Rhodopsin-mediated activation of alternative transduction pathways.

描述（由申请人提供）：我们研究的长期目标是阐明导致进行性致盲性疾病视网膜色素变性（RP）的分子机制。最近的工作表明，不适当的transducin信号转导活动引起的RP突变的一个子集的退化。转导素功能的遗传消除防止变性。然而，另一个子集的突变体退化的转导蛋白的独立的方式，并显示部分保护时，黑暗饲养。这表明，不适当的视紫红质介导的G蛋白非依赖性途径的激活可能是RP的原因。在过去的十年中，令人信服的证据已经积累表明，除了传统的G蛋白信号转导途径，激活的7-跨膜（7-TM）受体可以激活G蛋白非依赖性途径。对于一些受体，受体/抑制蛋白复合物介导细胞信号传导。MAP激酶和酪氨酸激酶信号转导通路已被牵连在这些替代途径。在这个R21的应用程序中，我们建议测试的假设，光激活视紫红质或视紫红质/抑制蛋白复合物激活G蛋白独立的信号通路。我们将使用在转导素α-亚基（Tra-/-）和/或抑制蛋白（Arr-/-）基因中具有无效突变的转基因小鼠分离替代途径。具体目标1将测试视紫红质/抑制蛋白信号传导复合物在Tra-/-杆中以光依赖性方式形成的预测。我们将通过Western印迹分析寻找arrestin与光暴露后的膜组分的关联。具体目标2将测试在不存在功能性转导蛋白的情况下，光激活视紫红质激活MAP激酶和/或酪氨酸激酶信号转导途径的预测。我们有针对性的调查17激酶信号分子，在感光细胞中富集。信号激活的标志性特征是蛋白质磷酸化。为了鉴定G蛋白独立的信号传导途径，我们将比较野生型、Tra-/-和Tra-/-、Arr-/-双突变小鼠的暗适应和光暴露视杆细胞外节制备物中信号激酶的磷酸化状态。我们预计只有在暴露于光的动物中才会激活。这些研究将为7-TM受体介导的G蛋白非依赖性信号转导提供基础.这为研究人类视网膜变性和其他7-TM受体疾病中的非传统信号转导途径提供了基础。

DESCRIPTION (provided by applicant): The long-term objective of our research is to elucidate the molecular mechanisms that cause the progressive blinding disease retinitis pigmentosa (RP). Recent work showed that inappropriate transducin signaling activity caused degeneration in a subset of RP mutations. Genetic ablation of transducin function protected from degeneration. However, another subset of mutants degenerated in a transducin-independent manner and showed partial protection when dark-reared. This suggests that inappropriate rhodopsin mediated activation of a G-protein independent pathway may be a cause of RP. Over the past decade, compelling evidence has accumulated to suggest that in addition to traditional G-protein signaling pathways, activated 7- transmembrane (7-TM) receptors can activate G-protein independent pathways. For a few receptors, receptor/arrestin complexes mediate cell signaling. MAP kinase and tyrosine kinase signal transduction pathways have been implicated in these alternative pathways. In this R21 application, we propose to test the hypothesis that light-activated rhodopsin or rhodopsin/arrestin complexes activate G-protein independent signaling pathways. We will isolate alternative pathway(s) using transgenic mice with null mutations in the transducin a-subunit (Tra-/-) and/or arrestin (Arr-/-) genes. Specific Aim 1 will test the prediction that rhodopsin/arrestin signaling complexes form in a light-dependent manner in Tra-/- rods. We will look for association of arrestin with membrane fractions after light exposure by Western blot analysis. Specific Aim 2 will test the prediction that in the absence of functional transducin, light- activated rhodopsin activates MAP kinase and/or tyrosine kinase signal transduction pathways. We have targeted for investigation 17 kinase-signaling molecules that are enriched in photoreceptor cells. A hallmark feature of signal activation is protein phosphorylation. To identify G-protein independent signaling pathways, we will compare the phosphorylation state of signal kinases in dark-adapted and light exposed rod outer segment preparations from wild-type, Tra-/- and Tra-/-, Arr-/- double mutant mice. We expect activation only in light-exposed animals. The proposed studies will provide a basic understanding of G- protein independent signaling by 7-TM receptors. This provides the basis for investigation of non-traditional signal transduction pathways in human retinal degenerations and other 7-TM receptor diseases.