Zhang Yong, Vogel Walter K, McCullar Jennifer S, Greenwood Jeffrey A, Filtz Theresa M
Molecular and Cellular Biology Program, 203 Pharmacy Building, Oregon State University, Corvallis, OR 97331-3507, USA.
Mol Pharmacol. 2006 Sep;70(3):860-8. doi: 10.1124/mol.105.021923. Epub 2006 Jun 8.
Phospholipase C-beta (PLC-beta) isoenzymes are key effectors in G protein-coupled signaling pathways. Prior research suggests that some isoforms of PLC-beta may exist and function as dimers. Using coimmunoprecipitation assays of differentially tagged PLC-beta constructs and size-exclusion chromatography of native PLC-beta, we observed homodimerization of PLC-beta3 and PLC-beta1 isoenzymes but failed to detect heterodimerization of these isoenzymes. Size-exclusion chromatography data suggest that PLC-beta3 and PLC-beta1 form higher affinity homodimers than PLC-beta2. Evidence supportive of limited PLC-beta monomer-homodimer equilibrium appears at < or =100 nM. Further assessment of homodimerization status by coimmunoprecipitation assays with differentially tagged PLC-beta3 fragments demonstrated that at least two subdomains of PLC-beta3 are involved in dimer formation, one in the catalytic X and Y domains and the other in the G protein-regulated carboxyl-terminal domain. In addition, we provide evidence consistent with the existence of PLC-beta homodimers in a whole-cell context, using fluorescent protein-tagged constructs and microscopic fluorescence resonance energy transfer assays.
磷脂酶C-β（PLC-β）同工酶是G蛋白偶联信号通路中的关键效应器。先前的研究表明，PLC-β的某些亚型可能以二聚体形式存在并发挥作用。通过对差异标记的PLC-β构建体进行共免疫沉淀分析以及对天然PLC-β进行尺寸排阻色谱分析，我们观察到PLC-β3和PLC-β1同工酶的同源二聚化，但未检测到这些同工酶的异源二聚化。尺寸排阻色谱数据表明，与PLC-β2相比，PLC-β3和PLC-β1形成具有更高亲和力的同源二聚体。在≤100 nM时出现支持有限的PLC-β单体 - 同源二聚体平衡的证据。通过对差异标记的PLC-β3片段进行共免疫沉淀分析进一步评估同源二聚化状态，结果表明PLC-β3的至少两个亚结构域参与二聚体形成，一个在催化X和Y结构域，另一个在G蛋白调节的羧基末端结构域。此外，我们使用荧光蛋白标记构建体和显微荧光共振能量转移分析，提供了与全细胞环境中PLC-β同源二聚体存在相一致的证据。
