The heat shock cognate protein hsc73 assembles with A(1) adenosine receptors to form functional modules in the cell membrane

A(1) adenosine receptors (A(1)Rs) are G protein-coupled heptaspanning recep tors that interact at the outer face of the plasma membrane with cell surfa ce ecto-adenosine deaminase (ecto-ADA). By affinity chromatography the heat shock cognate protein hsc73 was identified as a cytosolic component able t o interact with the third intracellular loop of the receptor. As demonstrat ed 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 f rom S-35-labeled guanosine-5'-O-(3-thio) triphosphate binding assays. Inter estingly this effect was stronger than that exerted by guanine nucleotide a nalogs, which uncouple receptors from G proteins, and was completely preven ted 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 colocaliza tion between A(1)R and hsc73 was detected in DDT1MF-2 cells by means of con focal microscopy, and no similar results were obtained for other G protein- coupled receptors. Colocalization between hsc73 and A(1)R was detected in s pecific regions of rat cerebellum and in the body of cortical neurons but n ot in dendrites or synapses. Remarkably, agonist-induced receptor internali zation leads to the endocytosis of A(1)Rs by two qualitatively different ve sicle types, one in which A(1)R and hsc73 colocalize and another in which h sc73 is absent. These results open the interesting possibility that signali ng via G protein-coupled receptors may be regulated by heat shock proteins.