From the Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel, and.
the Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-Einstein-Allee 11, 89081 Ulm, Germany.
J Biol Chem. 2010 Feb 5;285(6):3905-3915. doi: 10.1074/jbc.M109.085100. Epub 2009 Dec 10.
We combined fluorescence recovery after photobleaching (FRAP) beam-size analysis with biochemical assays to investigate the mechanisms of membrane recruitment and activation of phospholipase C-beta(2) (PLCbeta(2)) by G protein alpha(q) and betagamma dimers. We show that activation by alpha(q) and betagamma differ from activation by Rac2 and from each other. Stimulation by alpha(q) enhanced the plasma membrane association of PLCbeta(2), but not of PLCbeta(2)Delta, which lacks the alpha(q)-interacting region. Although alpha(q) resembled Rac2 in increasing the contribution of exchange to the FRAP of PLCbeta(2) and in enhancing its membrane association, the latter effect was weaker than with Rac2. Moreover, the membrane recruitment of PLCbeta(2) by alpha(q) occurred by enhancing PLCbeta(2) association with fast-diffusing (lipid-like) membrane components, whereas stimulation by Rac2 led to interactions with slow diffusing membrane sites. On the other hand, activation by betagamma shifted the FRAP of PLCbeta(2) and PLCbeta(2)Delta to pure lateral diffusion 3- to 5-fold faster than lipids, suggesting surfing-like diffusion along the membrane. We propose that these different modes of PLCbeta(2) membrane recruitment may accommodate contrasting functional needs to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)) in localized versus dispersed populations. PLCbeta(2) activation by Rac2, which leads to slow lateral diffusion and much faster exchange, recruits PLCbeta(2) to act locally on PtdInsP(2) at specific domains. Activation by alpha(q) leads to lipid-like diffusion of PLCbeta(2) accompanied by exchange, enabling the sampling of larger, yet limited, areas prior to dissociation. Finally, activation by betagamma recruits PLCbeta(2) to the membrane by transient interactions, leading to fast "surfing" diffusion along the membrane, sampling large regions for dispersed PtdInsP(2) populations.
我们将荧光恢复后光漂白（FRAP）束斑分析与生化测定相结合，研究了 G 蛋白α(q)和βγ二聚体募集和激活磷脂酶 C-β2(PLCβ2)的机制。我们发现，α(q)和βγ的激活与 Rac2 的激活以及彼此之间的激活不同。α(q)的刺激增强了 PLCβ2 的质膜结合，但缺乏与α(q)相互作用区域的 PLCβ2Δ则不然。虽然α(q)在增加 PLCβ2 的交换对 FRAP 的贡献和增强其膜结合方面类似于 Rac2，但后者的作用比 Rac2 弱。此外，α(q)对 PLCβ2 的膜募集是通过增强 PLCβ2 与快速扩散（类脂）膜成分的结合来实现的，而 Rac2 的刺激则导致与缓慢扩散的膜位点相互作用。另一方面，βγ的激活使 PLCβ2 和 PLCβ2Δ的 FRAP 比脂质快 3-5 倍，向纯侧向扩散，提示沿着膜呈冲浪样扩散。我们提出，这些不同的 PLCβ2 膜募集模式可能适应于水解磷脂酰肌醇 4,5-二磷酸（PtdInsP2）的局部与分散群体之间的对比功能需求。Rac2 激活 PLCβ2，导致缓慢的侧向扩散和更快的交换，将 PLCβ2 募集到特定结构域局部作用于 PtdInsP2。α(q)的激活导致 PLCβ2 与脂质类似的扩散伴随着交换，从而在解离之前能够对更大但有限的区域进行采样。最后，βγ的激活通过短暂的相互作用将 PLCβ2 募集到膜上，导致快速的“冲浪”沿膜扩散，对分散的 PtdInsP2 群体进行大区域采样。
