CHARACTERIZATION OF LIPID-PROTEIN BOUNDARY IN GPCR MODEL

Maryville College, Maryville TN

Investigators

Abstract

Membrane receptors for peptide hormones are found embedded in the outer membrane of cells where they transmit signals to proteins within the cell. Many of these receptors belong to a ubiquitous family of proteins which are important control points for many cellular events. These proteins are altered in many disease states and are believed to be one of the most important drug targets for medical therapy. We propose to initiate studies involving undergraduate student research to characterize the interactions between a model receptor and membrane lipids which we believe are modulators of receptor activity. The model receptor to be used in these studies is the well characterized yeast pheromone receptor, a protein with seven membrane-spanning domains. In preliminary studies with this system utilizing either lipid-modified cells or purified and reconstituted protein we have observed lipid-sensitive pheromone binding and receptor activation. Based on these findings and those reported for many other membrane receptors, we believe that this receptor interacts with specific lipids which can influence its structure and activity. Our approach will be to change the local environment surrounding this model protein and look for specific membrane components which may help regulate its activity. Initially, the membrane lipid composition of yeast cells will be altered by media supplementation, pharmacological agents, and through the use of mutants with defects in lipid metabolism. We will monitor receptor activity in cells and cellular membranes by following the binding and release of the hormone as well as downstream effects of the transduction pathway including coupling with internal proteins, gene expression and growth arrest. To examine the interactions under more defined conditions we will purify the protein by recently developed techniques and then return it to lipids of specified composition. Knowledge of the influence of the bilayer matrix on the structure and activity of this well characterized receptor will foster our understanding of how this important group of membrane proteins mediate signal transduction in health and disease. This is a multifaceted and multidisciplinary approach where we can address an important biological problem while training undergraduate students to develop the much needed process skills of science.