Membrane binding, a process initiated and sustained by lipid modifications, controls the function of Ras proteins as potently as does GTP binding. Despite this importance, very little is known of how membrane tethering affects signaling. Recent studies indicate that HRas trafficking to the plasma membrane proceeds through a previously unnoticed, and still uncharacterized, vesicular endomembrane pathway that requires farnesyl modification and nearby C-terminal amino acid residues. No specific proteins that might escort HRas from these internal membranes to its working location, the plasma membrane, have been identified. Upon reaching the plasma membrane, HRas is reported to localize into cholesterol-and sphingolipid-rich subdomains ("rafts "). In vitro methods predict that the different physical properties of the farnesyl and palmitoyl lipids will give these lipids opposing roles in regulating HRas raft insertion, but studies with HRas in intact cells have not yielded clear results. Raft partitioning is proposed to increase signaling efficiency, but there is conflicting information on whether partitioning limits or encourages access of particular regulatory or effector proteins to HRas while it is associated with the membrane. Novel variants of HRas have been made, and show that changes in the lipids and residues of the C-terminus can alter HRas signaling pathways in intact cells. This new evidence indicates that the C-terminal region and lipids attached to HRas are more than a passive anchor domain, and are likely to play much more active roles during trafficking and organizing of HRas in the plasma membrane than current models envision. The objective of this project is to examine how the C-terminal amino acids, farnesyl group, and palmitates of HRas individually participate in both the trafficking and plasma membrane interaction phases of HRas membrane binding . Three aims will be pursued. A. Variants of HRas will be designed to characterize features of HRas that control its exit out of endomembranes, and to identify the vesicular paths and proteins involved in moving HRas to the cell surface. B. The influence of the farnesyl group, palmitates and GTP binding on partitioning of HRas into subdomain sites of plasma membrane will be established by expressing proteins with distinct and novel combinations of these properties. C. The membrane locations in which activation and termination of HRas signaling occurs will be identified by co-localization or co-immunoprecipitation of HRas with regulatory proteins and effectors. These studies will use biochemical co-immunoprecipitation and membrane isolation techniques, as well as direct immunofluorescence imaging of membrane fragments. The primary emphasis will be intact cells, as this is the only currently available system where HRas palmitoylation can occur, and more importantly, enables the functional impact of changes in molecular properties to be gauged by monitoring the ability of variant proteins to cause focus formation in NIH3T3 cells. The results will generate a clearer view of how HRas becomes incorporated into cellular membranes and enable development of methods to manipulate the interaction of HRas with membrane subdomains. This information will also identify new subdomain-selective targeting sequences with which other proteins, such as HRas effectors, can be re-directed to specific sites on the cell surface. These studies will help us understand what properties of the membrane-binding domain of HRas can influence biological activity and lead towards the long-term goal of learning how to guide that interaction toward beneficial outcomes.

Agency
National Science Foundation (NSF)
Institute
Division of Molecular and Cellular Biosciences (MCB)
Application #
0110114
Program Officer
Eve Ida Barak
Project Start
Project End
Budget Start
2001-09-01
Budget End
2005-08-31
Support Year
Fiscal Year
2001
Total Cost
$396,399
Indirect Cost
Name
Iowa State University
Department
Type
DUNS #
City
Ames
State
IA
Country
United States
Zip Code
50011