Abstract
ADP-ribosylation factors (ARFs) regulate vesicular traffic through recruiting coat proteins. However, their functions in the anterograde transport of nascent G protein-coupled receptors (GPCRs) from the endoplasmic reticulum to the plasma membrane remain poorly explored. Here we show that treatment with brefeldin A, an inhibitor of guanine nucleotide exchange on ARFs, markedly attenuated the cell surface numbers of α2B-adrenergic receptor (AR), β2-AR, angiotensin II type 1 receptor, and chemokine (CXC motif) receptor 4. Functional inhibition of individual ARF GTPases by transient expression of the GDP-bound, GTP-bound, and guanine nucleotide-deficient mutants showed that the five human ARFs differentially modulated receptor cell surface expression and that the ARF1 mutants produced the most profound inhibitory effect. Furthermore, expression of the ARF1 GTPase-activating protein (GAP) ARFGAP1 significantly blocked receptor transport. Interestingly, the GDP- and GTP-bound ARF1 mutants arrested the receptors in distinct intracellular compartments. Consistent with the reduced receptor cell surface expression, extracellular signal-regulated kinase 1 and 2 activation by receptor agonists was significantly attenuated by the GDP-bound mutant ARF1T31N. Moreover, coimmunoprecipitation showed that α2B-AR associated with ARF1 and glutathione transferase pull-down assay indicated that the α2B-AR C terminus directly interacted with ARF1. These data show that ARF1 GTPase is involved in the regulation of cell surface expression of GPCRs at multiple transport steps.
Footnotes
- Received September 10, 2009.
- Accepted January 20, 2010.
C.D. and X.Z. contributed equally to this work.
This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grant GM076167].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.161489.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- ARF
- ADP-ribosylation factor
- GEF
- guanine nucleotide exchange factor
- ER
- endoplasmic reticulum
- ERGIC
- endoplasmic reticulum-Golgi intermediate complex
- TGN
- trans-Golgi network
- GAP
- GTPase-activating protein
- GPCR
- G protein-coupled receptor
- AR
- adrenergic receptor
- AT1R
- angiotensin II type 1 receptor
- CXCR4
- chemokine (CXC motif) receptor 4
- ERK1/2
- extracellular signal-regulated kinase 1 and 2
- HA
- hemagglutinin
- ISO
- isoproterenol
- UK-14,304
- 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine
- BFA
- brefeldin A
- Ang II
- angiotensin II
- SDF-1α
- stromal cell-derived factor 1α
- CGP-12177
- 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one
- RX 821002
- 2-(2-methoxy-1,4-benzodioxan-2yl)-2-imidazoline
- GFP
- green fluorescent protein
- GST
- glutathione transferase
- HEK
- human embryonic kidney
- DMEM
- Dulbecco's modified Eagle's medium
- FBS
- fetal bovine serum
- M3-MR
- M3-muscarinic receptor
- PBS
- phosphate-buffered saline
- PAGE
- polyacrylamide gel electrophoresis
- ARFGAP1
- ADP-ribosylation factor GTPase-activating protein 1.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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