RT Journal Article SR Electronic T1 Pharmacochaperoning of the A1 adenosine receptor is contingent upon the endoplasmic reticulum JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP mol.110.063511 DO 10.1124/mol.110.063511 A1 Laura Malaga-Dieguez A1 Qiong Yang A1 Jan Bauer A1 Halyna Pankevych A1 Michael Freissmuth A1 Christian Nanoff YR 2010 UL http://molpharm.aspetjournals.org/content/early/2010/03/10/mol.110.063511.abstract AB 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 carboxyterminus. The mutant receptors were rescued by incubation of cells with a pharmacochaperon, 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 pharmacochaperon action - was within bounds of the endoplasmic reticulum (ER). (i) The retained receptor co-localized with an ER marker; (ii) pharmacochaperoning initiated receptor transfer to Golgi stacks; (iii) 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 Hsp40 (heat-shock protein) family member DRiP78 (D1-receptor interacting protein, Mr~78×103) 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 chaperon ligands. Thus, occupancy of the binding pocket corrected the mutation-induced disorder indicating a mutual impingement of the transmembrane domain and the proximal carboxyterminus in establishing the stable receptor fold.The American Society for Pharmacology and Experimental Therapeutics