Optical Investigations of the Mast Cell MAFA Regulatory Protein

9807822 BARIAS本项目的长期目标是从生物物理学的角度了解肥大细胞功能相关抗原对大鼠2H3-RBL细胞的作用机制。MafA是一种具有C型凝集素胞外区和细胞质YSTL基序的II型膜蛋白。这种蛋白的交联会抑制I型FCE受体(FceR1)的信号传递，尽管每20个FceR1中只有一个MAFA。通过时间分辨磷光各向异性、荧光光漂白恢复和荧光能量转移等光学方法研究MAFA与FceR1和其他膜蛋白的相互作用。这些相互作用在接受不同刺激和抑制刺激的细胞之间的差异将被检测，例如通过抗原聚集FceR1或通过MAFA特异性抗体G63聚集MAFA。最后，这一全面的实验方法将扩展到来自MAFA的嵌合分子，并在能够传递FceR1信号的细胞中表达。这个项目提供了一个极好的机会来研究一种细胞表面分子如何调节数量更多的另一种分子的功能。使用分子生物学方法产生选择性修饰的受体和信号分子，结合生物物理方法在活细胞上测量这些分子的定量参数，为解决这一问题提供了一种强有力的途径。它还提供了一个机会来改进激光光学技术，以供更大的生物物理界使用，以检查细胞膜蛋白质的行为。这些方法是这个实验室的强项。

9807822 BARISAS The long-term goal of this project is to understand in biophysical terms the mechanism of function of the mast cell function-associated antigen(MAFA) on rat 2H3-RBL cells. MAFA is a type II membrane protein with a C-type lectin extracellular domain and a cytoplasmic YSTL motif. Crosslinking of this protein inhibits signaling by the type I Fce receptor (FceR1) despite the presence of only one MAFA per twenty FceR1. The interactions of MAFA with FceR1 and other membrane proteins will be examined through optical approaches including time-resolved phosphorescence anisotropy, fluorescence photobleaching recovery and fluorescence energy transfer. Differences in these interactions among cells receiving various stimulatory and inhibitory stimuli, such as clustering FceR1 by antigen or clustering MAFA by the MAFA-specific antibody G63 will be examined. Finally, this overall experimental approach will be extended to chimeric molecules derived from MAFA and expressed in cells capable of FceR1 signaling. This project provides an excellent opportunity to examine how cell surface molecules of one kind can regulate the function of another molecule present in much larger numbers. The use of molecular biological methods to produce selectively-modified receptors and signaling molecules in combination with biophysical approaches to measuring quantitative parameters of these molecules on living cells provides a powerful approach by which to address this problem. It also provides an opportunity to refine, for use by the larger biophysical community, laser optical techniques for examining the behavior of cell membrane proteins. These methods are a strength of this laboratory.