A functional comparison of mutations in integrin beta cytoplasmic domains:effects on the regulation of tyrosine phosphorylation, cell spreading, cell attachment and beta 1 integrin conformation

Cell adhesion is a multistep process that requires the interaction of integ rins with their ligands in cell attachment, the activation of integrin-trig gered signals, and cell spreading. Integrin beta subunit cytoplasmic domain s (beta tails) participate in regulating each of these steps; however, it i s not known whether the same or different regions within beta tails are req uired. We generated a panel of amino acid substitutions within the beta1 an d beta3 cytoplasmic domains to determine whether distinct regions within be ta tails regulate different steps in adhesion. We expressed these beta cyto plasmic domains in the context of interleukin 2 (IL-2) receptor (tac) chime ras and tested their ability to activate tyrosine phosphorylation, to regul ate beta1 integrin conformation and to inhibit PI integrin function in cell attachment and spreading. We found that many of the mutant beta1 and beta3 chimeras either had no effect on these parameters or dramatically inhibite d the function of the beta tail in most assays. However, one set of analogo us Ala substitutions in the beta1 and beta3 tails differentially affected t he ability of the tac-beta1 and tac-0 beta3 chimeras to activate tyrosine p hosphorylation. The tac-beta1 mutant containing Ala substitutions for the V TT motif did not signal, whereas the analogous tac-beta3 mutant was able to activate tyrosine phosphorylation, albeit not to wild-type levels. We also identified a few mutations that inhibited beta tail function in only a sub set of assays. Ala substitutions for the Val residue in the VTT motif of th e beta1 tail or for the conserved Asp and Glu residues in the membrane-prox imal region of the beta3 tail greatly diminished the ability of tac-beta1 a nd tac-beta3 to inhibit cell spreading, but had minimal effects in other as says. Ala substitutions for the Trp and Asp residues in the conserved WDT m otif in the beta1 tail had dramatic effects on the ability of tac-beta1 to regulate integrin conformation and function in cell spreading, but had no o r intermediate effects in other assays. The identification of mutations in the beta1 and beta3 tails that specifically abrogated the ability of these beta tails to regulate beta1 integrin conformation and function in cell spr eading suggests that distinct protein interactions with beta tails regulate beta cytoplasmic domain function in these processes.