Fondazione Santa Lucia, Rome, Italy.
Purinergic Signal. 2006 Nov;2(4):575-82. doi: 10.1007/s11302-006-9014-2. Epub 2006 Sep 28.
It is well established that several cell surface receptors interact with each other to form dimers and oligomers, which are essential for their activation. Since little is known about the quaternary structure of P2Y receptors, in the present work, we investigated the expression of the G-protein-coupled P2Y₄ subunit as monomeric or higher-order complex protein. We examined both endogenously expressed P2Y₄ subtype with the aid of specific anti-P2Y₄ antiserum, and heterologously transfected P2Y₄-tagged receptors with the use of antitag antibodies. In both cases, we found the P2Y₄ receptor displaying molecular masses corresponding to monomeric, dimeric and oligomeric structures. Experiments performed in the absence of reducing agents demonstrated that there is a strict correlation among the multiple protein bands and that the multimeric forms are at least partially assembled by disulphide bonds. The direct demonstration of P2Y₄ homodimerisation comes instead from co-transfection and differential co-immunoprecipitation experiments, with the use of differently tagged P2Y₄ receptors and antitag antibodies. The structural propensity of the P2Y₄ protein to form homo-oligomers may open the possibility of a novel regulatory mechanism of physiopathological functions for this and additional P2Y receptors.
已有充分证据表明，几种细胞表面受体相互作用形成二聚体和寡聚体，这对于它们的激活是必不可少的。由于对 P2Y 受体的四级结构知之甚少，因此在本工作中，我们研究了 G 蛋白偶联 P2Y₄ 亚基作为单体或更高阶复合物蛋白的表达。我们利用特异性抗 P2Y₄ 抗血清检查内源性表达的 P2Y₄ 亚型，并用抗标签抗体检查异源转染的 P2Y₄ 标记受体。在这两种情况下，我们都发现 P2Y₄ 受体显示出与单体、二聚体和寡聚体结构相对应的分子质量。在没有还原剂的情况下进行的实验表明，多个蛋白条带之间存在严格的相关性，并且多聚体形式至少部分通过二硫键组装。来自共转染和差异共免疫沉淀实验的直接证据表明 P2Y₄ 同源二聚化的发生，使用了不同标记的 P2Y₄ 受体和抗标签抗体。P2Y₄ 蛋白形成同源寡聚体的结构倾向可能为该受体和其他 P2Y 受体的生理病理功能提供一种新的调节机制。
