title: Recruitment of a cytoplasmic chaperone relay by the A2A adenosine receptor.
abstract: The adenosine A2A receptor is a prototypical rhodopsin-like G protein-coupled receptor but has several unique structural features, in particular a long C terminus (of >120 residues) devoid of a palmitoylation site. It is known to interact with several accessory proteins other than those canonically involved in signaling. However, it is evident that many more proteins must interact with the A2A receptor, if the trafficking trajectory of the receptor is taken into account from its site of synthesis in the endoplasmic reticulum (ER) to its disposal by the lysosome. Affinity-tagged versions of the A2A receptor were expressed in HEK293 cells to identify interacting partners residing in the ER by a proteomics approach based on tandem affinity purification. The receptor-protein complexes were purified in quantities sufficient for analysis by mass spectrometry. We identified molecular chaperones (heat-shock proteins HSP90α and HSP70-1A) that interact with and retain partially folded A2A receptor prior to ER exit. Complex formation between the A2A receptor and HSP90α (but not HSP90β) and HSP70-1A was confirmed by co-affinity precipitation. HSP90 inhibitors also enhanced surface expression of the receptor in PC12 cells, which endogenously express the A2A receptor. Finally, proteins of the HSP relay machinery (e.g. HOP/HSC70-HSP90 organizing protein and P23/HSP90 co-chaperone) were recovered in complexes with the A2A receptor. These observations are consistent with the proposed chaperone/coat protein complex II exchange model. This posits that cytosolic HSP proteins are sequentially recruited to folding intermediates of the A2A receptor. Release of HSP90 is required prior to recruitment of coat protein complex II components. This prevents premature ER export of partially folded receptors.
authors: From the Institute for Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria.
authors: Christian
authors: C
authors: Bergmayr
authors: Patrick
authors: P
authors: Thurner
authors: Simon
authors: S
authors: Keuerleber
authors: Oliver
authors: O
authors: Kudlacek
authors: Christian
authors: C
authors: Nanoff
authors: Michael
authors: M
authors: Freissmuth
authors: Christian W
authors: CW
authors: Gruber
mesh_terms: D000818:Animals; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism*; D057809:HEK293 Cells; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D019746:Intramolecular Oxidoreductases / Q000378:metabolism; D013058:Mass Spectrometry; D016716:PC12 Cells; D000071876:Prostaglandin-E Synthases; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D043705:Receptor, Adenosine A2A / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D012995:Solubility; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
chemical_list: D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D061988:Proteasome Inhibitors; D034741:RNA, Small Interfering; D043705:Receptor, Adenosine A2A; D011993:Recombinant Fusion Proteins; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases
keywords: 7-Helix Receptor; Adenosine Receptor; Adenylate Cyclase (Adenylyl Cyclase); Endoplasmic Reticulum (ER); G Protein-coupled Receptors (GPCR); G Proteins; Heat-Shock Protein
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