Identification of a Domain on the Alpha 5 Integrin Subunit Implicated in Cell Spreading and Signaling
Cao, Zuojun
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Permalink
https://hdl.handle.net/2142/84907
Description
Title
Identification of a Domain on the Alpha 5 Integrin Subunit Implicated in Cell Spreading and Signaling
Author(s)
Cao, Zuojun
Issue Date
1999
Doctoral Committee Chair(s)
Horwitz, Alan F.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Language
eng
Abstract
The alpha5beta1 integrin is a cell surface receptor for fibronectin. The primary sites at which the alpha5beta1 integrin interacts with fibronectin (Fn) are the RGD (Arg-Gly-Asp) amino acid sequence and PHSRN (Phe-His-Ser-Arg-Asn) synergy sequence. Based on previous crosslinking studies, sequence alignment, predicted conformation, and intron-exon boundaries, we identified a 144-residue region (223--367) on the alpha5 subunit as a putative binding region and divided it into four subdomains named domains I, II, III and IV. Chimeric receptors were prepared in which sequences on the alpha5 subunit were exchanged with the corresponding sequences on the alpha6 subunit, which is specific for laminin and does not bind via an RGD sequence. The mutated human alpha5 integrin gene was transfected into CHO B2 cells. Only chimeras of domains III or IV expressed on the cell surface. Both of these chimeras decreased the adhesion, spreading, focal contact assembly and migration on Fn. However, the mutants were recognized by a library of 4 adhesion and non adhesion perturbing MAbs suggesting that the phenotypic changes were not due to a major conformational change. The fibronectin RGD and PHSRN recognition remained almost intact. Finally, the affinity of soluble fibronectin to cells via the alpha5beta1 receptor decreased by only about 3 fold. This decrease is substantially less than the observed effects on migration and spreading, which were not altered by changes in substrate concentration. Thus, the alteration in binding sites does not easily account for the changes in cell spreading and focal adhesion assembly. The tyrosine phosphorylation and focal adhesion assembly that are seen when cells expressing the wild type alpha5 receptor adhere to fibronectin were inhibited in cells expressing the chimeric receptors. Therefore, while we cannot exclude the possibility that domains III and IV may participate indirectly in ligand binding, our results suggest that the chimeras of these domains likely interrupt alpha5-mediated conformational signaling.
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