Functional studies of integrin subunit 1 and its cytoplasmic domain

Abstract: Integrin subunit β1 can form functional heterodimeric receptors together with at least 11different a subunits. These integrins are the main cellular receptors for extracellular matrixproteins such as collagens, fibronectin, laminins, and others, and they are necessary fornormal embryonal development. In this thesis, the effects of lack of integrin subunit β1 onseveral adhesion-related events were studied in cell culture, and effects on formation oftumors and metastases were studied in vivo. Furthermore, the consequences of mutationsof potential phosphorylation sites in the cytoplasmic domain of β1 were analysed.Fibronectin matrix deposition was studied in a cell line, which was deficient inintegrin subunit β1 and in β1-transfected subclones of this line. The classical fibronectinreceptor α5β1 strongly promoted the process of fibronectin polymerisation. Surprisingly, the non-β1 integrin αVβ3 was found to take over this function in the absence of α5β1 but in a less efficient manner.To study the role of β1 integrins in malignancy, a β1 deficient fibroblastic cell linewas transformed with the ras and myc oncogenes. A β1-expressing counterpart wasestablished by transfecting the cells with β1 cDNA, and the two cell lines were examinedfor the ability to form tumors and metastases after subcutaneous or intravenous injectioninto mice. While the formation of primary tumors was not dependent on β1 integrins, theprocess of metastasis was dramatically enhanced by the presence of β1.Several cytoplasmic point mutations of potential phosphorylation sites did notdetectably affect β1 integrin function, but two of the mutants had marked effects. First,when two consecutive threonines were replaced with alanines, the integrin ligand bindingactivity was reduced, due to altered conformation of the extracellular domain. Second,when both of the two tyrosines were simultaneously exchanged to phenylalanines, theintegrin-mediated intracellular signaling was affected. Notably, tyrosine phosphorylationof focal adhesion kinase and paxillin in response to β1-mediated adhesion was prevented,leading to impaired cell spreading and migration.