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疾病色素性视网膜炎（RP）的分子机制。最近的研究表明，不适当的转导蛋白信号活动导致RP突变子集的变性。基因消融转导蛋白功能防止变性。然而，另一部分突变体以不依赖转导因子的方式退化，并在暗饲养时显示出部分保护。这表明不适当的视紫红质介导的g蛋白独立通路的激活可能是RP的原因之一。在过去的十年中，越来越多的证据表明，除了传统的g蛋白信号通路外，激活的7-跨膜（7- tm）受体还可以激活与g蛋白无关的信号通路。对于少数受体，受体/阻滞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与这些替代途径有关。在本R21应用中，我们提出验证光激活的视紫红质或视紫红质/阻滞蛋白复合物激活g蛋白独立信号通路的假设。我们将使用在转导蛋白a亚基（Tra-/-）和/或阻滞蛋白（Arr-/-）基因中具有零突变的转基因小鼠分离替代途径。特异性Aim 1将测试预测视紫红质/阻滞蛋白信号复合物在Tra-/-杆中以光依赖的方式形成。我们将在光暴露后用Western blot分析寻找抑制素与膜组分的关联。特异性Aim 2将验证在缺乏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。我们研究了富含光感受器细胞的17种激酶信号分子。信号激活的一个显著特征是蛋白磷酸化。为了确定与g蛋白无关的信号通路，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杆外段制剂中信号激酶的磷酸化状态。我们预计只有暴露在光线下的动物才会激活。提出的研究将为7-TM受体的G蛋白非依赖性信号传导提供基本的理解。这为研究人类视网膜变性和其他7-TM受体疾病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.