Adhesion of cells to extracellular matrices is a fundamental characteristic of allmulticellular organisms. Adhesion provides not only structural links between theintracellular cytoskeleton and the extracellular environment, but also provides sites ofsignal transduction that affect many aspects of cell behavior. This grant is aimed atunderstanding how signals from cell-matrix adhesions regulate members of the Rhofamily of GTPases, which are themselves key regulators of the cytoskeleton and manyintracellular processes. The goal of the first aim is to determine how adhesion to theextracellular matrix protein fibronectin stimulates RhoA. Having recently identifiedguanine nucleotide exchange factors, such as Lsc, that are involved in this process, weaim to determine the signaling pathways that lead to their activation. Our finding thatadhesion stimulates Lsc tyrosine phosphorylation and promotes Lsc binding to vinculinsuggests possible mechanisms of regulation and will be investigated further. We will alsoexamine how mechanical tension and substrate rigidity/pliability regulate RhoA activity.Here we have found that some but not all of the same exchange factors are involved.We will investigate the signaling pathways downstream from mechanical tension thatlead to exchange factor activation. In the second aim, we will follow up the discovery thatreactive oxygen species can activate Rho proteins by transient oxidation of cysteinesulfhydryls without involvement of exchange factors. Using redox-resistant mutants ofRho GTPases, we will determine the role of Rho protein activation by cysteine oxidationin events such as adhesion and in response to growth factor stimulation. In the finalaim, we will focus on the Rho GTPase sequestering protein RhoGDI, which we haverecently found contributes to crosstalk between different Rho family members.Competitive interactions of Rho proteins for binding to RhoGDI will be explored. We willinvestigate how Rho proteins are released from RhoGDI in response to adhesion anddetermine the pathways that lead to their degradation following displacement fromRhoGDI. Finally, we will explore the role of RhoGDI in mediating the trafficking of Rhoproteins from their sites of postranslational modification to their sites of action at theplasma membrane.

Public Health Relevance

The adhesive interactions of cells determine whether they will grow, die, migrate or stay in their current location. Our work aims to understand the signals that cells receive from their adhesions and how these signals regulate the Rho family of proteins that affect cell behavior. Deciphering adhesion-mediated signaling should increase our understanding of the role that adhesion plays in various diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029860-30
Application #
8143348
Study Section
Intercellular Interactions (ICI)
Program Officer
Gindhart, Joseph G
Project Start
1981-04-01
Project End
2014-08-31
Budget Start
2011-09-01
Budget End
2012-08-31
Support Year
30
Fiscal Year
2011
Total Cost
$423,592
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Physiology
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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Thompson, Peter M; Ramachandran, Srinivas; Case, Lindsay B et al. (2017) A Structural Model for Vinculin Insertion into PIP2-Containing Membranes and the Effect of Insertion on Vinculin Activation and Localization. Structure 25:264-275
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Scott, David W; Tolbert, Caitlin E; Graham, David M et al. (2015) N-glycosylation controls the function of junctional adhesion molecule-A. Mol Biol Cell 26:3205-14
Guilluy, Christophe; Burridge, Keith (2015) Nuclear mechanotransduction: forcing the nucleus to respond. Nucleus 6:19-22

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