Nini Lylia, Waheed Abdul A, Panicker Leelamma M, Czapiga Meggan, Zhang Jian-Hua, Simonds William F
Metabolic Diseases Branch, 10/8C-101, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
BMC Biochem. 2007 Sep 19;8:18. doi: 10.1186/1471-2091-8-18.
Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins), composed of G alpha, G beta, and G gamma subunits, are positioned at the inner face of the plasma membrane and relay signals from activated G protein-coupled cell surface receptors to various signaling pathways. G beta 5 is the most structurally divergent G beta isoform and forms tight heterodimers with regulator of G protein signalling (RGS) proteins of the R7 subfamily (R7-RGS). The subcellular localization of G beta 5/R7-RGS protein complexes is regulated by the palmitoylation status of the associated R7-binding protein (R7BP), a recently discovered SNARE-like protein. We investigate here whether R7BP controls the targeting of G beta 5/R7-RGS complexes to lipid rafts, cholesterol-rich membrane microdomains where conventional heterotrimeric G proteins and some effector proteins are concentrated in neurons and brain.
We show that endogenous G beta 5/R7-RGS/R7BP protein complexes are present in native neuron-like PC12 cells and that a fraction is targeted to low-density, detergent-resistant membrane lipid rafts. The buoyant density of endogenous raft-associated G beta 5/R7-RGS protein complexes in PC12 cells was similar to that of lipid rafts containing the palmitoylated marker proteins PSD-95 and LAT, but distinct from that of the membrane microdomain where flotillin was localized. Overexpression of wild-type R7BP, but not its palmitoylation-deficient mutant, greatly enriched the fraction of endogenous G beta 5/R7-RGS protein complexes in the lipid rafts. In HEK-293 cells the palmitoylation status of R7BP also regulated the lipid raft targeting of co-expressed G beta 5/R7-RGS/R7BP proteins. A fraction of endogenous G beta 5/R7-RGS/R7BP complexes was also present in lipid rafts in mouse brain.
A fraction of G beta 5/R7-RGS/R7BP protein complexes is targeted to low-density, detergent-resistant membrane lipid rafts in PC12 cells and brain. In cultured cells, the palmitoylation status of R7BP regulated the lipid raft targeting of endogenous or co-expressed G beta 5/R7-RGS proteins. Taken together with recent evidence that the kinetic effects of the G beta 5 complex on GPCR signaling are greatly enhanced by R7BP palmitoylation through a membrane-anchoring mechanism, our data suggest the targeting of the G beta 5/R7-RGS/R7BP complex to lipid rafts in neurons and brain, where G proteins and their effectors are concentrated, may be central to the G protein regulatory function of the complex.
异源三聚体鸟嘌呤核苷酸结合调节蛋白（G蛋白）由Gα、Gβ和Gγ亚基组成，位于质膜内表面，将来自活化的G蛋白偶联细胞表面受体的信号传递至各种信号通路。Gβ5是结构上差异最大的Gβ亚型，与R7亚家族的G蛋白信号调节蛋白（RGS）（R7-RGS）形成紧密的异二聚体。Gβ5/R7-RGS蛋白复合物的亚细胞定位受相关R7结合蛋白（R7BP）的棕榈酰化状态调节，R7BP是最近发现的一种类似SNARE的蛋白。我们在此研究R7BP是否控制Gβ5/R7-RGS复合物靶向脂筏，脂筏是富含胆固醇的膜微区，传统异源三聚体G蛋白和一些效应蛋白在神经元和大脑中集中于此。
我们发现内源性Gβ5/R7-RGS/R7BP蛋白复合物存在于天然神经元样PC12细胞中，且一部分靶向低密度、耐去污剂的膜脂筏。PC12细胞中内源性筏相关Gβ5/R7-RGS蛋白复合物的浮力密度与含有棕榈酰化标记蛋白PSD-95和LAT的脂筏相似，但与flotillin定位的膜微区不同。野生型R7BP的过表达，而非其棕榈酰化缺陷突变体，极大地富集了脂筏中内源性Gβ5/R7-RGS蛋白复合物的比例。在HEK-293细胞中，R7BP的棕榈酰化状态也调节共表达的Gβ5/R7-RGS/R7BP蛋白的脂筏靶向。内源性Gβ5/R7-RGS/R7BP复合物的一部分也存在于小鼠脑的脂筏中。
一部分Gβ5/R7-RGS/R7BP蛋白复合物靶向PC12细胞和大脑中低密度、耐去污剂的膜脂筏。在培养细胞中，R7BP的棕榈酰化状态调节内源性或共表达的Gβ5/R7-RGS蛋白的脂筏靶向。结合最近的证据，即通过膜锚定机制，R7BP棕榈酰化极大地增强了Gβ5复合物对GPCR信号的动力学效应，我们的数据表明，Gβ5/R7-RGS/R7BP复合物靶向神经元和大脑中G蛋白及其效应蛋白集中的脂筏，可能是该复合物G蛋白调节功能的核心。
