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.
