©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Breaking the Integrin Hinge
A DEFINED STRUCTURAL CONSTRAINT REGULATES INTEGRIN SIGNALING (*)

(Received for publication, January 22, 1996)

Paul E. Hughes (§) Federico Diaz-Gonzalez (¶) Lilley Leong Chuanyue Wu (1) John A. McDonald (1) Sanford J. Shattil Mark H. Ginsberg (**)

From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037 and theSamuel C. Johnson Medical Research Centre, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Integrins are heterodimeric (alpha, beta) cell adhesion receptors. We demonstrate that point mutations in the cytoplasmic domains of both the alpha and beta subunits promote constitutive signaling by the integrin alphabeta(3). By generating charge reversal mutations, we show these ``activating'' mutations may act by disrupting a potential salt bridge between the membrane-proximal portions of the alpha and beta subunit cytoplasmic domains. Thus, the modulation of specific interactions between the alpha and beta subunit cytoplasmic domains may regulate transmembrane signaling through integrins. In addition, these activating mutations induce dominant alterations in cellular behavior, such as the assembly of the extracellular matrix. Consequently, somatic mutations in integrin cytoplasmic domains could have profound effects in vivo on integrin-dependent functions such as matrix assembly, cell migration, and anchorage-dependent cell growth and survival.


INTRODUCTION

The integrin family of cell adhesion receptors are heterodimers of alpha and beta transmembrane subunits that play key roles in important biological processes such as inflammation, wound healing, and cell growth and survival. Integrins modulate their affinity for ligands via a process termed ``activation'' or ``inside-out'' signaling(1, 2) . Furthermore, ligand binding to integrins changes the activities of cytoplasmic kinases, GTPases, and phospholipases (``outside-in'' signaling)(2, 3, 4, 5) . Thus, integrins are bidirectional signaling receptors conducting information both into and out of the cell.

Inside-out signaling may involve the propagation of a conformational change from the integrin cytoplasmic domains to the extracellular domains resulting in high affinity ligand binding(6, 7) . Integrin alpha and beta subunit cytoplasmic domains share a similar membrane-proximal organization with apolar and polar sequences following sequentially after the membrane-cytoplasm interface (Fig. 1A). The conserved sequences for the alpha and beta subunits are -GFFKR and LLv-iHDR (highly conserved residues are uppercase, less conserved residues are lowercase, and dashes represent nonconserved residues). Deletion of these sequences in either alpha or beta subunit cytoplasmic domain ``activates'' integrins, locking them in the high affinity state(8, 9, 10, 11) . Consequently, we termed this region the integrin ``hinge.'' Hence, the capacity of the conserved membrane-proximal motifs to regulate the affinity state of integrins may depend on an interaction between them that constrains integrins to a low affinity state. Furthermore, studies on integrin assembly suggest that there may an interaction between the membrane-proximal portions of the alpha and beta subunit cytoplasmic domains(12) .


Figure 1: The Ala substitution of specific residues in the membrane-proximal regions of the alpha and beta(3) cytoplasmic domains activates alphabeta(3). A, schematic representation of the topology of the transmembrane and cytoplasmic domains of alphabeta(3). The conserved membrane-proximal sequences of the alpha and beta(3) are illustrated (highly conserved residues are uppercase, less conserved residues are lowercase). Integrin alpha and beta cytoplasmic domains share a similar membrane-proximal organization with apolar and polar sequences following on sequentially after the W-K membrane-cytoplasm interface. The conserved sequences for the alpha and beta subunits are -GFFKR and LLv-iHDR (dashes represent unconserved residues), respectively. Deletion of these sequences lock integrins in the high affinity state. The beta(4) and beta(8) subunits lack these conserved membrane-proximal sequences which suggests that they may signal via a different mechanism to other integrins. B, flow cytometry histograms illustrating PAC1 binding to alphabeta(3) and alpha(F992A)beta(3). Depicted are flow cytometry histograms illustrating PAC1 binding in the presence (open histogram) or absence (filled histogram) of competitive inhibitor, Ro 43-5054 to CHO cells expressing wild type alphabeta(3) and alpha(F992A)beta(3).The peptidomimetic Ro 43-5054, is a selective inhibitor of ligand binding to alphabeta(3)(32) . The alpha(F992A)beta(3) transfectants specifically bind PAC1, illustrating that this mutation activates alphabeta(3). In contrast, cells expressing wild-type alphabeta(3), which is in the low affinity state, can only bind PAC1 in the presence of an activating antibody (anti-LIBS6) that binds to the extracellular domain of beta(3)(13) . C, activation indices of the alphabeta(3) mutants. To obtain numerical estimates of integrin activation, an activation index (AI) was calculated for each of the alphabeta(3) mutants. PAC1 binding was measured in CHO cells expressing wild-type alphabeta(3), alpha(G991A)beta(3), alpha(F992A)beta(3), alpha(F993A)beta(3), alpha(K994A)beta(3), alpha(R995A)beta(3), and alphabeta(3)(D723A). Depicted are the mean activation indices ± S.D. of three independent experiments for each mutation. For each experiment, wild-type alphabeta(3) was included as a control. Transient transfection of CHO cells with activated forms of alphabeta(3) had surface expression levels similar to wild-type alphabeta(3). In 3 determinations, 5 different activated mutants were expressed at 75 ± 16% of wild-type alphabeta(3) (not shown).



In this paper, we describe point mutations in this hinge region that activate alphabeta(3). Moreover, by generating complementary charge reversal mutations, we show these activating mutations may act by disrupting a potential salt bridge between the membrane-proximal portions of the alpha and beta subunit cytoplasmic domains. In addition, we demonstrate that these active mutations can induce the constitutive outside-in signaling as assayed by the phosphorylation of pp125 and the ligand independent recruitment of alphabeta(3) to focal adhesions.


MATERIALS AND METHODS

Antibodies

The anti-alphabeta(3) antibody D57 (9) was biotinylated with biotin-N-hydroxysuccinimide (Sigma) according to the maufacturers directions. The isolation and characterization of anti-LIBS6 (13) and PAC1 (14) have been described previously.

cDNA Constructs

pCDM8 expression constructs encoding wild-type alpha and beta(3) were constructed as described(9) . The generation of expression constructs in pCDM8 encoding point mutation alpha and beta(3) cDNAs were undertaken using PCR (^1)mutagenesis(15) . CDM8 expression vectors encoding beta(3) mutants were constructed by cloning a 0.9-kb MluI and PstI cut PCR fragment encompassing the mutations into MluI and PstI cut pCDM8. This construct was then cut with AflII and DraIII, and the 3-kb fragment ligated with the 3.5-kb AflII-DraIII fragment of pCDbeta(3). pCDM8 expression vectors encoding alpha mutants were constructed by tripartite ligation of a 0.6-kb XbaI-BamHI cut PCR fragment encompassing the mutations, the 4.1-kb BamHI-DraIII fragment of pCDalpha, and the 2.6-kb DraIII-XbaI fragment of pCDM8. All constructs were verified by DNA sequencing and purified by CsCl centrifugation before transfection. Mutagenic oligonucleotides were synthesized on a model 391 DNA synthesizer.

PAC1 Binding

The cDNAs expressing alphabeta(3) variants were transiently transfected into Chinese hamster ovary (CHO-K1) as described(16) . PAC1 binding was then analyzed by two-color flow cytometry as described(9, 10) . Briefly, FITC-PAC1 binding was analyzed only on a gated subset of cells positive for alphabeta(3) expression detected with a biotinylated non-function blocking antibody to alphabeta(3) (D57) and phycoerythrin/strepavidin. To define affinity state, histograms depicting PAC1 staining in the absence or presence of the competitive inhibitor Ro 43-5054 (32) were compared. The activation index (AI) was defined as 100 times (F(o) - F(r))/(F(o) LIBS6 - F(r) LIBS6), where F(o) is the median fluorescence intensity of PAC1 binding and F(r) is the median fluorescence intensity of PAC1 binding in the presence of competitive inhibitor (Ro 43-5054, 1 µM). F(o) LIBS6 is the median fluorescence intensity of PAC1 binding in the presence of 2 µM anti-LIBS6, and F(r) LIBS6 is the median fluorescence intensity of PAC1 binding in the presence of 2 µM anti-LIBS6 and competitive inhibitor.

Analysis of pp125 Phosphorylation

100-mm tissue culture plates were coated with 5 mg/ml BSA or 100 µg/ml fibrinogen and blocked with 5 mg/ml BSA. The cells were harvested and resuspended in incubation buffer (137 mM NaCl, 2.7 mM MgCl(2), 5.6 mM glucose, 3.3 mM NaH(2)PO(4), and 20 mM HEPES, pH 7.4). 1 times 10^7 cells were added to each coated dish and incubated at 37 °C for 90 min. Cells were then washed in PBS and lysed with RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride, 1 mM Na(3)VO(4), and 100 kallikrein inactivating units/ml aprotinin. Lysates were clarified by centrifugation, and the protein content was determined with the BCA reagent. 300 µg of protein from each lysate were incubated overnight at 4 °C with the anti-pp125 rabbit polyclonal antibody BC3 (a generous gift from J. Thomas Parsons, Charlottesville, VA). Immune complexes were precipitated at 4 °C with protein A-Sepharose and washed extensively in ice-cold RIPA buffer containing 1 mM Na(3)VO(4). The immune complexes were extracted into Laemmli sample buffer containing 10% beta-mercaptoethanol, subjected to SDS-polyacrylamide gel electrophoresis on 7.5% polyacrylamide gels, and electrotransferred. Western blots were prepared and analyzed for phosphotyrosine-containing proteins as described (17) using a mixture of the anti-phosphotyrosine antibodies PY20 and PY72 (a generous gift from Bart Sefton, La Jolla, CA).

Immunofluorescence

CHO cells were transiently transfected as described (16) and, after 48 h, were cultured on fibronectin-coated coverslips for 2 h at 37 °C. The cells were fixed with 3.7% paraformaldehyde (methanol-free) and permeabilized with 0.2% Triton X-100. The coverslips were blocked in 10% normal goat serum, incubated with the rabbit polyclonal antibody 2308 (anti-human alpha) (^2)and the anti-hamster beta(1) mAb 7E2 (18) in 10% normal goat serum for 60 min, washed with PBS, and then incubated with the secondary antibodies, FITC-conjugated goat anti-mouse and FITC-conjugated goat anti-rabbit in 10% normal goat serum for an additional 30 min. The coverslips were washed in PBS and mounted using FITC-guard mounting medium. The slides were examined with a Leitz Orthoplan microscope with a 100times oil immersion objective. Photographs were taken on Kodak Tmax 400 film.

Fibronectin Matrix Assembly

Cells were suspended in alpha-minimal essential medium containing 10% fetal calf serum depleted of fibronectin by gelatin affinity adsorption and supplemented with purified human plasma fibronectin. All reagents were dialyzed against three changes of 100 volumes of alpha-minimal essential medium and passed through a 0.2-µm filter prior to addition to the culture medium. Cells were plated in 12-well HTC slides at a final density of 2.3 times 10^5 cells/ml and cultured for 41 h. The cells were then fixed with 3.7% paraformaldehyde and stained with polyclonal rabbit anti-human plasma Fn antibody MC54 (32 µg/ml) and Cy3-conjugated goat anti-rabbit IgG antibodies (7.5 µg/ml). Stained cell monolayers were observed using a Nikon FXA epifluorescence microscope, and representative fields were photographed using Kodak T-Max 400 or Ektachrome 1600 direct positive slide film.


RESULTS AND DISCUSSION

To define those membrane-proximal residues of the alpha subunit important for affinity modulation, we substituted specific residues in alpha with Ala. These variants were then co-expressed in Chinese hamster ovary (CHO) cells with a wild-type beta(3) and the binding of PAC1, an antibody specific for the active conformation of alphabeta(3) was used to define affinity states (14) (Fig. 1B). The Ala substitution of alpha(F992), alpha(F993), and alpha(R995) activated alphabeta(3) as determined by high affinity PAC1 binding (Fig. 1, B and C). In contrast, the Ala substitution of alpha(G991) and alpha(K994) had minimal effect (Fig. 1B). Therefore, Ala substitution of specific residues in the conserved membrane-proximal GFFKR motif of the alpha subunit can activate an integrin.

The Ala substitution of alpha(F992A), alpha(F993A), and alpha(R995A) may activate alphabeta(3) by the disruption of an interaction between the alpha and beta(3) cytoplasmic domains. The beta(3) cytoplasmic domain contains a highly conserved Asp residue that is at a similar displacement from the proposed cytoplasm-membrane interface as the highly conserved Arg-995 of alpha. This raises the possibility that the membrane-proximal regions may interact via a salt bridge formed between alpha(R995) and beta(3)(D723). To test this idea, we expressed alphabeta(3)(D723A); this integrin bound PAC1 with high affinity (Fig. 1C). To further test the proposed salt bridge we constructed the ``charge-reversal'' mutants, alpha(R995D) and beta(3)(D723R). Both the single mutations, alpha(R995D)beta(3) and alphabeta(3)(D723R), were in the high affinity state and exhibited spontaneous PAC1 binding (Fig. 2). However, the double charge reversal mutant alpha(R995D)beta(3)(D723R) complemented the activating effect of the individual mutations. We suggest that this double mutation may restore the potential salt bridge between the alpha and beta subunits, reforming the structural constraint which prevents the activation of the integrin. As a control, we examined the affinity state of double mutants alpha(R995A)beta(3)(D723A), alpha(R995A)beta(3)(D723R), and alpha(R995D)beta(3)(D723A). All of these variants bound PAC1 spontaneously (data not shown).


Figure 2: The membrane-proximal regions of the alpha and beta(3) cytoplasmic domains may interact via a salt bridge between alpha(R995) and beta(3)(D723). The activation indices of alpha(R995D)beta(3), alphabeta(3)(D723R), and alpha(R995D)beta(3)(D723R) are illustrated. The activation index of alpha(R995D)beta(3)(D723R) is significantly less (p leq 0.001) than alpha(R995D)beta(3) and alphabeta(3)(D723R). Depicted are the mean activation indices ± S.D. of three independent experiments for each mutation.



Ligand binding to integrins induces changes in cytoskeletal organization, intracellular pH, and protein tyrosine phosphorylation (outside-in signaling)(2, 3, 4, 5) . Above we described cytoplasmic domain mutations that result in constitutive inside-out signaling. To determine if these mutations caused constitutive intracellular signaling, we examined the tyrosine phosphorylation of focal adhesion kinase (pp125)(19, 20) . Stable CHO cell lines expressing the mutants alpha(F992A)beta(3) and alphabeta(3)(D723A) exhibited the constitutive phosphorylation of pp125 when in suspension (Fig. 3a). In contrast, cells expressing wild-type alphabeta(3) only phosphorylated pp125 when adherent to a fibrinogen matrix (Fig. 3a).


Figure 3: Membrane-proximal mutations in both the alpha and beta(3) cytoplasmic domains promote constitutive intracellular signaling. a, CHO cells expressing the activating mutations alpha(F992A)beta(3) and alphabeta(3)(D723A) constitutively phosphorylate pp125. CHO cell lines expressing alphabeta(3), alpha(F992A)beta(3), and alphabeta(3)(D723A) were incubated for 90 min at 37 °C on plates coated with either fibrinogen or BSA. The cells were in suspension on BSA-coated plates, but adhered to and spread on the fibrinogen-coated plates. The cells were then processed for analysis of pp125 tyrosine phosphorylation(17) . In contrast to cells expressing wild-type alphabeta(3), those expressing alpha(F992A)beta(3) and alphabeta(3)(D723A) exhibited tyrosine phosphorylation of pp125 when incubated in suspension over BSA. As expected, cells expressing alphabeta(3) phosphorylated pp125 when plated on fibrinogen-coated plates, as did cells expressing alpha(F992A)beta(3) or alphabeta(3)(D723A) (data not shown). Untransfected CHO cells did not phosphorylate pp125 when plated on BSA or fibrinogen-coated plates. b, recruitment of alpha(R995D)beta(3) and alphabeta(3)(D723R) to focal adhesions in a ligand independent manner. CHO cells were transiently transfected and, after 48 h, were cultured on fibronectin for 2 h. The cells were then stained with a mixture of anti-human alpha (panels A, B, C, and D) and anti-hamster beta(1) (mouse monoclonal 7E2). In all experiments, beta(1) was localized in punctuate structures along the cell edge and along the ventral surface, characteristic of focal adhesions (data not shown). alphabeta(3)(D119Y) had a uniform cell surface distribution when plated on fibronectin (panel A) and was not localized to focal adhesions. In contrast, alpha(R995D)beta(3)(D119Y) (panel B) and alphabeta(3)(D723R)(D119Y) (panel C) were recruited to the focal adhesions. alpha(R995D)beta(3)(D723R)(D119Y) (panel D) behaved similarly to alphabeta(3)(D119Y) and was not present in focal adhesions.



The association of integrins with specialized cytoskeletal structures termed focal adhesions, is regulated by ligand binding to their extracellular domains(21) . As another assay for outside-in signaling, we analyzed the effect of the activating mutations on integrin targeting to focal adhesions. To ensure that the targeting of the alphabeta(3) variants to focal adhesions was independent of ligand binding, each was expressed with a ligand binding-deficient beta(3) mutant, beta(3)(D119Y) (22) . When expressed in CHO cells plated on fibronectin, both alpha(R995D)beta(3)(D119Y) and alphabeta(3)(D723R)(D119Y) were spontaneously recruited to focal adhesions formed by endogenous hamster integrins (Fig. 3b). In contrast, the distribution of alpha(R995D)beta(3)(D723R)(D119Y) was diffuse, similar to that of alphabeta(3)(D119Y), and it was not recruited to focal adhesions formed by the endogenous integrins (Fig. 3b). Therefore, we conclude that these activating point mutations allow a spontaneous association of the integrin with the cytoskeleton in the absence of ligand binding. Thus, activating membrane-proximal point mutations in both integrin alpha and beta subunits can induce constitutive bidirectional transmembrane signaling.

There presently exists no three-dimensional structure of the native cytoplasmic domains of alphabeta(3). Furthermore, a high resolution structure of this transmembrane protein may be difficult to acquire. Consequently, a mutational analysis, similar to those conducted in bacterial chemoattractant receptors (24) and G protein-coupled receptors(23, 26) , can provide a viable alternative to develop a structural hypothesis of transmembrane signaling. The approach described here has led us to propose a plausible and testable mechanism for integrin signaling. Indeed, the present studies may provide insight into a general mechansim of signaling mediated by a variety of transmembrane receptors. Point mutations can constitutively activate such structurally diverse receptors such as G protein-coupled receptors, growth factor receptors, and bacterial chemoattractant receptors(23, 24, 25, 26) . However, only in the beta(2)-adrenergic receptor has constitutive bidirectional signaling been reported(26) . In common with integrins, the ability of specific mutations to activate these receptors has been ascribed to the release of a ``constraint'' that maintains the receptor in an off state. As the topography of integrins is comparatively simple, having two parallel membrane-spanning subunits, we have been able to identify such a constraint. Utilizing charge reversal mutations, we provide direct mutational evidence for a salt bridge constraining integrins into a nonsignaling state.

As reported here, membrane-proximal point mutations in both integrin alpha and beta subunits can cause constitutive bidirectional transmembrane signaling. Signals from integrins can influence cell growth and death, and the assembly of the extracellular matrix(27, 28, 29) . This raises the intriguing possibility that activating integrin mutations may produce dominant phenotypes in vivo. The assembly of a fibronectin matrix, a process important in wound healing and cell migration during development, is regulated by integrin affinity state(29, 30) . Therefore, fibronectin matrix assembly could be perturbed by activating integrin mutations. To test this idea, we used CHO B2 cells that are unable to assemble a fibronectin matrix due to a lack of the appropriate integrins(31) . Transfection of these cells with the constitutively active mutant alphabeta(3)(D723R) enabled them to assemble a fibronectin matrix (Fig. 4). In contrast, CHO B2 cells expressing wild-type alphabeta(3) failed to make a fibronectin matrix (Fig. 4). Thus, activating point mutations in the integrin cytoplasmic domains can influence the assembly of the extracellular matrix. It will be interesting to determine if such mutations could account for some of the increased deposition of extracellular matrix that characterizes certain pathological states.


Figure 4: Activating point mutations in alphabeta(3) promote fibronectin matrix assembly. alpha(5)-deficient CHO B2 cells expressing wild-type alphabeta(3) (A) and alphabeta(3)(D723R) (B) were cultured in medium supplemented with human plasma fibronectin (480 nM)(30) . CHO B2 cells expressing wild-type alphabeta(3) (A) failed to assemble a fibronectin matrix. In contrast, cells expressing alphabeta(3)(D723R) deposited an abundant fibronectin matrix (B). The alphabeta(3) specific peptide-mimetic Ro 44-883 (1 µM) inhibited fibronectin matrix assembly by cells expressing alphabeta(3)(D723R) (data not shown). Similar results were obtained with CHO B2 cells expressing the active mutant alpha(F992A)beta(3) (data not shown).




FOOTNOTES

*
This was work was supported by grants from the National Institutes of Health (to M. H. G., J. A. McD., and S. J. S.). This is Paper 9604-VB from the Scripps Research Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Postdoctoral fellow of the American Heart Association (California affiliate).

Recipient of a fellowship from the Minsterio di Sonidad, Spain. Present address, Servicio de Immunologia, Hospital de la Princesa, Diego de Leon, Madrid 28006, Spain.

**
To whom correspondence should be addressed. Tel.: 619-784-7118; Fax: 619-784-7343; ginsberg{at}scripps.edu.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; kb, kilobase(s); BSA, bovine serum albumin; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate.

(^2)
M. H. Ginsberg, unpublished data.


REFERENCES

  1. Williams, M. J., Hughes, P. E., O'Toole, T. E., and Ginsberg, M. H. (1994) Trends Cell Biol 4, 109-112 [CrossRef]
  2. Schwartz, M. A., Schaller, M. D., and Ginsberg, M. H. (1995) Annu. Rev. Cell Biol. 11, 549-99 [CrossRef][Medline] [Order article via Infotrieve]
  3. Hynes, R. O. (1992) Cell 69, 11-25 [Medline] [Order article via Infotrieve]
  4. Damsky, C. H., and Werb, Z. (1992) Curr. Opin. Cell Biol. 4, 772-776 [Medline] [Order article via Infotrieve]
  5. Sastry, S. K., and Horwitz, A. F. (1993) Curr. Opin. Cell Biol. 5, 819-824 [Medline] [Order article via Infotrieve]
  6. O'Toole T. E., Loftus, J. C., Du, X., Glass, A. A., Ruggeri, Z. M., Shattil, S. J., Plow, E. F., and Ginsberg, M. H. (1990) Cell Regul. 1, 883-893 [Medline] [Order article via Infotrieve]
  7. Sims, P. J., Ginsberg, M. H., Plow, E. F., and Shattil, S. J. (1991) J. Biol. Chem. 266, 7345-7352 [Abstract/Free Full Text]
  8. O' Toole, T. E., Mandelman, D., Forsyth, J., Shattil, S. J., Plow, E. F., and Ginsberg, M. H. (1991) Science 254, 845-847 [Medline] [Order article via Infotrieve]
  9. O'Toole, T. E., Katagiri, Y., Faull, R. J., Peter, K., Tamura, R. N., Quaranta, V., Loftus, J. C., Shattil, S. J., and Ginsberg, M. H. (1994) J. Cell Biol. 124, 1047-1059 [Abstract]
  10. Hughes, P. E., O'Toole, T. E., Ylanne, Y., Shattil, S. J., and Ginsberg, M. H. (1995) J. Biol. Chem 270, 12411-14217 [Abstract/Free Full Text]
  11. Crowe, D., Chiu H., Fong, S., and Weissman, I. (1994) J. Biol. Chem. 269, 14411-14418 [Abstract/Free Full Text]
  12. Briesewitz, R., Kern, A., and Marcantonio, E. E. (1995) Mol. Biol. Cell 6, 997-1010 [Abstract]
  13. Frelinger, A. L. III, Du, X. P., Plow, E. F., and Ginsberg, M. H. (1991) J. Biol. Chem. 266, 17106-17111 [Abstract/Free Full Text]
  14. Shattil, S. J., Hoxie, J. A., Cunningham, M., and Brass, L. F. (1985) J. Biol. Chem. 260, 11107-11114 [Abstract/Free Full Text]
  15. Kadowaki, H., Kadowaki, T., Wondisford, F. E., and Taylor, S. I. (1989) Gene (Amst.) 76, 161-166 [CrossRef][Medline] [Order article via Infotrieve]
  16. Chen, Y. P., O'Toole, T. E., Shipley, T., Forsyth, J., LaFlamme, S. E., Yamada, K. M., Shattil, S. J., and Ginsberg, M. H. (1994) J. Biol. Chem. 269, 18307-18310 [Abstract/Free Full Text]
  17. Huang M.-M., Lipfert, L., Cunningham, M., Brugge, J. S., Ginsberg, M. H., and Shattil, S. J. (1993) J. Cell Biol. 122, 473-483 [Abstract]
  18. Brown, P. J., and Juliano, R. L. (1988) Exp. Cell Res. 171, 303
  19. Schaller, M. D., and Parsons, J. T. (1994) Curr. Opin. Cell Biol. 6, 705-709 [Medline] [Order article via Infotrieve]
  20. Schaller, M. D., Borgman, C. A., Cobb, B. S., Vines, A. B., Reynolds, A. B., and Parsons, J. T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5192-5196 [Abstract]
  21. Burridge, K., Fath, K., Kelly, T., Nuckolls, G., and Turner, C. (1988) Annu. Rev. Cell Biol. 4, 487-525 [CrossRef]
  22. Loftus, J. C., O'Toole, T. E., Plow, E. F., Glass, A., Frelinger, A. L., and Ginsberg, M. H. (1990) Science 249, 914-917
  23. Hogger, P., Shockley, M. S., Lameh, J., and Sadee, W. (1995) J. Biol. Chem. 270, 7405-7410 [Abstract/Free Full Text]
  24. Yaghmai, R., and Hazelbauer, G. L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7890-7894 [Abstract]
  25. Bargmann, C. I., Hung, M.-C., and Weinberg, R. A. (1986) Cell 45, 649-657 [Medline] [Order article via Infotrieve]
  26. Pei, G., Samana, P., Lohse, M., Wang, M., Codina, J., and Lefkowitz, R. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2699-2702 [Abstract]
  27. Frisch, S. M., and Francis, H. (1994) J. Cell Biol. 124, 619-627 [Abstract]
  28. Meredith, J. E., Fazeli, B., and Schwartz, M. A. (1993) Mol. Biol. Cell 4, 953-961 [Abstract]
  29. Mosher, D. F., Sottile, J., Wu, C., and McDonald, J. A. (1992) Curr. Opin. Cell Biol. 4, 810-818 [Medline] [Order article via Infotrieve]
  30. Wu, C., Keivens, V. M., O' Toole, T. E., McDonald, J. A., and Ginsberg, M. H. (1995) Cell 83, 715-724 [Medline] [Order article via Infotrieve]
  31. Wu, C., Bauer, J., Juliano, R. L., and McDonald, J. A. (1993) J. Biol. Chem. 268, 21883-21888 [Abstract/Free Full Text]
  32. Alig, L., Edenhofer, A., Hadvary, P., Hurzeler, M., Knopp, D., Muller, M., Steiner, B., Trzeciak, A., and Weller, T. (1992) J. Med. Chem. 35, 4393-4407 [Medline] [Order article via Infotrieve]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.