Li Chunying, Roy Koushik, Dandridge Keanna, Naren Anjaparavanda P
Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.
J Biol Chem. 2004 Jun 4;279(23):24673-84. doi: 10.1074/jbc.M400688200. Epub 2004 Apr 1.
Based on electrophysiological measurements, it has been argued that the active form of cystic fibrosis trans-membrane conductance regulator (CFTR) Cl(-) channel is a multimer. It has also been demonstrated that this multimerization is likely due to PDZ domain-interacting partners. Here we demonstrate that although CFTR in vitro can self-associate into multimers, which depends on PDZ-based interactions, this may not be the case in cell membrane. Using chemical cross-linking, we demonstrated that CFTR exists as a higher order complex in cell membrane. However, this higher order complex is predominantly CFTR dimers, and the PDZ-interacting partners (Na(+)/H(+) exchanger regulatory factor-1 (NHERF1) and NHERF2) constitute approximately 2% of this complex. Interestingly solubilizing membrane expressing CFTR in detergents such as Triton X-100, Nonidet P-40, deoxycholate, and SDS tended to destabilize the CFTR dimers and dissociate them into monomeric form. The dimerization of CFTR was tightly regulated by cAMP-dependent protein kinase-dependent phosphorylation and did not depend on the active form of the channel. In addition, the dimerization was not influenced by either the PDZ motif or its interacting partners (NHERF1 and NHERF2). We also demonstrated that other signaling-related proteins such as Gbeta and syntaxin 1A can be in this higher order complex of CFTR as well. Our studies provide a deeper understanding of how the CFTR assembly takes place in native cell membrane.
基于电生理测量结果，有人认为囊性纤维化跨膜传导调节因子（CFTR）氯离子通道的活性形式是一种多聚体。也有研究表明这种多聚化可能是由于与PDZ结构域相互作用的蛋白所致。在此我们证明，虽然CFTR在体外能够自组装形成多聚体，且这种组装依赖于基于PDZ的相互作用，但在细胞膜中情况可能并非如此。通过化学交联，我们证明CFTR在细胞膜中以更高阶的复合物形式存在。然而，这种更高阶的复合物主要是CFTR二聚体，与PDZ相互作用的蛋白（钠/氢交换调节因子-1（NHERF1）和NHERF2）在该复合物中约占2%。有趣的是，用Triton X-100、Nonidet P-40、脱氧胆酸盐和SDS等去污剂溶解表达CFTR的膜，往往会使CFTR二聚体不稳定并使其解离成单体形式。CFTR的二聚化受环磷酸腺苷依赖性蛋白激酶依赖性磷酸化的严格调控，且不依赖于通道的活性形式。此外，二聚化不受PDZ基序或其相互作用蛋白（NHERF1和NHERF2）的影响。我们还证明，其他与信号传导相关的蛋白，如Gβ和 syntaxin 1A也可存在于CFTR的这种更高阶复合物中。我们的研究为深入理解CFTR在天然细胞膜中的组装方式提供了依据。
