Distinct Functions of Integrin ct and/3 Subunit Cytoplasmic Domains in Cell Spreading and Formation of Focal Adhesions

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. J. Yl~nne's present address is Department of Biochemistry, University of Helsinki, Helsinki, Finland. This is publication Y'/148-CVB from the Scripps Research Institute. Integrin cytoplasmic domains are topographically accessible to intracellular cytoplasmic components. Moreover, the B1 cytoplasmic domain binds to focal adhesion proteins such as talin (22) and o~-actinin (41). The/31 cytoplasmic domain is required for localization of recombinant as~ to existing focal adhesions (19, 31, 56), but the presence of endogenous /~ precluded analysis of initiation in the published work. Preliminary studies reported that the or5 subunit cytoplasmic domain is not absolutely required for focal adhesion formation (Juliano, R. L., J. S. Bauer, L. J. Kornberg, and J. Varner. 1992. MoL BioL Cell. 3[Suppl]:94a). Integrin Otm~3 (platelet GPIIb--lIIa) is a prototype integrin that mediates cell-cell and cell-substratum interaction (43, 44), signals cell spreading (61) and initiates and enters focal adhesions (64, 65). Moreover this integrin is required for the retraction of fibrin clots (5, 37), wherein intraeeilular contractile forces are transmitted to extraceilular fibrin polymers. tXir~3 offers several advantages in the studies of integrin domains which function in cell adhesion. (a) Certain ligands such as fibrinogen (fg)~ and von Wdlebrand factor promote adhesion via am~3, but not through the majority of other integrins. Thus, in many cell lines, events mediated by transfected otn~3, such as cell adhesion, spreading, and focal adhesion formation, can be analyzed with minimal confounding effects of endogenous integrins. (b) Well character1. Abbre~ation used in this paper: fg, fibrinogen. © The Rockefeller University Press, 0021-9525/93/07/223/11 $2.00 The Journal of Cell Biology, Volume 122, Number 1, July 1993 223-233 223 ized quantitative ligand binding assays for Otm~3 are available (32), again without significant contributions from endogenous integrins. (c) An extensive library of otm~3 rnAbs exists. Antibodies specific for occupied (13,14) or activated (54) conformations of CXabB3 are available and they permit precise in situ analysis of receptor function. Other antibodies which inhibit or enhance (13, 14) receptor function can be used for otr.B3 ligand binding studies. In the present work, we examined the effects of truncations of Otm~3 cytoplasmic domains on initiation of cell spreading and focal adhesion formation. We found that the B subunit cytoplasmic domain is necessary and sufficient for these cytoskeleton-related processes and for recruitment of integrins to existing focal adhesions. Further, the ~3 cytoplasmic domain was also required for the contraction of fibrin clots, i.e., for the transmission of contractile events to a model extracellular matrix. In contrast, the ot subunit cytoplasmic domain, while not required for initiation, regulates the integrin specificity of recruitment to focal adhesions. Materials and Methods