Date of Award

5-2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Molecular Sciences

Research Advisor

Jie Zheng, Ph.D.

Committee

Stephan W. Morris, M.D. David R. Nelson, Ph.D. Susan E. Senogles, Ph.D. Stephen W. White, Ph.D.

Keywords

NMR, structure, GIT, FAK, paxillin, interaction, focal adhesion, signal, regulation

Abstract

The G protein coupled receptor (GPCR)-kinase (GRK) interacting protein 1 (GIT1) is a multidomain protein that plays an important role in cell adhesion, motility, cytoskeletal remodeling, and membrane trafficking. GIT1 mediates the localization of p21-activated kinase (PAK) and PAK-interactive exchange factor (PIX) to focal adhesions, and its activation is regulated by the interaction between its C terminal paxillin-binding domain (PBD) and the LD motifs of paxillin.

In this dissertation, we determined the solution structure of rat GIT1 PBD by nuclear magnetic resonance (NMR) spectroscopy. The PBD folds into a four-helix bundle, which is structurally similar to the focal adhesion targeting (FAT) domain and the vinculin tail (Vt) domain. The PBD is more stable than the FAT domain and there is no evidence of helix 1 swapping.

Previous studies showed that GIT1 interacts with paxillin through the LD4 motif. However, studies in this dissertation demonstrated that in addition to the LD4 motif, the GIT1 PBD can also bind to the paxillin LD2 motif; and both LD2 and LD4 motifs competitively target the same site on the PBD surface. This dissertation also probed the function of paxillin splice variants by comparing their interaction with GIT1 PBD. It seems the paxillin isoforms did not play an important role in determining the affinity to GIT1. We also revealed that paxillin S272 phosphorylation does not influence GIT1 PBD binding in vitro. These results are in agreement with the notion that phosphorylation of paxillin S272 plays an essential role in regulating focal adhesion turnover.

This dissertation also computationally derived the complex structures of GIT1 PBD bound with either LD2 peptide or LD4 peptide, based on the experimental binding site information. The LD2 and LD4 peptides bound to GIT1 PBD in a manner similar to the crystal structure of FAT-LD2 complex. The complex structures visualized the reason why both LD2 and LD4 can bind to the same GIT1 binding site. It also addressed the specificity problem in determining paxillin binding to GIT1 versus FAK.

Our finding reconciles the controversial observations of earlier studies and provides a clearer picture of focal adhesion regulation. The structural studies of GIT1 PBD presented in this dissertation shed more light on the understanding of GIT functions. The novel findings also allow us to propose a working model regarding FA disassembly.

DOI

10.21007/etd.cghs.2008.0379

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