A functional comparison of mutations in integrin β cytoplasmic domains

Abstract

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 β subunit cytoplasmic domains (β tails) participate in regulating each of these steps; however, it is not known whether the same or different regions within β tails are required. We generated a panel of amino acid substitutions within the β1 and β3 cytoplasmic domains to determine whether distinct regions within β tails regulate different steps in adhesion. We expressed these β cytoplasmic domains in the context of interleukin 2 (IL-2) receptor (tac) chimeras and tested their ability to activate tyrosine phosphorylation, to regulate β1 integrin conformation and to inhibit β1 integrin function in cell attachment and spreading. We found that many of the mutant β1 and β3 chimeras either had no effect on these parameters or dramatically inhibited the function of the β tail in most assays. However, one set of analogous Ala substitutions in the β1 and β3 tails differentially affected the ability of the tac-β1 and tac-β3 chimeras to activate tyrosine phosphorylation. The tac-β1 mutant containing Ala substitutions for the VTT motif did not signal, whereas the analogous tac-β3 mutant was able to activate tyrosine phosphorylation, albeit not to wild-type levels. We also identified a few mutations that inhibited β tail function in only a subset of assays. Ala substitutions for the Val residue in the VTT motif of the β1 tail or for the conserved Asp and Glu residues in the membrane-proximal region of the β3 tail greatly diminished the ability of tac-β1 and tac-β3 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 β1 tail had dramatic effects on the ability of tac-β1 to regulate integrin conformation and function in cell spreading, but had no or intermediate effects in other assays. The identification of mutations in the β1 and β3 tails that specifically abrogated the ability of these β tails to regulate β1 integrin conformation and function in cell spreading suggests that distinct protein interactions with β tails regulate β cytoplasmic domain function in these processes.

Cas phosphorylation regulates focal adhesion assembly

“…For example, the integrin β1 tail may bind to Yes1 that is associated with Cas ( Arias-Salgado et al, 2005 ), or membrane microdomains could mediate co-clustering of Yes1 with integrins ( Lietha and Izard, 2020 ). Whatever the mechanism for cluster formation, cluster growth could then activate associated SFKs by increasing transphosphorylation ( Arias-Salgado et al, 2003 ; Berrier et al, 2002 ; Bodeau et al, 2001 ; Buensuceso et al, 2003 ). Further understanding of the precise mechanism will require additional in vivo and in vitro analysis.…”

Integrin α2β1 modulates EGF stimulation of Rho GTPase‐dependent morphological changes in adherent human rhabdomyosarcoma RD cells

“…The integrin signaling mechanism is complex, involving the cytoplasmic domains of both α and β subunits (O'Toole et al, 1994; Briesewitz et al, 1995). The cytoplasmic domain of the β subunit plays a critical role in binding cytoskeletal‐associated proteins, cell attachment, spreading, and migration (Horwitz et al, 1986; Otey et al, 1993; Sakai et al, 1998; Bodeau et al, 2001). However, studies of chimeric integrins containing different α cytoplasmic domains reveal both a quantitative as well as a qualitative difference in their signaling properties for various cellular functions (Chan et al, 1992; Kassner et al, 1995; Ivaska et al, 1999; Sastry et al, 1999; Liu et al, 2000; Wang et al, 2000).…”