensitization in HEK-293 cells, although it attenuated the resensitization of the β2-adrenergic receptor. However, in C6 glioma cells, both DynK44A and Arr2(319–418) significantly reduced 5-HT2A receptor resensitization. Taken together, these results provide the first convincing evidence of cell-type-specific roles for endocytosis inhibitors in regulating GPCR activity. Additionally, these results imply that novel GRK and arrestin-independent mechanisms of 5-HT2A receptor desensitization and resensitization exist in HEK-293 cells.
Footnotes
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This work was supported in part by National Institutes of Health Grants RO1-MH61887, RO1-MH57635, and KO2-MH01366 and by a NARSAD independent investigator award (to B.L.R.).
- Abbreviations:
- 5-HT
- 5-hydroxytryptamine (serotonin)
- PI
- phosphoinositide
- GPCR
- G protein-coupled receptor
- GRK
- G protein-coupled receptor kinase
- HEK
- human embryonic kidney
- DynK44A
- Dynamin K44A
- Arr2(319–418)
- arrestin-2 (319–418)
- IBMX
- 3-isobutyl-1-methylxanthine
- PAO
- phenylarsine oxide
- ConA
- concanavalin A
- HRP
- horseradish peroxidase
- BODIPY-FL
- 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid, succinimidyl ester
- DMEM
- Dulbecco's modified essential medium
- F12
- Ham's F12 nutrient mixture
- PBS
- phosphate-buffered saline
- β2AR
- β2-adrenergic receptor
-
- Received March 22, 2001.
- Accepted July 25, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
