RasGRP4-dependent Responses in Mast Cells

描述（由申请人提供）：我们最近克隆了编码信号蛋白Ras鸟嘌呤核苷酸释放蛋白（RasGRP）家族的第四个成员的cDNA和基因。RasGRP 4是肥大细胞（MC）限制性、阳离子依赖性鸟嘌呤核苷酸交换因子。它也是一种二酰基甘油/佛波酯受体，在决定MC系中表达哪些蛋白酶、细胞因子和类花生酸介质中起着重要作用。已经鉴定了RasGRP 4的等位基因变体，以及RasGRP 4的功能不同的同种型，其是其基因的差异剪接的结果。早期对C3 H/HeJ小鼠的基因连锁研究揭示了染色体7A 3-B1上的一个显著位点，该位点控制其固有的气道对乙酰甲胆碱的反应性。我们最近发现，从这种低反应小鼠中产生的MCs产生了一种有缺陷的mRasGRP 4亚型。大量的研究表明RasGRP 4在MC的发育过程中起着至关重要的作用，RasGRP 4的异常表达可导致MC功能障碍。互补的方法将用于拟议的研究，以测试这些假设，并扩大我们的知识，最近确定的RasGRP家族的信号蛋白。在具体目标1中，将确定调节小鼠和人RasGRP 4表达的转录和转录后机制。在具体目标2中，将评估RasGRP 4的等位基因亚型及其不同结构域的功能重要性。RasGRP 4调控的MCs中的主要基因和信号通路也将被确定，以及抑制MCs中RasGRP 4依赖性信号事件的控制机制。在特定目标3中，将创建RasGRP 4缺陷的非转化MC，以评估该信号蛋白在体内MC发育和功能中的重要性。 由于RasGRP 4是一种MC限制性信号蛋白，似乎作用于酪氨酸激酶受体Kit的下游，因此预计所获得的数据将显着推进我们关于主要控制MC发育和功能的定义不清的Kit依赖性途径的知识。还预期使用药物和其他方法靶向破坏人类中的RasGRP 4表达和/或活性将有益于MC依赖性病症。

DESCRIPTION (provided by applicant): We recently cloned the cDNAs and genes that encode the fourth member of the Ras Guanine Nucleotide Releasing Protein (RasGRP) family of signaling proteins. RasGRP4 is a mast cell (MC) restricted, cation-dependent, guanine nucleotide exchange factor. It also is a diacylglycerol/phorbol ester receptor that plays a prominent role in dictating which protease, cytokine, and eicosanoid mediators are expressed in MC lines. Allelic variants of RasGRP4 have been identified, as well as functionally different isoforms of RasGRP4 that are the result of differential splicing of its gene. Earlier gene-linkage studies of C3H/HeJ mice revealed a prominent site on chromosome 7A3-B1 that controls its intrinsic airway reactivity to methacholine. We recently found that the MCs developed from this hyporesponsive mouse produce a defective isoform of mRasGRP4. The accumulated data suggest that RasGRP4 is of critical importance in MC development and that the expression of abnormal isoforms of RasGRP4 can lead to MC dysfunction. Complementary approaches will be used in the proposed studies to test these hypotheses and to expand our knowledge concerning the recently identified RasGRP family of signaling proteins. In Specific Aim 1, the transcriptional and posttranscriptional mechanisms that regulate the expression of mouse and human RasGRP4 will be identified. In Specific Aim 2, the functional importance of allelic isoforms of RasGRP4 and its different domains will be evaluated. The predominant genes and signaling pathways in MCs that are regulated by RasGRP4 also will be identified, as well as the control mechanisms that dampen RasGRP4- dependent signaling events in MCs. In Specific Aim 3, RasGRP4-deficient, non transformed MCs will be created to evaluate the importance of this signaling protein in MC development and function in vivo. Because RasGRP4 is a MC-restricted signaling protein that appears to act downstream of the tyrosine kinase receptor Kit, it is anticipated that the obtained data will significantly advance our knowledge concerning the poorly defined Kit-dependent pathway that dominantly controls MC development and function. It also is anticipated that targeted disruption of RasGRP4 expression and/or activity in humans using pharmaceutical and other approaches will be beneficial in MC-dependent disorders.