David D. Chang
Integrin Mediated Cell - Matrix Interaction
Integrins are heterodimeric cell surface receptors for extracellular matrix proteins. The attachment of cells to extracellular matrix is a dynamic process requiring affinity up-regulation of integrins and cytoskeletal restructuring. My laboratory is interested in studying the regulation of integrin mediated cell adhesion and the mechanism of cell activation that follows the cell adhesion.
One approach we have undertaken is to identify and characterize the proteins that bind to the cytoplasmic domain of integrins. Using a yeast two hybrid technique, we have identified two proteins interacting with the cytoplasmic domain of integrin beta chain. The first protein which belongs to the Gbeta protein family binds to the membrane proximal portion with a presumed alpha-helical structure conserved in all integrin beta chains. The interaction of this protein with integrins requires a phorbol ester stimulation, a treatment known to cause integrin affinity up-regulation. The second protein is a novel protein of 200 amino acids that binds to a conserved and functionally important NPXY sequence motif found in the C-terminus of beta1 integrin. This protein undergoes phosphorylation that is subject to a regulation during integrin dependent cell adhesion and cell spreading. The ongoing work is focused on characterizing the functions of these two proteins in the cell-matrix interaction.
The interaction between integrins on cell surface and the extracellular matrix proteins are stabilized by cytoskeletal actin-stress fibers. Actin-stress fiber formation is regulated by rho-family GTPases (cdc42H/rac1/rhoA) which control the intracellular concentration of phosphatidylinositol(4,5) bisphosphate (PIP2) and the phosphorylation state of myosin light chain (MLC). In an attempt to define the role of cytoskeletal structures on cell adhesion and cell growth, we have generated cell lines expressing various RhoA mutants under the control of an inducible promoter. In addition, we have engineered the expression of myosin phosphatase, a downstream target of RhoA involved in dephosphorylation of MLC, in some of these cell lines. The availability of these cell lines will allow us to characterize the role cytoskeletal actin-stress fibers on cell adhesion and growth.