TY - JOUR T1 - Pharmacochaperoning of the A<sub>1</sub> Adenosine Receptor Is Contingent on the Endoplasmic Reticulum JF - Molecular Pharmacology JO - Mol Pharmacol SP - 940 LP - 952 DO - 10.1124/mol.110.063511 VL - 77 IS - 6 AU - Laura Málaga-Diéguez AU - Qiong Yang AU - Jan Bauer AU - Halyna Pankevych AU - Michael Freissmuth AU - Christian Nanoff Y1 - 2010/06/01 UR - http://molpharm.aspetjournals.org/content/77/6/940.abstract N2 - 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. Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics ER -