The Heat Shock Cognate Protein hsc73 Assembles with A 1 Adenosine Receptors To Form Functional Modules in the Cell Membrane

Abstract

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.