title: The heat shock cognate protein hsc73 assembles with A(1) adenosine receptors to form functional modules in the cell membrane.
abstract: A(1) adenosine receptors (A(1)Rs) are G protein-coupled heptaspanning receptors that interact at the outer face of the plasma membrane with cell surface ecto-adenosine deaminase (ecto-ADA). By affinity chromatography the heat shock cognate protein hsc73 was identified as a cytosolic component able to interact with the third intracellular loop of the receptor. As demonstrated by surface plasmon resonance, purified A(1)Rs interact specifically with hsc73 with a dissociation constant in the nanomolar range (0.5 +/- 0.1 nM). The interaction between hsc73 and A(1)R led to a marked reduction in the binding of the ligands and prevented activation of G proteins, as deduced from (35)S-labeled guanosine-5'-O-(3-thio)triphosphate binding assays. Interestingly this effect was stronger than that exerted by guanine nucleotide analogs, which uncouple receptors from G proteins, and was completely prevented by ADA. As assessed by immunoprecipitation a high percentage of A(1)Rs in cell lysates are coupled to hsc73. A relatively high level of colocalization between A(1)R and hsc73 was detected in DDT(1)MF-2 cells by means of confocal microscopy, and no similar results were obtained for other G protein-coupled receptors. Colocalization between hsc73 and A(1)R was detected in specific regions of rat cerebellum and in the body of cortical neurons but not in dendrites or synapses. Remarkably, agonist-induced receptor internalization leads to the endocytosis of A(1)Rs by two qualitatively different vesicle types, one in which A(1)R and hsc73 colocalize and another in which hsc73 is absent. These results open the interesting possibility that signaling via G protein-coupled receptors may be regulated by heat shock proteins.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Barcelona, 08028 Barcelona, Spain.
authors.forename: S
authors.initials: S
authors.lastname: Sarrió
authors.forename: V
authors.initials: V
authors.lastname: Casadó
authors.forename: M
authors.initials: M
authors.lastname: Escriche
authors.forename: F
authors.initials: F
authors.lastname: Ciruela
authors.forename: J
authors.initials: J
authors.lastname: Mallol
authors.forename: E I
authors.initials: EI
authors.lastname: Canela
authors.forename: C
authors.initials: C
authors.lastname: Lluis
authors.forename: R
authors.initials: R
authors.lastname: Franco
mesh_terms: D000243:Adenosine Deaminase / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques; D001921:Brain / Q000378:metabolism; D016466:CHO Cells / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells / Q000378:metabolism; D008297:Male; D008647:Mesocricetus; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D010660:Phenylisopropyladenosine / Q000378:metabolism; D011233:Precipitin Tests; D011817:Rabbits; D051381:Rats; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D013462:Sulfur Radioisotopes
chemical_list: D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494371:HSPA8 protein, human; D006360:Heat-Shock Proteins; C494373:Hspa8 protein, rat; D010446:Peptide Fragments; D018047:Receptors, Purinergic P1; D013462:Sulfur Radioisotopes; D010660:Phenylisopropyladenosine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000243:Adenosine Deaminase; D019204:GTP-Binding Proteins