Article |
Address correspondence to Fiona M. Watt, Keratinocyte Laboratory, Cancer Research UK London Reasearch Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK. Tel.: 44-20-7269-3528. Fax: 44-20-7269-3078. E-mail: fiona.watt{at}cancer.org.uk
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Abstract |
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Key Words: integrin; keratinocyte; differentiation; adhesion; tumor
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Introduction |
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When cultured epidermal keratinocytes are deprived of contact with an adhesive substratum, they initiate terminal differentiation. This can be partly inhibited by ligation of ß1 integrins with antibodies or high concentrations of extracellular matrix proteins (Adams and Watt, 1989; Watt, 2002). To investigate the mechanism by which integrins regulate differentiation, we have previously introduced a series of wild-type and mutant chick ß1 integrin subunits into primary human keratinocytes and tested the ability of chick-specific anti-integrin antibodies to block suspension-induced terminal differentiation (Levy et al., 2000). Those studies established that the differentiation signal can still be transduced by ß1 cytoplasmic domain mutants that fail to localize to focal adhesions. A point mutation that abolishes ß1 ligand binding is inactive in regulating suspension-induced differentiation and this led us to conclude that the differentiation signal is "do not differentiate," transduced by ligand-occupied receptors, rather than "do differentiate," transduced by unoccupied receptors.
Introduction of the wild-type chick ß1 subunit into primary human keratinocytes has no effect on the proportion of cells that undergo spontaneous terminal differentiation; this is also true for several lines of human keratinocytes derived from squamous cell carcinomas (Levy et al., 2000). However, transduction of one such line, SCC4, does have a strong positive effect on differentiation (Levy et al., 2000). SCC4 are derived from a squamous cell carcinoma of the tongue (Rheinwald and Beckett, 1981). They are poorly differentiated, with only 1% of cells expressing the terminal differentiation marker, involucrin, in culture (Levy et al., 2000). Introduction of the wild-type ß1 subunit into SCC4 cells increases the percentage of involucrin-positive cells to 10%, which is similar to the percentage in preconfluent primary keratinocytes (Levy et al., 2000).
Because poorly differentiated squamous cell carcinomas have a worse prognosis than tumors exhibiting a moderate or high degree of differentiation (Lacy et al., 1999; Petter and Haustein, 2000), the nature of the differentiation defect in SCC4 cells could have broad significance for our understanding of how integrins influence the course of the disease. The failure of SCC4 cells to differentiate does not reflect integrin loss or overexpression, because the level and range of integrins (predominantly 2ß1,
3ß1,
5ß1,
6ß4, and
vß5) expressed are similar to primary human keratinocytes (Levy et al., 2000). To test the hypothesis that the SCC4 ß1 integrin subunit is inherently defective in differentiation control, we sequenced the complete ß1 cDNA. Here we describe a point mutation in the ß1 subunit and report on its functional consequences.
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Results |
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The affinity of recombinant vß1 for fibronectin is lower than that of
5ß1, and the transfection efficiency of the
vß1 Fc chimeras was also lower (Fig. 2 b; unpublished data). Hence, it was not possible to obtain an indication of the relative affinity of mutant and wild-type
vß1 for fibronectin. Nevertheless, over a broad range of concentrations, T188I
vß1 showed increased binding to fibronectin when compared with wild-type
vß1 (Fig. 2 b). The solid phase assays thus demonstrate that T188I increased the binding of two different ß1 integrin heterodimers to fibronectin.
The T188I mutation promotes ß1 integrinmediated adhesion to a greater extent than the wild-type subunit
Threonine 188 is conserved between human and chick ß1, and to compare the T188I mutation with the wild-type subunit in intact cells, we engineered the mutation in the chick ß1 subunit. We used the chick subunit to allow detection with species-specific antibodies in both human and mouse cells. The wild-type chick ß1 subunit forms functional heterodimers with human 2,
3, and
5 in primary human keratinocytes and behaves in the same way as human ß1 in assays of keratinocyte differentiation, extracellular matrix adhesion, and Erk MAPK activation (Levy et al., 1998, 2000; Zhu et al., 1999).
A ß1-null mouse embryo fibroblast cell line, GD25 (Wennerberg et al., 1996), was retrovirally transduced with either the wild-type (Fig. 3 a) or mutant (Fig. 3 b) integrin. The use of high titre retroviral packaging cells enabled us to study mixed populations, avoiding clonal selection (Levy et al., 1998). The level of cell surface expression of wild-type and T188I ß1 was the same (Fig. 3, a and b). Parental GD25 cells express only the vß3 integrin (vitronectin receptor); when the ß1 subunit is introduced, it is expressed as heterodimers with
3 and
6 (laminin receptors) and with
5 (fibronectin receptor) (Wennerberg et al., 1996).
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Enhanced ligand binding and ligand-independent ß1 activation in SCC4 cells compared with keratinocytes homozygous for wild-type ß1 integrin subunit
In the solid phase and GD25 assays, the T188I mutation promoted ligand binding when tested in the absence of wild-type ß1. Because SCC4 cells are heterozygous for the mutation, we examined whether the mutant also promoted adhesion when coexpressed with the wild-type subunit. We compared the adhesion of SCC4 cells with other squamous cell carcinomaderived keratinocytes (SCC13) that are homozygous for wild-type ß1. In the presence of magnesium or manganese ions, the proportion of SCC4 cells that adhered to fibronectin or collagen was significantly greater than the proportion of SCC13 cells (P < 0.05; Fig. 4, a and b). Thus, T188I promoted adhesion when expressed as the sole form of ß1 (Fig. 2; Fig. 3, c and d) or when coexpressed with wild-type ß1 (Fig. 4, a and b).
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Effects of the T188I mutation on cell behavior
To study the effects of the T188I mutation on cell behavior, we monitored the spreading, motility, and invasiveness of parental GD25 cells and cells transduced with the wild-type or mutant ß1 subunit (Figs. 5 and 6). Both the wild-type and mutant integrins localized to focal adhesions (Fig. 5 a), but the mutant promoted more rapid cell spreading than the wild type (Fig. 5 b). Both forms of the ß1 subunit promoted the random motility of GD25 cells on fibronectin equally (P = 0.34; Fig. 6, a and b). The wild-type subunit promoted invasiveness through Matrigel to a greater extent than the mutant (Fig. 6 c), probably reflecting the inverse relationship between integrin affinity and the optimal ligand concentration for cell movement (Palecek et al., 1997).
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The T188I mutation is inactive in regulating keratinocyte differentiation
To examine whether the T188I mutation was responsible for the failure of SCC4 keratinocytes to undergo terminal differentiation (Levy et al., 2000), SCC4 cells were transduced with the wild-type or T188I mutant chick ß1 integrin. The level of expression of each chick integrin subunit, determined using species-specific antibodies, was equivalent and corresponded to that of the endogenous subunits (Fig. 7 a; Levy et al., 2000). Neither subunit had any major effect on the growth rate of SCC4 cells (Fig. 7 b). However, whereas the wild-type subunit stimulated terminal differentiation (as measured by the proportion of cells expressing involucrin or cornifin), the mutant subunit had no effect (Fig. 7 c). To test whether activation of ß1 integrins also inhibited differentiation of primary keratinocytes, we incubated preconfluent adherent cultures for 48 h with the activating antibody TS2/16 (Takada and Puzon, 1993) or control anti-ß1 antibodies. At the end of the incubation period, the proportion of differentiated keratinocytes was 13% in the presence of control antibodies, but was only 7% in TS2/16-treated cultures (P < 0.05; Fig. 7 d). Thus antibody-induced activation of the wild-type ß1 subunit can suppress keratinocyte terminal differentiation. This is consistent with the observation that high concentrations of fibronectin can inhibit suspension-induced terminal differentiation of human keratinocytes (Adams and Watt, 1989; Watt, 2002).
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Discussion |
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The adhesion-promoting effect of the mutation was observed when T188I was expressed as 5ß1 (Fig. 2 a; Fig. 3 c; Fig. 4 a),
vß1 (Fig. 2 b),
3ß1 or
6ß1 (Fig. 3 d), and
2ß1 (collagen receptor; Fig. 4 b) heterodimers. Nevertheless, the extent to which adhesion was activated did appear to depend on the
partner and the assay conditions (e.g., Figs. 2 and 3). By modeling the ß1 I-like domain on the crystal structures of
vß3 (Xiong et al., 2001, 2002), we can speculate about how the T188I mutation might increase ligand binding (Fig. 1 a). The threonine may interact with other residues to stabilize a low-affinity form of the receptor; replacing the threonine with isoleucine might prevent this interaction. Alternatively, the presence of isoleucine may increase ligand binding by altering the normal movement of the loop that occurs on activation.
Consistent with our observations, replacement of the ß2 specificity loop with the ß3 loop activates binding of Lß2 to ICAM1 (Kamata et al., 2002). Swapping the specificity loops of the ß1 and ß3 subunits determines the ability of each subunit to regulate Rho GTPases, demonstrating its importance in translating information from ligand binding into intracellular signaling events (Miao et al., 2002). It is possible that in addition to, or as a consequence of, increasing ligand binding, the T188I mutation might alter the interactions of ß1 integrins with other membrane proteins or affect organization of the extracellular matrix (Schwartz, 2001; Leitinger and Hogg, 2002); however, this remains to be investigated.
The evidence that T188I is indeed responsible for the failure of SCC4 to differentiate rests on the observation that transduction of SCC4 cells with wild-type ß1 increases differentiation, whereas transduction with T188I does not (Fig. 7 c). Furthermore, antibody-mediated activation of the wild-type ß1 subunit, to mimic the effect of the T188I mutation, suppresses differentiation of normal keratinocytes (Fig. 7 d). We have previously shown that the mechanism by which ß1 integrins regulate keratinocyte differentiation is via occupied receptors transducing a "do not differentiate" signal (Levy et al., 2000). The properties of the T188I mutation support this conclusion, in that by strongly promoting ligand binding, cells expressing the mutant integrin receive a strong "do not differentiate" message. The enhanced activation of Erk MAPK by T188I independent of cell spreading adds weight to the view that activation of this pathway downstream of ß1 integrins plays a role in suppressing keratinocyte differentiation (Zhu et al., 1999; Haase et al., 2001). It is interesting that the decision to differentiate appears to be an all or nothing response to relatively modest changes in integrin-mediated adhesion, both in the case of the T188I mutation and when a dominant negative integrin mutation is introduced into keratinocytes (Zhu et al., 1999).
Changes in integrin expression, whether loss, de novo expression, or overexpression, are all reported to influence the prognosis of epithelial tumors (van Waes et al., 1991; Jones et al., 1997; Bagutti et al., 1998), the best-documented mechanism being by promoting invasion (Giancotti and Ruoslahti, 1999; Lochter et al., 1999; Thomas et al., 2001). In contrast, the T188I mutation is not correlated with altered integrin expression levels (Levy et al., 2000) and contributes to the neoplastic phenotype by inhibiting integrin-regulated differentiation. It is now of interest to determine the overall frequency of integrin mutations in tumors, to uncover the extent to which such mutations affect the onset and outcome of the disease.
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Materials and methods |
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Solid phase assays with recombinant integrins
Integrin cDNAs were cloned into the vector pEE12.2h, which contains the human 1 Fc domain as a Sal1EcoR1 genomic fragment. Separate vectors encoding the extracellular regions of the human
5,
v, wild-type ß1, and T188I ß1 integrin subunits were constructed, essentially as described previously (Stephens et al., 2000). The appropriate
and ß integrin vectors were transiently coexpressed in CHOL761h cells, and integrin chimeras were captured from the culture supernatant with goat antihuman-Fc antiserum. Solid phase binding to fibronectin-coated plates was performed as follows. 96-well plates (Nunc) were coated with the 50-kD fragment of fibronectin at 2 µg/ml in 0.1 M sodium bicarbonate, pH 8.5. Plates were blocked with 5% (wt/vol) BSA, 1% (vol/vol) Tween-20 in PBS. Supernatants containing soluble integrins were pretreated with 25 mM EDTA and then extensively dialyzed against 20 mM Tris-HCl, 150 mM NaCl, 1 mM MgCl2, pH 7.5. Titrations of integrins were performed in 1% (wt/vol) BSA, 20 mM Tris-HCl, 150 mM NaCl, 1 mM MgCl2, pH 7.5, and incubated on the fibronectin for 2 h at room temperature. Bound integrins were detected with an HRP-labeled goat antihuman IgG-Fc serum (Jackson ImmunoResearch Laboratories).
Cell culture and retroviral infection
Primary human keratinocytes and SCC4, SCC13, and GD25 cells were cultured as described previously (Levy et al., 2000; Wennerberg et al., 1996). The pBabe-puro ß1 T188I construct was made using the pBabe-puro chick ß1 wild-type vector (Levy et al., 2000) as a template via the Quikchange mutagenesis protocol (Stratagene). Retroviral packaging cell lines were generated as described previously (Levy et al., 2000) or by transfecting Phoenix packaging cells (G. Nolan, Stanford University, Stanford, CA) and then using the virus-containing supernatant either to directly infect GD25 cells or to make stable amphotropic virus AM12 producers (Levy et al., 2000). The AM12 producer lines were used to infect SCC4 cells as described previously (Levy et al., 2000).
Cell attachment, spreading, motility, and invasion
96-well flat-bottomed assay plates (Dynex) were coated with fibronectin as described previously (Levy et al., 2000). Preconfluent 75-cm2 flasks of cells were washed twice in serum-free medium and incubated with 5 ml of serum-free medium containing 5 µM CellTracker green dye (Molecular Probes) for 20 min at 37°C. The cells were washed twice, placed in serum-containing medium for a further 20 min, and then harvested. Cells were resuspended at 3 x 105 cells/ml in TBS containing Mg2+ (10 mM), Mn2+ (1 mM), or Ca2+ (1 mM). 100 µl of cell suspension was added per well to triplicate wells and incubated at 37°C for 15 min. The plate was then washed twice with TBS. The fluorescence value from each well was measured and compared with a standard curve prepared from serial dilutions of the same cell suspension.
To quantitate cell motility and spreading, subconfluent cultures were harvested and plated onto dishes coated in 10 µg/ml fibronectin, at a density that allowed tracking of individual cells. The cells were filmed for 25 h. Individual cells were tracked using Kinetic Imaging Tracking software and the results analyzed using Mathematica (Wolfram Research). Cell spreading was measured during the first 3 h of filming: the number of unspread cells and the total number of cells in every third frame were recorded, and the percentage of spread cells was calculated. Any cells that failed to spread over the period of the film (24 h) were discounted as nonviable.
Invasion assays were performed as described previously (Thomas et al., 2001). GD25 cells were mixed with Matrigel (Becton Dickinson) to give a final concentration of 3 x 105 cells/ml in a 1:2 dilution of Matrigel. 100 µl of the mixture was placed in 0.8-µm pore size 24-well cell culture inserts (Becton Dickinson) and left to gel for 1 h at 37°C. 100 µl serum-free medium was placed above the gel and 750 µl medium containing 10% donor calf serum was placed in the lower compartment. After 2 d, the inserts were removed from their wells, the Matrigel was removed, and the membranes were fixed in methanol and stained with crystal violet. The center of each membrane was photographed, and the number of cells was counted using NIH Image analysis software.
Measurement of Erk MAPK activation
GD25 cells expressing wild-type or mutant ß1 integrins were plated on 10 µg/ml fibronectin in the presence or absence of 10 ng/ml EGF for various times and then lysed and immunoblotted as described previously (Haase et al., 2001). The antibody specific for phosphorylated Erk1/2 was purchased from New England Biolabs, Inc., and the antibody to total Erk was from Santa Cruz Biotechnology, Inc. Protein bands were visualized with HRP-conjugated secondary antibodies using ECL (Amersham Biosciences). The level of Erk phosphorylation was quantitated using the Scion Image package, dividing the value for each phosphoErk band with the value for the corresponding total Erk band.
Cell differentiation assays
The proportion of terminally differentiated cells was determined essentially as described previously (Levy et al., 2000). Involucrin was detected with SY-5 or DH-1 antibodies (Levy et al., 2000) and cornifin with SQ37C, a generous gift of A. Jetten (National Institute of Environmental Health Sciences, Research Triangle Park, NC) (Fujimoto et al., 1997).
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Acknowledgments |
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F.M. Watt and R.D. Evans are supported by Cancer Research UK.
Submitted: 3 September 2002
Revised: 6 December 2002
Accepted: 30 December 2002
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References |
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---|
Adams, J.C., and F.M. Watt. 1989. Fibronectin inhibits the terminal differentiation of human keratinocytes. Nature. 340:307309.[CrossRef][Medline]
Bagutti, C., P.M. Speight, and F.M. Watt. 1998. Comparison of integrin, cadherin, and catenin expression in squamous cell carcinomas of the oral cavity. J. Pathol. 186:816.[CrossRef][Medline]
Bishop, L.A., W.J. Kee, A.J. Zhu, and F.M. Watt. 1998. Lack of intrinsic polarity in the ligand-binding ability of keratinocyte ß1 integrins. Exp. Dermatol. 7:350361.[Medline]
Fujimoto, W., G. Nakanishi, J. Arata, and A.M. Jetten. 1997. Differential expression of human cornifin and ß in squamous differentiating epithelial tissues and several skin lesions. J. Invest. Dermatol. 108:200204.[Abstract]
Giancotti, F.G., and E. Ruoslahti. 1999. Integrin signaling. Science. 285:10281032.
Haase, I., R.M. Hobbs, M.R. Romero, S. Broad, and F.M. Watt. 2001. A role for mitogen-activated protein kinase activation by integrins in the pathogenesis of psoriasis. J. Clin. Invest. 108:527536.
Hogg, N., and P.A. Bates. 2000. Genetic analysis of integrin function in man: LAD-1 and other syndromes. Matrix Biol. 19:211222.[CrossRef][Medline]
Jones, J., F.M. Watt, and P.M. Speight. 1997. Changes in the expression of v integrins in oral squamous cell carcinomas. J. Oral Pathol. Med. 26:6368.[Medline]
Kamata, T., K.K. Tieu, T. Tarui, W. Puzon-McLaughlin, N. Hogg, and Y. Takada. 2002. The role of the CPNKEKEC sequence in the ß(2) subunit I domain in regulation of integrin (L)ß(2) (LFA-1). J. Immunol. 168:22962301.
Kunicki, T.J. 2001. The role of platelet collagen receptor (glycoprotein Ia/IIa; integrin 2ß1) polymorphisms in thrombotic disease. Curr. Opin. Hematol. 8:277285.[CrossRef][Medline]
Lacy, P.D., E.L. Spitznagel, Jr., and J.F. Piccirillo. 1999. Development of a new staging system for recurrent oral cavity and oropharyngeal squamous cell carcinoma. Cancer. 86:13871395.[CrossRef][Medline]
Leitinger, B., and N. Hogg. 2002. The involvement of lipid rafts in the regulation of integrin function. J. Cell Sci. 115(Pt. 5):963972.
Levy, L., S. Broad, A.J. Zhu, J.M. Carroll, I. Khazaal, B. Péault, and F.M. Watt. 1998. Optimised retroviral infection of human epidermal keratinocytes: long-term expression of transduced integrin gene following grafting on to SCID mice. Gene Ther. 5:913922.[CrossRef][Medline]
Levy, L., S. Broad, D. Diekmann, R.D. Evans and F.M. Watt. 2000. ß1 integrins regulate keratinocyte adhesion and differentiation by distinct mechanisms. Mol. Biol. Cell. 11:453466.
Lochter, A., M. Navre, Z. Werb, and M.J. Bissell. 1999. 1 and
2 integrins mediate invasive activity of mouse mammary carcinoma cells through regulation of stromelysin-1 expression. Mol. Biol. Cell. 10:271282.
Luque, A., M. Gómez, W. Puzon, Y. Takada, F. Sánchez-Madrid, and C. Cabañas. 1996. Activated conformations of very late activation integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355-425) of the common ß1 chain. J. Biol. Chem. 271:1106711075.
Mercurio, A.M., and I. Rabinovitz. 2001. Towards a mechanistic understanding of tumor invasionlessons from the 6ß4 integrin. Semin. Cancer Biol. 11:129141.[CrossRef][Medline]
Miao, H., S. Li, Y.L. Hu, S. Yuan, Y. Zhao, B.P. Chen, W. Puzon-McLaughlin, T. Tarui, J.Y. Shyy, Y. Takada, et al. 2002. Differential regulation of Rho GTPases by ß1 and ß3 integrins: the role of an extracellular domain of integrin in intracellular signaling. J. Cell Sci. 115:21992206.
Palecek, S.P., J.C. Loftus, M.H. Ginsberg, D.A. Lauffenburger, and A.F. Horwitz. 1997. Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature. 385:537540.[CrossRef][Medline]
Petter, G., and U.F. Haustein. 2000. Histologic subtyping and malignancy assessment of cutaneous squamous cell carcinoma. Dermatol. Surg. 26:521530.[CrossRef][Medline]
Rheinwald, J.G., and M.A. Beckett. 1981. Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultured from human squamous cell carcinomas. Cancer Res. 41:16571663.[Abstract]
Schwartz, M.A. 2001. Integrin signaling revisited. Trends Cell Biol. 11:466470.[CrossRef][Medline]
Stephens, P.E., S. Ortlepp, V.C. Perkins, M.K. Robinson, and H. Kirby. 2000. Expression of a soluble functional form of the integrin 4ß1 in mammalian cells. Cell Adhes. Commun. 7:377390.[Medline]
Takada, Y., and W. Puzon. 1993. Identification of a regulatory region of integrin ß1 subunit using activating and inhibiting antibodies. J. Biol. Chem. 268:1759717601.
Takagi, J., T. Kamata, J. Meredith, W. Puzon-McLaughlin, and Y. Takada. 1997. Changing ligand specificities of vß1 and
vß3 integrins by swapping a short diverse sequence of the ß subunit. J. Biol. Chem. 272:1979419800.
Thomas, G.J., M.P. Lewis, I.R. Hart, J.F. Marshall, and P.M. Speight. 2001. vß6 integrin promotes invasion of squamous carcinoma cells through up-regulation of matrix metalloproteinase-9. Int. J. Cancer. 92:641650.[CrossRef][Medline]
van der Flier, A., and A. Sonnenberg. 2001. Function and interactions of integrins. Cell Tissue Res. 305:285298.[CrossRef][Medline]
van Waes, C., K.F. Kozarsky, A.B. Warren, L. Kidd, D. Paugh, M. Liebert, and T.E. Carey. 1991. The A9 antigen associated with aggressive human squamous carcinoma is structurally and functionally similar to the newly defined integrin 6ß4. Cancer Res. 51:23952402.[Abstract]
Watt, F.M. 2002. Role of integrins in regulating epidermal adhesion, growth and differentiation. EMBO J. 21:39193926.
Wennerberg, K., L. Lohikangas, D. Gullberg, M. Pfaff, S. Johansson, and R. Fässler. 1996. ß1 integrin-dependent and -independent polymerization of fibronectin. J. Cell Biol. 132:227238.[Abstract]
Xiong, J.P., T. Stehle, B. Diefenbach, R. Zhang, R. Dunker, D.L. Scott, A. Joachimiak, S.L. Goodman, and M.A. Arnaout. 2001. Crystal structure of the extracellular segment of integrin vß3. Science. 294:339345.
Xiong, J.P., T. Stehle, R. Zhang, A. Joachimiak, M. Frech, S.L. Goodman, and M.A. Arnaout. 2002. Crystal structure of the extracellular segment of integrin vß3 in complex with an Arg-Gly-Asp ligand. Science. 296:151155.
Zhu, A.J., I. Haase, and F.M. Watt. 1999. Signaling via ß1 integrins and mitogen-activated protein kinase determines human epidermal stem cell fate in vitro. Proc. Natl. Acad. Sci. USA. 96:67286733.