The Heat Shock Cognate Protein hsc73 Assembles with A1 Adenosine Receptors To Form Functional Modules in the Cell Membrane

Abstract

A₁ adenosine receptors (A₁Rs) 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 A₁Rs interact specifically with hsc73 with a dissociation constant in the nanomolar range (0.5 ± 0.1 nM). The interaction between hsc73 and A₁R led to a marked reduction in the binding of the ligands and prevented activation of G proteins, as deduced from ³⁵S-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 A₁Rs in cell lysates are coupled to hsc73. A relatively high level of colocalization between A₁R and hsc73 was detected in DDT₁MF-2 cells by means of confocal microscopy, and no similar results were obtained for other G protein-coupled receptors. Colocalization between hsc73 and A₁R 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 A₁Rs by two qualitatively different vesicle types, one in which A₁R 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.