Regulation of Fibronectin Receptor Distribution

To determine the role of each intracellular domain of the fibronectin receptor in receptor distribution, chimeric receptors were constructed containing the human interleukin-2 receptor (gp55 subunit) as the extracellular and transmembrane domains, in combination with either the a 5 or ß, intracellular domain of the fibronectin receptor as the cytoplasmic domain . These chimeric receptors were transiently expressed in normal fibroblasts, and their localization on the cell surface was determined by immunofluorescence using antibodies to the human interleukin-2 receptor. The a5 chimera was expressed diffusely on the plasma membrane . The 0 1 chimera, however, colocalized with the endogenous fibronectin receptor at focal contacts of cells spread on fibronectin . On cells spread in the presence of serum, the 01 chimera colocalized both with the fibronectin receptor at sites of extracellular fibronectin fibrils and with the vitronectin receptor at focal contacts . The 0 1 intracellular domain alone, therefore, contains sufficient information to target the chimeric receptor to regions of the cell where ligandoccupied integrin receptors are concentrated . The findTHE integrins, a family of transmembrane heterodimeric receptors consisting of a and ß subunits, play a central role in cell adhesion and migration . These processes are important in development, wound healing, metastasis, and other biological events . Integrins function in both cell-cell and cell-substratum adhesion . Integrin heterodimers can be classified into subfamilies based on the different ß subunits . Different combinations of a and ß subunits give rise to receptors with different ligand specificities, including receptors for fibronectin, vitronectin, collagen, and laminin. Although some integrins are cell-type specific, most function in many cell types, and most cell types express a variety ofintegrin receptors, allowing them to interact with many extracellular matrix components (recent reviews include Akiyama et al ., 1990 ; Albelda and Buck, 1990 ; Hemler, 1990 ; Hynes, 1990 ; Mosher, 1989; Ruoslahti, 1991) . Integrin receptors involved in cell-substratum adhesion are generally believed to function as transmembrane links between the extracellular matrix and thecytoskeleton . When cells form stable adhesions, integrin receptors concentrate in adhesion sites termed focal contacts . Via their intracellu© The Rockefeller University Press, 0021-9525/92/04/437/11 $2 .00 The Journal of Cell Biology, Volume 117, Number 2, April 1992 437-447 ing that the 0 1 chimeric protein behaves like a ligandoccupied receptor, even though the 0 1 chimera cannot itself bind extracellular ligand, suggests an intracellular difference between occupied and unoccupied receptors, and predicts that the distribution of integrin receptors can be regulated by ligand occupancy. We tested this prediction by providing a soluble cell-binding fragment of fibronectin to cells spread on laminin . Under conditions preventing further ligand adsorption to the substrate, this treatment nevertheless resulted in the relocation of diffuse fibronectin receptors to focal contacts . Similarly, a redistribution of diffuse vitronectin receptors to focal contacts occurred on cells spread on laminin after the addition of the small soluble peptide GRGDS. We conclude that the propensity for receptor redistribution to focal contacts driven by the 0 1 cytoplasmic domain alone is suppressed in heterodimeric unoccupied fibronectin receptors, and that ligand occupancy can release this constraint . This redistribution of integrin receptors after the binding of a soluble substrate molecule may provide a direct means of assembling adhesion sites . lar domains, integrins are thought to interact directly with some of the cytoskeletal proteins that colocalize with them at these sites, such as talin and a-actinin (Singer, 1982 ; Chen et al ., 1985, Damsky et al ., 1985 ; Horwitz et al ., 1986 ; Burridge et al ., 1988 ; Tapley et al ., 1989 ; Otey et al ., 1990) . The integrin 01 intracellular domain is particularly well conserved in vertebrates from humans to amphibians, suggesting its importance in receptor function (DeSimone and Hynes, 1988) . This role has been supported by transfection experiments . By assaying the function ofheterodimers consisting of transfected normal and mutant 01 subunits and endogenous a subunits, it has been shown that the ß intracellular domain is required for the localization of 01 integrin receptors in focal contacts (Solowska et al ., 1989 ; Hayashi et al ., 1990 ; Marcantonio et al ., 1990), as well as its function in cell adhesion (Hayashi et al ., 1990) . However, it is still not known whether the 0 1 intracellular domain alone is sufficient for receptor interaction with the cytoskeleton, or whether in analogy to the requirements for ligand-receptor interaction, both the a and ß subunits are required for receptor interaction with the cytoskeleton (Buck et al ., 1986) . 437 on Jne 6, 2017 D ow nladed fom Published April 15, 1992