The applicant proposes a program of research to prepare him for a career in the laboratory investigation of diseases of the kidney. The research will be conducted in the laboratory of Dr. Dennis A. Ausiello at the Massachusetts General Hospital. The applicant will study the intracellular mechanisms by which an epithelial cell maintains its polarity. This topic has broad implications for the pathophysiology of many acute and chronic renal failure syndromes including acute tubular necrosis, obstructive nephropathy, and polycystic kidney disease as well as vasopressin regulation of renal water reabsorption during diabetes insipidus and the syndrome of inappropriate secretion of antidiuretic hormone. How a cell regulates and directs the intracellular trafficking of its proteins to become a differentiated polarized epithelial cell has implications not only for cells of the kidney but for all cells in the human body. In particular, the applicant will study the structure and function of a recently discovered class of intracellular signalling proteins, the RGS (Regulators of G-protein Signaling) proteins, and their role in the regulation of the secretory pathways of intracellular protein trafficking. This application will present preliminary data showing a novel direct interaction between RGS4 and RGS-GAIP with the COPI coatomer protein beta'-COP. In addition, when RGS4 and RGS-GAIP are overexpressed in the kidney epithelial cell line LLC-PK1, a functional disruption of the constituitive secretory pathway occurs resulting in redistribution of beta'-COP as well as redistribution of the membrane protein aquaporin 2, without gross disruption of the Golgi apparatus. The applicant proposes to build on these observations and to pursue several lines of investigation to further explore and clarify the exact molecular mechanisms by which the RGS proteins, heterotrimeric G-proteins and the coatomer proteins interact to control the intracellular protein trafficking of a polarized cell.
The Specific Aims of this project will include 1) generation of mutants of RGS4, RGS- GAIP, and beta'-COP to identify the regions of the molecules responsible for interaction with each other; 2) assay of these mutants in epithelial cells for their ability to regulate protein trafficking; 3) crystallize RGS4 and GAIP with beta'-COP; 4) identify novel interactions between RGS proteins and other proteins in the secretory pathway using biochemical, genetic, and molecular biological techniques. These studies will have broad implications to scientists working in the areas of both signal transduction and protein trafficking. Understanding the molecular details of the constituitive and regulated secretory pathways may provide novel therapeutic approaches for modulating cellular responses in renal and other diseases.
