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

Abstract

The ubiquitous nucleoside adenosine exerts multiple physiological actions via specific receptors, four of which (A 1 , A 2a , A 2b , and A 3 ) have been cloned (see reference 25 for a review). Activation of A 1 adenosine receptors (A 1 Rs) by adenosine or synthetic analogs, in turn regulates the activity of several membrane and intracellular proteins, such as adenylate cyclase, Ca 2ϩ channels, K ϩ channels and phospholipase C (see reference 25 for a review). Towards the intracellular side of the plasma membrane, A 1 Rs are functionally coupled to members of the pertussis toxin-sensitive family of G proteins, G i1 , G i2 , G i3 , and G o (13,20). For this coupling Nanoff et al. (23) have shown that a proteic factor of 30 to 40 kDa is necessary (23). On the other side of the plasma membrane, A 1 Rs interact with ecto-adenosine deaminase (ecto-ADA) an interaction which is needed for efficient signaling (11,27). Moreover, in response to agonists, A 1 Rs and ADA are internalized together via the same endocytic pathway (28).There is recent evidence indicating that members of the superfamily of G protein-coupled receptors interact with cytosolic proteins. As an example a recent report has described the interaction of the C terminus of metabotropic glutamate receptor type 1␣ with glyceraldehyde-3-phosphate dehydrogenase, tubulin, and an unknown protein (9). The aim of this study was to identify proteins interacting with the third intracellular loop of A 1 R. By means of affinity chromatography, coimmunoprecipitation, and biosensor technology we have found that the heat shock cognate protein hsc73 is able to bind to A 1 R. Interestingly, the interaction between this protein and the A 1 R results in reduced binding of ligands, thus suggesting a role for hsc73 in the regulation of the receptor function. MATERIALS AND METHODSMaterials. ADA (EC 3.5.4.4), electrophoresis reagents, fetal bovine serum and 5Ј-guanylylimidodiphosphate [Gpp(NH)p] were purchased from Boehringer Mannheim (Barcelona, Spain). Activated thiol Sepharose-4B, activated CH-Sepharose 4B, Sephadex G-25, and protein-A Sepharose CL4B were from Pharmacia Biotech (Barcelona, Spain). digitonin,

Monoclonal antibody to novel cell surface epitope on Hsc70 promotes morphogenesis of bile ducts in newborn rat liver

“…Although the mechanism for this effect is at present unclear, the results suggest that binding of MAb OC.10 to surface Hsc70 is stimulating morphogenic signaling pathways. Supporting this assumption is an emerging regulatory role for Hsc70 in cell signaling, a role suggested by signaling complexes composed of Hsc70 and CXCR4 (Triantafilou et al 2001), CXCR2 (Fan et al 2002), and the G protein-coupled A1 adenosine receptor (Sarrio et al 2000). Hsc70 binding has also been shown to be required for the activation of glucocorticoid receptors in the presence of steroids (Rajapandi et al 2000).…”

A1R–A2AR heteromers coupled to Gs and Gi/0 proteins modulate GABA transport into astrocytes

“…Competition experiments were performed by incubating (120 min) membranes (0.18 mg protein/ml) at 25°C in 50 mM Tris-HCl buffer, pH 7.4, containing 10 mM MgCl 2 and 0.2 U/ml adenosine deaminase with 0.8 nM Free and membrane-bound ligand were separated by rapid filtration of 500 μl aliquots in a cell harvester (Brandel, Gaithersburg, MD, USA) through Whatman GF/C filters embedded in 0.3 % polyethylenimine that were subsequently washed for 5 s with 5 ml of ice-cold Tris-HCl buffer. The filters were incubated with 10 ml of Ecoscint H scintillation cocktail (National Diagnostics, Atlanta, GA, USA) overnight at room temperature and radioactivity counts were determined using a Tri-Carb 1600 scintillation counter (PerkinElmer, Boston, MA, USA) with an efficiency of 62 % [18]. Radioligand displacement curves were analyzed by nonlinear regression using commercial program GRAFIT (Erithacus Software, Surrey, UK) as indicated previously [13].…”

Identification of Dopamine D1–D3 Receptor Heteromers

“…The filters were incubated with 10 ml of Ecoscint H scintillation mixture (National Diagnostics, Atlanta, GA) overnight at room temperature, and radioactivity counts were determined using a Tri-Carb 1600 scintillation counter (PerkinElmer Life Sciences) with an efficiency of 62% (20). Binding data from competition experiments were analyzed by nonlinear regression to the previously described equations (20,21), using the commercial Grafit curve-fitting software (Erithacus Software, Surrey, UK). Goodness of fit was tested according to reduced 2 value given by the nonlinear regression program and biphasic over monophasic competition curves were selected according to a p Ͻ 0.05 using an F test (22).…”