Distinct functions of integrin alpha and beta subunit cytoplasmic domains in cell spreading and formation of focal adhesions

Abstract

Abstract. Integrin-mediated cell adhesion often results in cell spreading and the formation of focal adhesions. We exploited the capacity of recombinant human o~/~3 integrin to endow heterologous cells with the ability to adhere and spread on fibrinogen to study the role of integrin cytoplasmic domains in initiation of cell spreading and focal adhesions. The same constructs were also used to analyze the role of the cytoplasmic domains in maintenance of the fidelity of the integrin repertoire at focal adhesions. Truncation mutants of the cytoplasmic domain of oe~ did not interfere with the ability of Ottr~3 to initiate cell spreading and form focal adhesions. Nevertheless, deletion of the tXnb cytoplasmic domain allowed indiscriminate recruitment of O~m~3 to focal adhesions formed by other integrins. Truncation of the/33 subunit cytoplasmic domain abolished cell spreading mediated by o~m/~3 and also abrogated recruitment of c~m,/~3 to focal adhesions. This truncation also dramatically impaired the ability of O~nb/~3 to mediate the contraction of fibrin gels. In contrast, the/33 subunit cytoplasmic truncation did not reduce the fibrinogen binding affinity of t~m~3. Thus, the integrin/~3 subunit cytoplasmic domain is necessary and sufficient for initiation of cell spreading and focal adhesion formation. Further, the/33 cytoplasmic domain is required for the transmission of intraceUular contractile forces to fibrin gels. The o~ subunit cytoplasmic domain maintains the fidelity of recruitment of the integrins to focal adhesions and thus regulates their repertoire of integrins. C ELL adhesion is controlled by binding affinity and kinetics of interaction between adhesive ligands and cell surface receptors. In addition, events such as lateral diffusion of receptors (7) and interactions with and reorganization of the cytoskeleton strengthen adhesion (16,29). Cell adhesion induces changes of cell shape and cytoskeietai organization and regulates cell growth and patterns of gene expression (24,48,53). Integrins are transmembrane heterodimeric glycoprotein adhesion receptors present in almost all ceils (23). These receptors mediate all of the consequences of cell adhesion enumerated above. Moreover, after initial cell adhesion, many integrins are concentrated at sites of close approximation between cell and substrate. These sites, termed focal adhesions or focal contacts (4), are also sites of end-on insertion of actin microfilaments into the plasma membrane and at which certain cytoskeletal and signaling molecules concentrate (53). Ligand binding to many integrins triggers focal adhesion formarion. In addition to initiating focal adhesions, certain mutant integrins (58) or receptor chimeras containing the El cytoplasmic domain (27, 49) are recruited to existing focal adhesions without evident ligand binding. Thus, analysis of the role of integrins in formation of focal adhesions may be divided into initiation and recruitment functions. Integrin cytoplasmic domains are topographically accessible to intracellular cytoplasmic...

β1-Integrin Cytoplasmic Subdomains Involved in Dominant Negative Function

“…contains a default signal for interaction with cytoskeletal molecules that is masked by the ␣ subunit cytoplasmic domain (Briesewitz et al, 1993;Ylanne et al, 1993). In response to matrix ligands or to multivalent antibody binding, the inhibitory effect of the ␣ subunit can be released, and integrins can interact with cytoskeletal and signaling molecules (Miyamoto et al, 1995a,b).…”

“…In vivo, the ␤1 cytoplasmic domain is sufficient for its localization to preformed focal adhesions (LaFlamme et al, 1992, Akiyama et al, 1994 and for the initiation of signaling to FAK (Lukashev et al, 1994). A model has been proposed in which the ␤1 subunit cytoplasmic domain ‡ Corresponding author.© 1998 by The American Society for Cell Biology 715contains a default signal for interaction with cytoskeletal molecules that is masked by the ␣ subunit cytoplasmic domain (Briesewitz et al, 1993;Ylanne et al, 1993). In response to matrix ligands or to multivalent antibody binding, the inhibitory effect of the ␣ subunit can be released, and integrins can interact with cytoskeletal and signaling molecules (Miyamoto et al, 1995a,b).…”

Membrane Targeting and Coupling of NHE1-IntegrinαIIbβ3-NCX1 by Lipid Rafts following Integrin-Ligand Interactions Trigger Ca2+ Oscillations

“…Calcium oscillations could also be induced in human platelets by plating the cells on a substrate coated with Fg or rhodostomin (rho), a disintegrin protein isolated from snake venom (10,11). Like platelets, Chinese hamster ovary cells expressing exogenous integrin ␣ IIb ␤ 3 on their plasma membrane (CHO ␣ IIb ␤ 3 ) also exhibit active calcium oscillations when plated on substrates coated with Fg or rho (12). Using CHO ␣ IIb ␤ 3 cells as an experimental model, we report here that calcium oscillations downstream of integrin ␣ IIb ␤ 3 signaling could be readily triggered by the combined function of two ionic exchangers, the sodiumproton exchanger NHE1 and the sodium-calcium exchanger NCX1, which are actively recruited from intracellular vesicles to plasma membranes and form molecular complexes with integrin ␣ IIb ␤ 3 by a lipid microdomain-or lipid raft-dependent mechanism.…”

During human thymic development, β1 integrins regulate adhesion, motility, and the outcome of RHAMM/hyaluronan engagement

“…ECM proteins have been implicated in thymic differentiation [24][25][26][27][28][29][30] and thymic stromal cells express ␤ 1 integrins [31] as well as provide an ECM substrate [27,31,32] for cell adhesion and migration [11,12,30,[33][34][35][36]. Pro-T cells utilize the integrin ␣ 6 ␤ 1 [37,38] and the fibronectin receptor ␣ 5 ␤ 1 [30] in colonization of the thymic rudiment, a process involving migratory behavior.…”

“…However, during the earliest developmental stages of the immune system, progenitor lymphocytes are likely to require anchoring within a nurturing microenvironment of ECM, growth factors, and stromal cells. Although ␤ 1 integrins have been widely characterized as receptors mediating stable and strong adhesion [6][7][8][9][10][11][12][13], they also participate in weakly adhesive motile behavior [14][15][16][17][18]. Human thymocytes express ␤ 1 integrins [2,[19][20][21], receptors known to bind to ECM components fibronectin, laminin, and collagen [5,22,23], and a substantial proportion of post-natal thymocytes spontaneously bind to fibronectin, utilizing ␣ 4 ␤ 1 [20].…”