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Molecular and Cellular Biology, July 2000, p. 5164-5174, Vol. 20, No. 14
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Sara Sarrió, Vicent Casadó, Marisol Escriche, Francisco Ciruela, Josefa Mallol, Enric I. Canela, Carmen Lluis, and Rafael Franco*

Department of Biochemistry and Molecular Biology, University of Barcelona, 08028 Barcelona, Spain

Received 17 February 2000/Accepted 28 April 2000

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.

* Corresponding author. Mailing address: Dept. Bioquímica i Biologia Molecular, Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain. Phone: 34 934021208. Fax: 34 934021219. E-mail: r.franco{at}sun.q.ub.es.

Molecular and Cellular Biology, July 2000, p. 5164-5174, Vol. 20, No. 14
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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