Specification of the Direction of Adhesive Signaling by the Integrin β Cytoplasmic Domain

Abstract

Integrin adhesion receptors can signal in two directions: first, they can regulate cellular behaviors by modulating cellular signaling enzymes ("outside-in signaling"); second, cells can regulate the affinity of integrins ("inside-out signaling") by such pathways. Integrin ␤ cytoplasmic domains (tails) mediate both types of signaling, and Src family kinases (SFKs) and talin, which bind to ␤ tails, are important for integrin signaling. Here, we utilized "homology scanning" mutagenesis to identify ␤ tail mutants selectively defective in c-Src binding and found that amino acid exchanges affecting a combination of an Arg and Thr residue in the integrin ␤3 tail control the binding specificity for SFKs but have no effect on talin binding. Using ␤ tail mutants at these residues, we found that SFK binding to integrin ␤ tails is dispensable for inside-out signaling but is obligatory for cell spreading, a marker of outside-in signaling. Conversely, we found that point mutations that disrupt talin binding abolish integrin activation, but they do not inhibit SFK binding to the ␤3 tail or the initiation of outside-in signaling once the integrins are in a high affinity form. Thus, we show that inside-out and outside-in integrin signaling are mediated by distinct and separable interactions of the integrin ␤ tails. Furthermore, based on our results, it is possible to discern the relative contributions of the direction of integrin signaling on biological functions in cell culture and, ultimately, in vivo.

Tyrosine Phosphorylation of the Integrin β3 Subunit Regulates β3 Cleavage by Calpain

“…This requirement is consistent with the findings that c-Src binds to the ␤ 3 C-terminal domain and that inhibition of Src family of protein kinases inhibited cell spreading (27)(28)(29). Calpain cleavage of ␤ 3 cytoplasmic domain disrupts the c-Src binding site in the C-terminal domain of ␤ 3 , which potentially explains why calpain cleavage of integrins plays important roles in detaching the rear end of a cell during migration (30), and in cell detachment during apoptosis (31).…”

Cysteine protease cathepsin X modulates immune response via activation of β2 integrins

“…36 Therefore, the b 2 subunit, through which the adhesion properties of LFA-1 and Mac-1 are regulated, may have different roles in the function of these two receptors. 8 Within both, a and b subunits, there appear to be several potential proteolytic cleavage sites that have different effects on these receptors.…”

Structure and mechanics of integrin-based cell adhesion

“…A prominent biochemical event in integrin outside-in signaling is protein tyrosine phosphorylation due to activation of Src and FAK family protein tyrosine kinases [52,64,90,91]. Src and its inhibitory Csk are constitutively bound to the integrin β-tails: Src through its SH3 domain to the β-tail c-terminus.…”

“…Integrin binding to ligand induces Src activation, through Csk dissociation, Src transphosphorylation (in clustered integrins) and/or recruitment of tyrosine phosphatases (e.g. receptor tyrosine phosphatase RPTPα, or PTP-1B [91,92]. Syk kinases, which are involved in inducing Rac-dependent lamellepodia formation, are recruited to clustered integrins by direct interaction of its SH2 domain with the integrin β-tail c-terminus and are activated by Src within seconds after integrin-fibrinogen interaction in platelets [93].…”