Transmembrane Signal Transduction by Integrin Cytoplasmic Domains Expressed in Single-subunit Chimeras*

Integrins are heterodimeric, transmembrane cell ad- hesion receptors that have recently been shown to function in transmembrane signal transduction. To examine the specific role of integrin intracellular domains in signal transduction, chimeric receptors containing various integrin intracellular domains coupled to a reporter consisting of the transmembrane and extracellular domains of the small, non-signaling subunit of the inter-leukin-2 receptor were expressed in cultured human fi- broblasts and assayed for their ability to trigger tyrosine phosphorylation of the 125-kDa cytoplasmic ty- rosine kinase, ~ ~ 1 2 5 ~ ~ . Tyrosine phosphorylation of pp12SFm was induced in cultured fibroblasts that tran- siently expressed chimeric receptors containing either the pl, p,, or Pa integrin intracellular domain and were selected by magnetic bead sorting. However, expression of chimeric receptors containing either the a5 or an alternatively spliced form of the p, intracellular domain (&), as well as those lacking an intracellular domain, failed to induce tyrosine phosphorylation of ~ ~ 1 2 5 ~ ~ . These results indicate that information contained in the pl, ps, or pa integrin intracellular domain is sufficient to stimulate integrin-mediated tyrosine phosphorylation of specific intracellular proteins and that integrin extracellular and transmembrane domains are not required for inducing tyrosine phosphorylation. Our

Integrins are heterodimeric, transmembrane cell adhesion receptors that have recently been shown to function in transmembrane signal transduction. To examine the specific role of integrin intracellular domains in signal transduction, chimeric receptors containing various integrin intracellular domains coupled to a reporter consisting of the transmembrane and extracellular domains of the small, non-signaling subunit of the interleukin-2 receptor were expressed in cultured human fibroblasts and assayed for their ability to trigger tyrosine phosphorylation of the 125-kDa cytoplasmic tyrosine kinase, ~~1 2 5~~.
Tyrosine phosphorylation of pp12SFm was induced in cultured fibroblasts that transiently expressed chimeric receptors containing either the pl, p,, or Pa integrin intracellular domain and were selected by magnetic bead sorting. However, expression of chimeric receptors containing either the a5 or an alternatively spliced form of the p, intracellular domain (&), as well as those lacking an intracellular domain, failed to induce tyrosine phosphorylation of ~~1 2 5~~. These results indicate that information contained in the pl, ps, or pa integrin intracellular domain is sufficient to stimulate integrin-mediated tyrosine phosphorylation of specific intracellular proteins and that integrin extracellular and transmembrane domains are not required for inducing tyrosine phosphorylation. Our results also indicate that alternative splicing can regulate the ability of p integrin intracellular domains to participate in signal transduction, and they further suggest that the carboxyl-terminal region of specific fl integrins may play a role in the signal transduction pathway involving extracellular matrix molecules.
Integrins are members of a large family of more than 20 receptors that function in cell-cell and cell-substrate adhesion and in cell migration (1-6). All integrins consist of noncovalently linked a and p subunits, both of which are transmembrane. None of the integrin intracellular domains is known to have any enzymatic activity, Except for the p4 integrin, all integrins have relatively short cytoplasmic domains of 60 amino acids or less. The integrins that function in cell-sub-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Present address: Dept. of Physiology and Cell Biology, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208. strate adhesion are often localized to specialized structures on the ventral surface of cells termed focal contacts or focal adhesions, where they link extracellular matrix proteins such as fibronectin, laminin, vitronectin, and collagens with intracellular structures such as the cytoskeleton (7).
The hypothesis that integrins form the transmembrane link through which adhesive proteins can modulate cellular behavior is supported by recent studies demonstrating the involvement of integrins in several signal transduction pathways, including those leading to changes in protein tyrosine phosphorylation, intracellular pH, intracellular free calcium concentration, and inositol lipid turnover (8-13). Adhesion of cultured cells to extracellular matrix proteins results in the tyrosine phosphorylation of a number of intracellular proteins, including p~1 2 5~~, paxillin, and tensin (14"18), some of which can be localized in focal contacts.
pp12EiFAK (also known as focal adhesion kinase, FAK,l or FadK) is a 125-kDa cytoplasmic protein tyrosine kinase that is localized in focal contacts of cultured cells (19,201 that can also be tyrosine-phosphorylated in response to the clustering of integrins (21) through an as yet unknown mechanism. Similarly, the adhesion of platelets to adhesive ligands and the binding of fibrinogen to platelets via the aI& integrin results in tyrosine phosphorylation of ~~1 2 5~~ in a process that also appears to require either platelet adhesion or aggregation (22-24). ~~1 2 5~~ may represent a common intermediate in multiple signal transduction pathways, suggesting that processes such as cell adhesion that lead to tyrosine phosphorylation of ~~1 2 5~~ can modulate diverse cellular functions through multiple pathways. Tyrosine phosphorylation of ~~1 2 5~~ is also modulated by cellular transformation by pp60' "" (19,25,26). Furthermore, treatment of mouse 3T3 cells with the neuropeptide bombesin and aggregation of the IgE receptor of basophilic leukemia cells can each stimulate tyrosine phosphorylation of ~~1 2 5~~ (27)(28)(29).
The cytoplasmic tails of p1 integrins are required for their localization in focal contacts (30)(31)(32). Expression of chimeric receptors derived by fusing the intracellular domains of either the ab or p1 subunit of the fibronectin receptor integrin to a reporter domain consisting of extracellular and transmembrane regions of the small, low affinity, non-signaling subunit of the human interleukin-2 (IL-2) receptor (33,34) demonstrated that the p1 intracellular domain alone was sufficient to target the reporter domain to focal contacts (35). Similar observations were made using integrin-cadherin chimeras (36).
Because the p1 chimeric receptors could not bind an extracellular matrix ligand, these experiments suggested that an intracellular mechanism is also involved in receptor distribution. Such an accumulation of integrins in focal contacts occurs in endogenous receptors as a response to ligand occupancy (35). In this study we have examined the ability of chimeric receptors containing different integrin p intracellular domains to participate in the signal transduction pathway that results in tyrosine phosphorylation of FAK. We have found that only specific intracellular domains can participate in signal transduction and that this participation may be regulated by alternative splicing of p integrin intracellular domains.
MATERIALS AND METHODS Cells-Noma1 human foreskin fibroblasts were obtained and cultured as described previously (37). Cells were transfected by electroporation at 170 V and 960 microfarads with a Gene Pulser (Bio-Rad) as described (35,38). The cell line 7G7B6 secreting anti-IL-2 receptor monoclonal antibody was obtained from the American Type Culture Collection (Rockville, MD) and cultured as described (39).
Chimeric Receptors-Chimeric receptors were constructed using standard molecular biological techniques as described' (35,40). Cells transiently expressing chimeric receptors were isolated by magnetic bead sorting after 48 h of culture as described (41).
Protein Reagents-Fibronectin was purified from outdated human plasma obtained from the Department of Transfusion Medicine, National Institutes of Health using citric acid elution from a gelatin-Sepharose affinity column (42). Anti-IL-2 receptor antibody 7G7B6 was purified from ascites generated in BALB/c mice using sequential ammonium sulfate precipitation, DE52 (Whatman) ion exchange chromatography (39), and protein A-Sepharose affinity chromatography. Fluorescein-labeled anti-IL-2 receptor antibody was obtained from Boehringer Mannheim, anti-tyrosine phosphate monoclonal antibody PY20 from ICN, a n t i -p~l 2 5~~ monoclonal antibody from Upstate Biotechnology, Inc., lZsI-anti-mouse IgG from Amersham Corp., anti-mouse IgG coupled to agarose from Sigma, and BioMag anti-mouse IgGcoupled magnetic beads from Advanced Magnetics, Inc.
Assays-Tyrosine phosphorylation was determined by lysing cells in RIPA buffer and Western immunoblotting as described by Guan et al. (14). Protein concentrations were estimated using the Micro-BCAassay (Pierce Chemical Co.) using bovine serum albumin as a standard. ~~1 2 5~~~ was immunoprecipitated as described (29) and detected by Western immunoblotting using anti-phosphotyrosine monoclonal antibodies.
Fluorescence-activated Cell Sorting-Human fibroblasts transiently expressing the p1 chimeric receptor were harvested after 1 day of culture, treated with fluorescein-labeled anti-IL-2 receptor antibody (Boehringer Mannheim) diluted 1/100 in 1% crystalline bovine serum albumin (Calbiochem) in Dulbecco's phosphate-buffered saline, and sorted by fluorescence-activated cell sorting under sterile conditions using a Becton Dickinson FACStar Plus fluorescence-activated cell sorter. Cells selected for expression of cell surface IL-2 receptor reporter domain were cultured for an additional day, harvested, and either analyzed immediately for tyrosine phosphorylation or collected by binding to magnetic beads prior to analysis.

RESULTS AND DISCUSSION
In the present study, we have used a series of chimeric human integrin-11-2 receptor proteins transiently expressed in cultured human foreskin fibroblasts to probe the mechanism of integrin-mediated signal transduction. As shown in Fig. 1, there is enhanced tyrosine phosphorylation of ~~1 2 5~~ in cultured human foreskin fibroblasts expressing the human p1 chimeric receptor collected by binding to magnetic beads using an antidL-2-receptor antibody (lane 4). In contrast, there is a relatively low background level of tyrosine phosphorylation of ~~1 2 5~~ in control cells expressing the IL-2 extracellular and transmembrane domains with no integrin intracellular domain (lune 3 ) and in cells expressing the a5 intracellular domain (lane 5). For reference, the background level of protein tyrosine phosphorylation in nontransfected human foreskin fibroblasts allowed to attach for 20 min on poly-L-lysine (lane 1) and the relatively high level of tyrosine phosphorylation of ~~1 2 5~~ resulting from adhesion of nontransfected cells on fibronectin (lane 2 ) are also shown. We confirmed that the major tyrosine-  5) were transfected, cultured for 2 days, isolated by magnetic bead sorting, lysed, and analyzed for tyrosine phosphorylation by Western blotting. The relative mobilities of molecular size markers are shown on the left, and the position of ~~1 2 5~~ is indicated by the arrowhead. As negative and positive controls, respectively, nontransfected human foreskin fibroblasts were allowed to attach to poly-L-lysine (lane l ) and fibronectin (lane 2) substrates for 20 min prior to analysis for tyrosine phosphorylation. All lanes were loaded with equal amounts of total cellular protein, representing approximately 5 x io5 cellsilane.
for signal transduction, suggesting that the only integrin-specific interactions required involve the p1 intracellular domain.
Cells transiently expressing the p1 chimeric receptor were also purified by fluorescence-activated cell sorting and assayed for signal transduction. In this purified population of transfected cells expressing chimeric receptors, there was no induction of tyrosine phosphorylation of ~~1 2 5~~~ unless they were subsequently treated with magnetic beads containing monoclonal anti-IL-2 receptor antibodies (Fig. 2, compare lanes 3  and 4). This result is consistent with previous studies showing that some form of extracellular interaction is required for inducing the tyrosine phosphorylation of ~~1 2 5~~ (14-18, 21).
The participation of other human p integrin intracellular domains in the signal transduction pathway was also examined. As shown in Fig. 3 A , chimeric receptors containing the p1 (lane 2), p3 (lane 3), and p5 (lane 5) integrin intracellular domains also have the ability to induce tyrosine phosphorylation of ~~1 2 5~~ when compared to the chimera lacking an intracellular domain (lane 1 ). The ability to trigger tyrosine phosphorylation of pp12EiFAK appears to be specific to the p,, P3, and p5 intracellular domains. When the alternatively spliced form of the p3 intracellular domain, designated /33B (43), was assayed, it did not induce tyrosine phosphorylation of pp12FiFAK (Fig. @,   lane 4 ) . The lanes in Fig. 3A were loaded with equal amounts of total protein solubilized from cells bound to magnetic beads containing anti-IL-2 antibodies and, therefore, expressing chimeric receptors on their surfaces. Furthermore, in separate control experiments, flow cytometry analysis indicated that the a5, pl, p3, and the P3* chimeric receptors, as well as the control receptor lacking an intracellular domain, were all expressed at similar levels on the cell surface (not shown), making it unlikely that the results shown were due to artifactually low expression of the /33B receptor. The sequences of the human pl, p3, /33B, and p5 intracellular domains are compared in Fig. 3B  (43-48). The inability of the p3B intracellular domain to induce tyrosine phosphorylation of ~~1 2 5~~ suggests that alternative splicing may be a mechanism to regulate the ability of integrins to participate in the transmembrane signaling process.
Our findings indicate that all the integrin-specific structural information required for transmembrane signaling in the form  integrin that is nonconserved with respect to the intracellular domains of those integrins capable of participating in transmembrane signaling. of tyrosine phosphorylation of ~~1 2 5~~~ is entirely contained within the pl, p3, and ps intracellular domains, which are as small as 47 amino acid residues. The cytoplasmic domains of these integrins can mediate signaling even in the absence of their transmembrane and extracellular domains, which can be replaced by the non-integrin transmembrane and extracellular domains from the low affinity subunit of the IL-2 receptor. These domains of the IL-2 receptor by themselves cannot mediate pp12!jFAK phosphorylation (Fig. 1). Several laboratories have demonstrated by deletion analysis that the presence or absence of particular intracellular domains can alter integrin function (14,30,(49)(50)(51)(52)(53)(54)(55), including observations that the p1 intracellular domain is required for signal transduction (14), whereas the as intracellular domain is not (55). Our results represent the first direct evidence that an integrin intracellular domain by itself contains sufficient structural information to transfer information to a cytoplasmic signal transduction pathway.

K L L M I I H D + R E F A . K . F E K E~A N A K W D T G E~. P . !~: K S A V T T V V~N
The inability of the p3B integrin to trigger tyrosine phosphorylation suggests that integrin-mediated signal transduction can be regulated by alternative splicing that generates differ-ent isoforms of integrin intracellular domains. Our results suggest a function for the carboxyl-terminal end of the PI, p3, and ps intracellular domains in the coupling of integrins into the tyrosine phosphate signal transduction cascade. This region may function either by binding or by regulating the binding of ~~1 2 5~~~ or an as yet unidentified protein. Alternatively, the carboxyl-terminal end of the p3B may have a negative effect on interactions required for the induction of the pathway involving tyrosine phosphorylation of ~~1 2 5~~~.
Three separate regions of the p1 intracellular domain, designated cyto-1, cyto-2, and cyto-3 (Fig. 3B), have already been identified as important for cell surface distribution (56). It is not yet known which, if any, of these regions are involved in signal transduction, although the cyto-2 and cyto-3 regions are spliced out of the p3B integrin. Since both integrins and pp12EiFAK can be localized in focal contacts, perhaps interactions required for integrin clustering in these specialized structures may also be important for tyrosine phosphorylation of ~~1 2 5~"~. This notion is consistent with the observation that pl, p3, and ps chimeric receptors concentrate in focal contacts but the P3B chimera does not.' The exact sequence of interactions that results in tyrosine phosphorylation of pp12tjFAK is currently unclear. Recently published studies indicate that pp12ijFAK either is autophosphorylated or is phosphorylated by pp60"'"" or another 60-kDa phosphoprotein (19,(22)(23)(24)(25)57). Whatever the mechanism, interactions involving integrin p intracellular domains appear to be of central importance.
Molecular Growth Regulation, NICHHD, National Institutes of Health