Structural and functional characterization of the focal adhesion protein FAP52
|Organizations:||University of Oulu, Faculty of Medicine, Department of Pathology
University of Helsinki, Department of Pathology
|Online Access:||PDF Full Text (PDF, 1.1 MB)|
|Persistent link:|| http://urn.fi/urn:isbn:9514274555
|Publish Date:|| 2004-12-01
|Thesis type:||Doctoral Dissertation
|Defence Note:||Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium 101 A of the Faculty of Medicine (Aapistie 5 A), on December 11th, 2004, at 12 noon.
Docent Pekka Lappalainen
Docent Jari Ylänne
FAP52 (focal adhesion protein, 52 kDa) is a focal adhesion-associated protein composed of a highly α-helical NH2-terminus containing a poorly characterized FCH (Fes/CIP4 homology) domain, unstructured linker region and the COOH-terminal SH3 domain. FAP52 is also known as PACSIN 2 or syndapin II. Together with other PACSINs and syndapins FAP52 shares a common domain architecture.
The aim of this study was to characterize FAP52 in structural and functional terms. The function was pursued by identifying binding partners for FAP52, and by overexpressing the recombinant FAP52 in cultured cells. For the structural studies, various physico-chemical methods, such as chemical cross-linking, gel filtration chromatography, circular dichroism and X-ray crystallography were applied. In addition, the histological distribution of FAP52 in chicken tissues was explored.
FAP52 binds filamin, a protein that regulates the dynamics of the cytoskeleton by crosslinking actin filaments. The binding site in FAP52 was mapped to the NH2-terminal 184 amino acids, of which the residues 146–184 form the core of the binding. In filamin, the binding site resides in the repeats 15–16 in the rod-like molecule encompassing 24 such repetitive domains. Overexpression of FAP52 or its filamin-binding domain in chicken embryo heart fibroblasts induced the formation of filopodial extensions on the cell surface and reduced the number of focal adhesions, suggesting a role in the organization of the cellular cytoskeleton and in cell adhesion machinery.
Experiments utilizing surface plasmon resonance analysis, size exclusion chromatography and chemical cross-linking showed that FAP52 self-associates in vitro and in vivo. The region responsible for the self-association was mapped to the amino acids 146–280, which is predicted to fold into a coiled-coil arrangement.
FAP52 was crystallized by using the hanging-drop vapor-diffusion method and ammonium sulfate grid screen. Native dataset was collected from two crystals, which diffracted to 2.8 Å and 2.1 Å resolution. For one form of crystals, phasing was performed using the native dataset and the datasets from two xenon-derivatized crystals. X-ray crystallography studies revealed a dimer in asymmetric unit.
Histological and in vitro studies showed that, in liver, FAP52 is preferentially expressed in bile canaliculi. In other tissues, FAP52 showed a specific staining pattern in gut, kidney, brain and gizzard.
Together, these data show that FAP52 self-associates in vivo and, probably via its interaction with its binding partner filamin, participates in the organization of the cytoskeletal architecture, especially of the cell surface protrusions, such as filopodia and microvilli of bile canaliculi.
Acta Universitatis Ouluensis. D, Medica
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