Abstract
Exchanging each of the conserved aromatic residues of the NPxxY(x)5,6F sequence (at the boundary of helices 7 and 8) generated variants of the A1 adenosine receptor that were retained within the cell. The mutations disconnected a link between α-helix 7 and cytosolic helix 8, likely destabilizing the structure of the proximal carboxyl terminus. The mutant receptors were rescued by incubation of cells with a pharmacochaperone, a membrane-permeable ligand that homosterically binds to the receptor; pharmacochaperoning restored the density of functional receptors at the plasma membrane. The following observations support the assumption that retention and the site of pharmacochaperone action were within bounds of the endoplasmic reticulum (ER): 1) the retained receptor colocalized with an ER marker; 2) pharmacochaperoning initiated receptor transfer to Golgi stacks; and 3) the inhibitor of glycoprotein synthesis tunicamycin suppressed receptor chaperoning. Our data are consistent with the hypothesis that pharmacochaperoning stabilizes the structure of late folding intermediates and lifts a block on maturation, allowing the receptors to exit from the ER. We suggest that the ER-associated 40-kDa heat shock protein family member D1 receptor interacting protein 78 (DRiP78; Mr, ∼78,000) represents a model executor of quality control. Overexpressed DRiP78 interacted physically with the A1 receptor, inhibited export to the plasma membrane, and in this action was selective for the mutants relative to the wild-type receptor. Both agonist and antagonist were effective chaperone ligands. Thus, occupancy of the binding pocket corrected the mutation-induced disorder, indicating a mutual impingement of the transmembrane domain and the proximal carboxyl terminus in establishing the stable receptor fold.
Footnotes
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported by the Austrian Science Fund [Grants 16083-B11, P18150-B11].
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.063511.
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ABBREVIATIONS:
- ER
- endoplasmic reticulum
- CPA
- N6-cyclopentyladenosine
- DPCPX
- 8-cyclopentyl-1,3-dipropylxanthine
- IBMX
- 3-isobutyl-1-methylxanthine
- DRiP78
- D1-receptor-interacting protein 78
- A1R
- A1 adenosine receptor
- C-tail
- cytoplasmic carboxyl-terminal tail
- wt
- wild type
- HA
- hemagglutinin
- CFP
- cyan fluorescent protein
- ECFP
- enhanced CFP
- YFP
- yellow fluorescent protein
- GFP
- green fluorescent protein
- HEK
- human embryonic kidney
- IP
- immunoprecipitation
- PDI
- protein disulfide isomerase
- ICQ
- intensity correlation quotient
- HME
- HEPES/MgCI2/EDTA
- HRP
- horseradish peroxidase
- DSP
- dithiobis[succinimidylpropionate]
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- BFA
- brefeldin A
- Hsp
- heat shock protein.
- Received January 7, 2010.
- Accepted March 9, 2010.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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