Integrins are cell-surface receptors that mediate and coordinate cellular responses to the extracellular matrix (ECM). Cellular signalling pathways can regulate cell adhesion by altering the affinity and avidity of integrins for ECM. The Ras family of small G proteins, which includes H-ras, R-ras and Rap, are important elements in cellular signalling pathways that control integrin function.

Abstract

Control of integrin affinity for ligands (integrin activation) is essential for normal cell adhesion, migration, and assembly of an extracellular matrix. Integrin activation is usually mediated through the integrin β subunit cytoplasmic tail and can be regulated by many different biochemical signaling pathways. We report that specific binding of the cytoskeletal protein talin to integrin β subunit cytoplasmic tails leads to the conformational rearrangements of integrin extracellular domains that increase their affinity. Thus, regulated binding of talin to integrin β tails is a final common element of cellular signaling cascades that control integrin activation.

The α4 integrins are indispensable for embryogenesis, haematopoiesis and immune responses1, 2, possibly because α4 regulates cellular functions differently from other integrins through its cytoplasmic tail3. We used novel mimics4 of the α4 tail to identify molecules that could account for α4-specific signalling. Here we report that the α4 tail, but not several other α-subunit tails, binds tightly to the signalling adaptor paxillin. Paxillin physically associated with α4 integrins in Jurkat T cells at high stoichiometry, and joining the α4 tail to αIIb resulted in a complex of integrin αIIbβ3 with paxillin. This association markedly enhanced the rates of αIIbβ3-dependent phosphorylation of focal adhesion kinase and cell migration. It also reduced cell spreading, focal adhesion and stress fibre formation. A point mutation within the α4 tail that disrupts paxillin binding reversed all of these effects. Furthermore, α4β1-dependent adhesion to VCAM-1 led to spreading of mouse embryonic fibroblasts derived from paxillin-null but not from wild-type mice. Thus, the tight association of paxillin with the α4 tail leads to distinct biochemical and biological responses to integrin-mediated cell adhesion.

The integrin family of adhesion receptors are involved in cell growth, migration and tumour metastasis1. Integrins are heterodimeric proteins composed of an and a subunit, each with a large extracellular, a single transmembrane, and a short cytoplasmic domain. The dynamic regulation of integrin affinity for ligands in response to cellular signals is central to integrin function2. This process is energy dependent and is mediated through integrin cytoplasmic domains3. However, the cellular machinery regulating integrin affinity remains poorly understood. Here we describe a genetic strategy to disentangle integrin signalling pathways. Dominant suppression occurs when overexpression of isolated integrin 1 cytoplasmic domains blocks integrin activation. Proteins involved in integrin signalling were identified by their capacity to complement dominant suppression in an expression cloning scheme. CD98, an early T-cell activation antigen that associates with functional integrins4, was found to regulate integrin activation. Furthermore, antibody-mediated crosslinking of CD98 stimulated 1 integrin-dependent cell adhesion. These data indicate that CD98 is involved in regulating integrin affinity, and validate an unbiased genetic approach to analysing integrin signalling pathways.