Regulation of ct 6 B 1 Integrin Laminin Receptor Function by the Cytoplasmic Domain of the ct 6 Subunit

The tx6~l integrin is expressed on the macrophage surface in an inactive state and requires cellular activation with PMA or cytokines to function as a laminin receptor (Shaw, L. M., J. M. Messier, and A. M. Mercurio. 1990. J. Cell Biol. 110:2167-2174). In the present study, the role of the ix6 subunit cytoplasmic domain in ot6/51 integrin activation was examined. The use of P388D1 cells, an ct6-integrin deficient macrophage cell line, facilitated this analysis because expression of either the ot6A or ot6B subunit cDNAs restores their activation responsive laminin adhesion (Shaw, L. S., M. Lotz, and A. M. Mercurio. 1993. J. Biol. Chem. 268:11401-11408). A truncated cz6 cDNA, ot6-ACYT, was constructed in which the human cytoplasmic domain sequence was deleted after the GFFKR pentapeptide. Expression of this cDNA in P388D1 cells resulted in the surface expression of a chimeric o~6-ACYT/31 integrin that was unable to mediate laminin adhesion or increase this adhesion in response to PMA under normal conditions, i.e., in medium that contained physiological concentrations of Ca ++ and Mg ++. The a6A-ACYT transfectants adhered to laminin, however, when Ca÷+/Mg ÷÷ was replaced with 150 #M Mn ÷÷. We also assessed the role of serine phosphorylation in the regulation of o~6A~l integrin function by site-directed mutagenesis of the two serine residues present in the ot6A cytoplasmic domain because this domain is phosphorylated on serine residues in response to stimuli that activate the laminin receptor function of ot6A/~l. Point mutations were introduced in the ot6A cDNA that changed either serine residue #1064 (M1) or serine residue #1071 (M2) to alanine residues. In addition, a double mutant (M3) was constructed in which both serine residues were changed to alanine residues. P388D1 transfectants which expressed these serine mutations adhered to laminin in response to PMA to the same extent as cells transfected with wild-type ot6A cDNA. These findings provide evidence for a novel mode of integrin regulation that is distinct from that reported for other regulated integrins (O'Toole, T. E., D. Mandelman, J. Forsyth, S. J. Shattil, E. E Plow, and M. H. Ginsberg. 1991. Science (Wash. DC). 254:845-847. Hibbs, M. L. , H. Xu, S. A. Stacker, and T. A. Springer. 1991. Science (Wash. DC). 251:1611-1613), and they demonstrate that serine phosphorylation of the ot6A cytoplasmic domain is not involved in this regulation. T HE rapid activation of integrin function by signal transduction pathways constitutes an important regulatory mechanism for cell-cell and cell-matrix interactions (for review see Ginsberg et al., 1992; Hynes, 1992). This hypothesis is substantiated by the finding that several integrins are expressed on the cell surface in an inactive state and require cellular activation with a variety of agonists to acquire the capability to mediate adhesion to their appropriate ligands (e.g., Duet al., 1991; Dustin and Springer, 1991; Shaw et al., 1990). This process has been termed "insideout" integrin signaling, and the integrins that are the targets of signaling pathways are often referred to as "activationdependent" integrins (Ginsberg et al., 1992; Hynes, 1992). Address all correspondence to Arthur M. Mercurio, Laboratory of Cancer Biology, Deaconess Hospital, Harvard Medical School, 50 Binney Street, Boston, MA 02115. Examples of such activation-dependent integrins include the leukocyte specific/52 integrins (Dustin and Springer, 1991; Hermanowski-Vosatka et al., 1992), the aIIbl$3 platelet integrin (Duet al., 1991), and the tx6B1 integrin on macrophages (Shaw et al., 1990, 1993) and T-cells (Shimizu et al., 1990). It is clear that kinase activation is a critical component of inside-out integrin signaling (Shaw et al., 1990; Dustin and Springer, 1991; Shattil and Brugge, 1991). G proteins (Shattil et al., 1992) and the production of specific lipids (Hermanowski-Vosatlo et al., 1992) have also been implicated in this process. Although the details of these signaling pathways have not been elucidated, it appears that they may induce a conformational change in the integrin extraceliular domain which facilitates ligand binding (Du et al., 1991; Neugebauer and Reichardt, 1991; Diamond and Springer, 1993). The cytoplasmic domains of the activatio'n-dependent inte© The Rockefeller University Press, 0021-9525/93/11/1017/9 $2.00 The Journal of Cell Biology, Volume 123, Number 4, November 1993 1017-1025 1017 grins are likely targets of intracellular signaling pathways. For example, the cytoplasmic domains of the/Y2 (Chatila et al., 1989), 83 (Hillery et al., 1991), and ~6 (Shaw et al., 1990; Hogervorst et al., 1993) integrin subunits are phosphorylated on serine residues in response to the appropriate agonists. Such information, although suggestive, is correlative and it does not confirm a role for the cytoplasmic domain in integrin regulation. For this reason, mutagenesis of the cytoplasmic domains of ctL82 and allb83 integrins has been used to examine their role in inside-out signaling. From these studies, it appears that the 82 cytoplasmic domain of the oiL82 integrin is critical for its regulation because deletion of the 82 cytoplasmic domain, but not the oiL cytoplasmic domain, resulted in an inactive receptor that could no longer respond to PMA activation (Hibbs et al., 1991a). Site-directed mutagenesis of the serine residue in the 82 subunit that is phosphorylated in response to PMA did not alter the activation of the o~L82 receptor, indicating that phosphorylation may not be an important mechanism for activation of receptor function (Hibbs et al., 1991b). In contrast to the results obtained with ~L82, it was observed for odlb83 that deletion of the cdlb cytoplasmic domain, but not the 83 cytoplasmic domain, created a constitutively active receptor that did not require cellular activation of its ligandbinding function (O'Toole et al., 1991). The existing data, therefore, indicate distinct modes of regulation for c~L82 and otfib83 because each of these integrins differ not only in which subunit cytoplasmic domain is critical for activation of function, but also in how the specific cytoplasmic domain influences receptor regulation. We have reported that the o~681 integrin requires insideout signaling to function as a macrophage laminin receptor (Shaw et al., 1990). This activation-dependent function correlates with the association of this integrin with the actin cytoskeleton and serine phosphorylation of the or6 subunit (Shaw et al., 1990). Based on these findings and the disparate results obtained with the otL82 (Hibbs et al., 1991a,b) and cdlb83 integrins (O"l'oole et al., 1991), we thought it was important to investigate the role of the or6 subunit cytoplasmic domain in the regulation of ot681 function. This analysis was facilitated by our recent finding that the activation responsive adhesion of the o~681 integrin is maintained when the o~6 subunit is expressed by cDNA transfection in P388Dt cells, an o~6-deficient macrophage cell line (Shaw et al., 1993). In this report, we provide evidence for a mechanism of integrin regulation that is distinct from o~L82 and o~IIbB3 because deletion of the , 6 cytoplasmic domain abolished laminin adhesion under physiological conditions as well as in response to PMA. We also observed that sitedirected mutagenesis of the two serine residues in the ~6 cytoplasmic domain did not inhibit cell attachment to laminin. Materials and Methods