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首页> 外文期刊>Molecular and Cellular Biology >The Heat Shock Cognate Protein hsc73 Assembles with A1 Adenosine Receptors To Form Functional Modules in the Cell Membrane
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The Heat Shock Cognate Protein hsc73 Assembles with A1 Adenosine Receptors To Form Functional Modules in the Cell Membrane

机译：热休克同源蛋白hsc73与A1腺苷受体组装以在细胞膜中形成功能模块

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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.
机译：A 1 腺苷受体（A 1 Rs）是G蛋白偶联的庚烷受体，它们在质膜的外表面与细胞表面胞外腺苷脱氨酶（ecto- ADA）。通过亲和层析，热休克同源蛋白hsc73被鉴定为能够与受体的第三细胞内环相互作用的胞质组分。如表面等离子体共振所证实，纯化的A 1 Rs与hsc73特异性相互作用，其解离常数在纳摩尔范围（0.5±0.1 nM）。从 35 S标记的鸟苷-5推导，hsc73与A 1 R之间的相互作用导致配体的结合显着减少，并阻止了G蛋白的活化。 '- O -（3-硫代）三磷酸结合试验。有趣的是，这种作用比鸟嘌呤核苷酸类似物所产生的作用要强得多，鸟嘌呤核苷酸类似物使受体与G蛋白脱钩，并被ADA完全阻止。通过免疫沉淀评估，细胞裂解物中高百分比的A 1 Rs与hsc73偶联。通过共聚焦显微镜在DDT 1 MF-2细胞中检测到A 1 R和hsc73之间较高的共定位水平，其他G未获得类似结果蛋白偶联受体。 hsc73和A 1 R在大鼠小脑的特定区域和皮质神经元体内共定位，但在树突或突触中未检测到。值得注意的是，激动剂诱导的受体内在化导致A 1 Rs被两种性质上不同的囊泡内吞，一种是A 1 R和hsc73共定位，另一种是hsc73缺席。这些结果打开了有趣的可能性，即通过G蛋白偶联受体的信号传导可能受热激蛋白调节。

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