Control Of G Protein Signaling: Role Of The Rgss

我们发现了一个被称为RGSs的蛋白质家族，它通过使用七种跨膜受体和异源三聚体G蛋白的途径损害信号转导。这些受体在与配体（如激素或趋化因子）结合后被激活，触发G α亚基将GTP交换为GDP；这导致G α和G β - γ亚基的分离和下游信号传导。RGS蛋白结合G α亚基并作为GTPase激活蛋白（gap）发挥作用，从而使G α亚基失活并促进其与G β - γ的重新结合。我们已经证明RGS蛋白通过趋化因子受体调节信号，并且它们可以抑制趋化性。表达RGS1的B淋巴细胞在趋化因子SDF- 1的作用下无法迁移。相反，从RGS1基因被基因靶向破坏的小鼠中获得的RGS1 -/- B细胞对SDF-1的趋化反应增强，并且在暴露于趋化因子后不能正常脱敏。这可能是RGS1 -/-小鼠免疫反应受损和淋巴组织结构改变的结果。我们还发现某些RGS蛋白可以直接抑制腺苷酸环化酶的激活，从而提供了这些蛋白抑制RGS诱导的cAMP产生的机制。这些发现与嗅觉系统有关。气味激活RGS家族成员Golf，从而激活腺苷酸环化酶III型（AC III）和cAMP的产生。RGS2有效抑制AC III介导的cAMP生成。嗅觉神经元同时表达RGS2和RGS3，将RGS2抗体微注射到嗅觉神经元中可显著增强气味诱导的信号转导。我们发现RGS3基因位于另一个称为C2PA的编码区附近。C2PA和RGS3编码区结合在一起产生几种不同的蛋白质，包括PDZ-RGS3，一种最近发现的干扰小脑神经元CXCL12信号的蛋白质。CXCL12是胚胎发育过程中这些神经元募集的重要趋化因子。这些组合的基因座现在统称为RGS3基因。我们已经将绿色荧光蛋白的编码区融合到5个RGS3亚型上，并正在研究它们在细胞内的位置。我们还通过质谱分析共免疫沉淀蛋白，分离出几种与RGS3的C2PA部分相互作用的蛋白。我们通过直接共免疫沉淀实验证实了它们的相互作用。我们现在正在研究RGS3同工异构体与这些新发现的相互作用物之间相互作用的功能重要性。我们继续研究RGS14及其在中心体功能中的潜在作用。我们已经在正常细胞和细胞系中记录了内源性RGS14在中心体中的表达。中心体在细胞分裂过程中组织有丝分裂纺锤体。我们发现RGS14的过表达导致细胞质分裂、DNA复制后细胞分裂和染色体分离的严重缺陷，从而导致多核细胞。目前的研究主要集中在分析降低细胞中RGS14水平的后果。小鼠RGS13、RGS3、RGS18和RGS5基因已被分离，小鼠RGS3和RGS5位点的基因靶向正在进行中。最初的瞄准尝试不成功，重新设计的瞄准结构现在正在测试中。我们对RGS5进行了一系列的生化研究，表明RGS5是一种有效的Gqalpha GAP，参与心血管功能的调节。此外，我们已经证明RGS5是周细胞和血管平滑肌细胞的新标记物。PDGF-B和pdgfr - β缺失胚胎中RGS5表达的缺失与这些动物中已知的周细胞丢失位点相关。我们已经证明另一种RGS蛋白RGS13是生发中心B淋巴细胞的优秀标记物。RGS13抑制Gqalpha和Gialpha介导的信号传导，并抑制B淋巴细胞趋化因子CXCL12和CXCL13的趋化性。RGS18在巨核细胞和血小板中强烈表达，在单核细胞和未成熟树突状细胞中表达水平较低。分化为成熟树突状细胞后，RGS18水平显著下降，表明成熟树突状细胞流动性增强可能与RGS18水平下降有关。我们用RGS1、RGS2、RGS3、RGS4、RGS5、RGS13、RGS14、RGS18制备了GFP融合蛋白。这些结构被用于制造永久性细胞系，以研究这些蛋白质在一系列不同激动剂刺激下的细胞内定位。最近发现了两个编码Gsalpha gap的人类基因RGS-PX1和RGS-PX2。这些复杂的蛋白质除了具有rgs样结构域外还具有许多结构域。我们已经分离出两个与人类基因同源的小鼠基因和第三个基因RGS-PX3。已经确定了人类和小鼠RGS-PX3的编码区。我们最近完成了它们在各种小鼠组织中的表达研究。我们还鉴定了一种酵母蛋白，与RGS-PX蛋白具有相当大的同源性。我们计划研究该蛋白在酵母中的功能，并尝试用哺乳动物基因补充酵母中缺乏RGS-PX的酵母。此外，还鉴定出一株秀丽隐杆线虫RGS-PX。该基因在秀丽隐杆线虫中的作用将通过RNAi抑制其表达来探讨。为了寻求另一种方法来鉴定在调节GPCR信号传导中重要的分子，我们选择了对趋化因子刺激反应高或低的B细胞系。通过反复选择那些在趋化性试验中有反应或没有反应的细胞，我们开发了B细胞系，其中大多数细胞对CXCL12有反应，或者很少细胞移动。使用类似的方法，我们已经开发出对CXCL13高或低反应的B细胞系。我们利用基因芯片分析了高反应细胞和低反应细胞中的基因表达。有趣的是，RGS1和RGS13在低反应细胞系中与高反应细胞系相比均显著上调。其他一些有趣的基因的表达也被改变了。我们目前正在通过Northern blot和RT-PCR验证这些变化。

We have discovered a protein family termed RGSs that impair signal transduction through pathways that use seven trans- membrane receptors and heterotrimeric G proteins. Such receptors, when activated following the binding of a ligand such as a hormone or chemokine, trigger the G alpha subunit to exchange GTP for GDP; this causes the dissociation of G alpha and G beta-gamma subunits and downstream signaling. RGS proteins bind G alpha subunits and function as GTPase activating proteins (GAPs), thereby deactivating the G alpha subunit and facilitating their re-association with G beta-gamma. We have shown that RGS proteins modulate signaling through chemokine receptors and that they can inhibit chemotaxis. RGS1 expressing B lymphocytes fail to migrate in response to the chemokine SDF- 1. Conversely, RGS1 -/- B cells obtained from mice in which the RGS1 gene has been disrupted by gene targeting have an enhanced chemotaxic response to SDF-1 and fail to desensitize properly following exposure to chemokines. Likely as consequnce the RGS1 -/- mice have impaired immune respones and altered lymphoid tissue architecture. We have also shown that certain RGS proteins can directly inhibit the activation of adenylyl cyclase, thereby providing a mechanism by which these proteins can inhibit RGS induced cAMP production. These findings are relevant to the olfactory system. Odorants activate the RGS family member Golf, which leads to activation of adenylyl cyclase type III (AC III) and the production of cAMP. RGS2 potently inhibits AC III mediated cAMP production. Olfactory neurons express both RGS2 and RGS3 and the microinjection of an antibody to RGS2 into olfactory neurons profoundly enhances odorant induced signal transduction. We have found that the RGS3 gene is situtated close to another coding region termed C2PA. Together the C2PA and RGS3 coding regions combine to produce several distinct proteins including PDZ-RGS3, a recently described protein that interferes with the CXCL12 signaling in cerebellar neurons. CXCL12 is a chemokine important for the recruitment of these neurons during embryonic development. The combined loci are now collectively referred to as the RGS3 gene. We have fused the coding region for green fluorescent protein to 5 of the RGS3 isoforms and are studying their intracellular location. We have also isolated several interacting proteins with the C2PA portion of RGS3 by analyzing co-immunoprecipitating proteins by mass spectroscopy. We have confirmed their interaction by direct co-immunoprecipitation experiments. We are now studying the functional importance of the interaction between the RGS3 isoforms and these newly identified interactors. We have continued our studies of RGS14 and its potential role in centrosome function. We have documented endogenous RGS14 expression in centrosomes in both normal cells and in cell lines. Centrosomes organize the mitotic spindle during cell division. We have found that overexpression of RGS14 leads to profound defects in cytokinesis, the splitting of cells following DNA replication and chromosomal segration, thereby causing multinucleated cells. Current studies are focused on analyzing the consequences of reducing RGS14 levels in cells. The murine RGS13, RGS3, RGS18, and RGS5 genes have been isolated and gene targeting of the mouse RGS3 and RGS5 loci is in progress. Initial targeting attempts have been unsuccessful and re-designed targeting constructs are now being tested. We have carried out a series of biochemical studies of RGS5, which indicate that RGS5 is a potent Gqalpha GAP and takes part in the regulation of cardiovascular function. In addition, we have shown that RGS5 is a novel marker for pericytes and vascular smooth muscle cells. The absence of RGS5 expression in PDGF-B and PDGFR-beta null embryos correlated with known sites of pericyte loss in these animals. We have shown that another RGS protein, RGS13, is an excellent marker for germinal center B lymphocytes. RGS13 inhibits both Gqalpha and Gialpha mediated signaling and inhibits chemotaxis in response to the B lymphocyte chemokines, CXCL12 and CXCL13. RGS18 is strongly expressed in megakaryocytes and platelets and at lower levels in monocytes and immature dendritic cells. Upon differentiation to mature dendritic cells RGS18 levels dramatically decline suggesting that enhanced mobility of mature dendritic cells may be related to the fall in RGS18 levels. We have made GFP fusion proteins with RGS1, RGS2, RGS3, RGS4, RGS5, RGS13, RGS14, and RGS18. These constructs are being used to make permanent cell lines to study the intracellular localization of these proteins following stimulation by a panel of different agonists. Recently two human genes, RGS-PX1 and RGS-PX2, which encode GAPs for Gsalpha have been identified. These are complicated proteins that possess a number of domains besides their RGS-like domain. We have isolated two murine genes that are orthologues of the human genes and a third gene, RGS-PX3. Coding regions for both human and mouse RGS-PX3 have been identified. We have recently completed a study of their expression in various mouse tissues. We have also identifed a yeast protein that shares considerable homology with the RGS-PX proteins. We plan to study the function of this protein in yeast and attempt to complement yeast deficient in RGS-PX with mammalian genes. In addition a C. elegans RGS-PX has also been identified. The role of this gene in C. elegans will be approached by using RNAi to inhibit its expression. In order to pursue another approach to identifying molecules important in regulating GPCR signaling, we have selected B cell lines that are either hyper- or hypo-responsive to chemokine stimulation. By repetitively selecting those cells that responded or did not respond in chemotaxis assays we have developed B cell lines in which the majority of the cells move in response to CXCL12 or in which very few of the cells move. Using a similar approach we have developed B cell lines hyper- or hypo-responsive to CXCL13. Using gene chips we have analyzed the gene expression in the hyper and hypo-responsive cells. Interesting both RGS1 and RGS13 were significantly upregulated in the hypo-responsive cell lines as compared to the hyper-responsive lines. The expression of a number of other interesting genes were also modified. We are currently verifying these changes by Northern blot and RT-PCR.