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RESEARCH ARTICLE |
The Center for Cell Biology and Cancer Research, Albany Medical College,
Albany, NY 12208, USA
*
Author for correspondence (e-mail:
laflams{at}mail.amc.edu
)
Accepted April 23, 2001
Cell adhesion is a multistep process that requires the interaction of integrins with their ligands in cell attachment, the activation of integrin-triggered signals, and cell spreading. Integrin ß subunit cytoplasmic domains (ß tails) participate in regulating each of these steps; however, it is not known whether the same or different regions within ß tails are required. We generated a panel of amino acid substitutions within the ß1 and ß3 cytoplasmic domains to determine whether distinct regions within ß tails regulate different steps in adhesion. We expressed these ß cytoplasmic domains in the context of interleukin 2 (IL-2) receptor (tac) chimeras and tested their ability to activate tyrosine phosphorylation, to regulate ß1 integrin conformation and to inhibit ß1 integrin function in cell attachment and spreading. We found that many of the mutant ß1 and ß3 chimeras either had no effect on these parameters or dramatically inhibited the function of the ß tail in most assays. However, one set of analogous Ala substitutions in the ß1 and ß3 tails differentially affected the ability of the tac-ß1 and tac-ß3 chimeras to activate tyrosine phosphorylation. The tac-ß1 mutant containing Ala substitutions for the VTT motif did not signal, whereas the analogous tac-ß3 mutant was able to activate tyrosine phosphorylation, albeit not to wild-type levels. We also identified a few mutations that inhibited ß tail function in only a subset of assays. Ala substitutions for the Val residue in the VTT motif of the ß1 tail or for the conserved Asp and Glu residues in the membrane-proximal region of the ß3 tail greatly diminished the ability of tac-ß1 and tac-ß3 to inhibit cell spreading, but had minimal effects in other assays. Ala substitutions for the Trp and Asp residues in the conserved WDT motif in the ß1 tail had dramatic effects on the ability of tac-ß1 to regulate integrin conformation and function in cell spreading, but had no or intermediate effects in other assays. The identification of mutations in the ß1 and ß3 tails that specifically abrogated the ability of these ß tails to regulate ß1 integrin conformation and function in cell spreading suggests that distinct protein interactions with ß tails regulate ß cytoplasmic domain function in these processes.
Key words: Integrin ß cytoplasmic domains, Tyrosine phosphorylation, Cell attachment, Cell spreading
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