Molecular mechanisms of signal transduction via guanine nucleotide binding proteins (G proteins) are crucial in regulation of cardiovascular, endocrine and neural function. Despite the recent advances in understanding of structural basis of G protein-receptor interaction by powerful molecular biological tools, there is much less known about the dynamics of this interaction. The availability of high level expression systems will permit spectroscopic analysis of the specifically modified receptor and G protein subunits so that the rate and equilibrium constraints of individual activation steps can be defined. The combination of a newly developed flow cytometry method and high efficiency energy transfer measurements will allow me to resolve conformational changes and physical interactions within the receptor-G protein ternary complex by monitoring both processes in real time. Defining the mechanisms of receptor-coupled G protein subunit activation in a native membrane environment is our long-term objective.
The specific aims of proposed research are: 1) To develop fluorescently labeled receptor-G protein subunit pair with efficient fluorescence energy transfer (FRET); 2) To obtain kinetic parameters of G protein transformation in response to receptor activation.
