Truncation of the A1 adenosine receptor reveals distinct roles of the membrane-proximal carboxyl terminus in receptor folding and G protein coupling.
The carboxyl terminus (C-tail) of G protein-coupled receptors is divergent in length and structure and may represent an individualized cytoplasmic domain. By progressively truncating the A1 adenosine receptor, a Gi/o-coupled receptor with short cytoplasmic stretches, we identify two inherent functions of the C-tail, namely a role in receptor export from the endoplasmic reticulum (ER) and a role in G protein coupling. Deletion of the last 22 and 26 amino acids (of 36) reduced and completely abolished surface expression of the receptor, respectively. The severely truncated receptors were retained in the ER and failed to bind ligands. If overexpressed, even a substantial portion of the full-length receptor was retained in the ER in a form that was not functional. These data indicate that folding is rate limiting in export from the ER and that the proximal segment of the carboxyl terminus provides a docking site for the machinery involved in folding and quality control. In addition, the proximal portion is also important in G protein coupling. This latter role was unmasked when the distal portion of the C-tail (the extreme 18 amino acids, including a palmitoylated cysteine) had been removed; the resulting receptor was functional and transferred the agonist-mediated signal more efficiently than the full-length receptor. Signaling was enhanced because the coupling affinity increased (by 3-fold), which translated into a higher agonist potency. Thus, the distal portion of the carboxyl terminus provides for an autoinhibitory restraint, presumably by folding back and preventing G protein access to the proximal part of the C-tail.
Pankevych
Halyna
H
Institute of Pharmacology, University of Vienna, Währinger Strasse 13A, A-1090 Vienna, Austria.
Korkhov
Volodymir
V
Freissmuth
Michael
M
Nanoff
Christian
C
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008024:Ligands; D020935:MAP Kinase Signaling System; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051381:Rats; D018047:Receptors, Purinergic P1 / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors
D018076:DNA, Complementary; D008024:Ligands; D018047:Receptors, Purinergic P1; D000242:Cyclic AMP; D019204:GTP-Binding Proteins