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DEPARTMENT OF VASCULAR BIOLOGY


STAFF

David J. Loskutoff, Ph.D. Member and Chairman
David A. Cheresh, Ph.D.* Member
Kenneth R. Chien, M.D., Ph.D. Adjunct Member
Linda K. Curtiss, Ph.D.* Associate Member
Gregory J. del Zoppo, M.D.** Associate Member
Thomas S. Edgington, M.D.* Member
Carol A. Fulcher, Ph.D.** Associate Member
Mark H. Ginsberg, M.D. Member
John H. Griffin, Ph.D.** Member
Mary Jo Heeb, Ph.D.** Assistant Member
Thomas J. Kunicki, Ph.D.** Associate Member
Eugene G. Levin, Ph.D.** Associate Member
Joseph C. Loftus, Ph.D. Adjunct Assistant Member
Nigel Mackman, Ph.D.* Associate Member
Edwin L. Madison, Jr., Ph.D. Associate Member
Lindsey Miles, Ph.D. Associate Member
Timothy E. O'Toole, Ph.D. Assistant Member
Wolfram Ruf, Ph.D.* Associate Member
Zaverio Ruggeri, M.D.** Member
Raymond R. Schleef, Ph.D. Associate Member
Martin A. Schwartz, Ph.D. Associate Member
Dietmar Seiffert, M.D.*** Dupont-Merck, Wilmington, DE
Sanford J. Shattil, M.D. Member
Yoshikazu Takada, M.D., Ph.D. Associate Member
Jerry L. Ware, Ph.D.** Associate Member
Mark J. Yeager, M.D., Ph.D. ****
Associate Member


SENIOR RESEARCH ASSOCIATES

Fahumiya Samad, Ph.D.


POSTDOCTORAL RESEARCH FELLOWS

Sarvesh Adda, Ph.D.
Elizabeth K. Baker, Ph.D.
Grigory I. Belogrudov, Ph.D.
Charito S. Buensuceso, Ph.D.
David A. Calderwood, Ph.D.
Gary Coombs, Ph.D.
Joseph L. Chuang, Ph.D.
Gang Deng, M.D., Ph.D.*** Berlex Biosciences, Richmond, CA
Martin Eigenthaler, M.D.*** Medizinische Universitätsklinik Bau 4, Würzburg, Germany
Csilla A. Fenczik, Ph.D.
Kazuhiko Fujisawa, M.D.
Jingbo Gao, M.D.
Yun Gong, M.D.
Stephen B. Hawley, Ph.D.
Laurence Hervio, Ph.D.
Sibylle Hess, Ph.D.
Liane Hofferer, Ph.D.*** IMMUNO AG, Orth/Donau, Austria
Geng Hu, Ph.D.
Paul Edward Hughes, Ph.D.
Hayato Ihara, Ph.D.
Fred E. Indig, Ph.D.*** The Salk Institute, La Jolla, CA
Atsushi Irie, Ph.D.*** Kumamoto University, Kumamoto, Japan
Tetsuji Kamata, M.D.
Hirokazu Kashiwagi, M.D.
Song-Hua Ke, Ph.D.*** Novex, San Diego, CA
Jean M. Lewis, Ph.D.
Johann Y. Lin, M.D., Ph.D.
Souchun Liu, Ph.D.
Jere E. Meredith, Jr., Ph.D.
Martin Pfaff, Ph.D.*** INSERM U331, Lyon Cedex, France
Leo S. Price, Ph.D.
Gregory B. Quinn, Ph.D.
Joe W. Ramos, Jr., Ph.D.
Xiang-Dong Ren, M.D., Ph.D.
Mark W. Renshaw, Ph.D.
Marc Roesel, Ph.D.
David M. Rose, Ph.D.
Ann Stiko, M.D., Ph.D.
Eileen Collins Tozer, Ph.D.
Song Xue, M.D., Ph.D.
Koji Yamamoto, M.D., Ph.D.*** Nagoya University School of Medicine, Nagoya Aichi, Japan
Ana-Belen Ybarrondo, Ph.D.*** Pharmingen, San Diego, CA
Kenji Yokoyama, M.D.
Qi Zhang, M.D., Ph.D.
Yanliang Zhang, Ph.D.
Roy Zent, M.D., Ph.D.


VISITING INVESTIGATORS

Victor Marder, M.D.*** University of Rochester Medical Center, Rochester, NY
Tor Ny, Ph.D.*** University of Umea, Umea, Sweden
Tariq Jabbar Sethi, M.D., Ph.D.
Jan van Mourik, Ph.D.*** Central Laboratory of the Netherlands Red Cross, Amsterdam, the Netherlands
Anton van Zonneveld, Ph.D.*** University of Amsterdam, Amsterdam, the Netherlands


* Joint appointment in Department of Immunology
** Joint appointment in Department of Molecular and Experimental Medicine
*** Appointment completed; new location shown
**** Joint appointments in Departments of Cell Biology and Molecular Biology
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Chairman's Overview

David J. Loskutoff, Ph.D.

When considered in its entirety, the vascular system is by far the largest organ in the body, and vascular diseases are the most common cause of death and disability in Western societies. Members of the Department of Vascular Biology seek to define the basic molecular and cellular mechanisms that (1) control cell signaling, gene expression, and growth in the walls of normal and abnormal vessels and (2) regulate thrombosis, inflammation, flow, and other processes that guarantee vascular homeostasis. Cell adhesion biology and extracellular proteolysis are two common themes of this work, and these two areas of interest are rapidly converging. In this overview, I briefly highlight some of the accomplishments of our investigations and point out how our basic studies of molecules, cells, and mice are leading to the development of novel strategies for treatment of human vascular disease. Although no overview can provide the interesting detail of the individual studies, I hope this summary will stimulate you to read the more complete reports that follow.

Cell adhesion is a major research theme of our work, and several world leaders and promising young investigators in this field are members of the department. For example, Mark Ginsberg, a senior investigator in the department, was one of the pioneers in the discovery of the integrin family of adhesion receptors and in the analysis of the ligand binding and signaling mechanisms of integrins. His studies with peptide inhibitors have led to the development of a new class of drugs for prevention of heart attacks and complications of angioplasty that are now in clinical trials. More recently, his studies on the relationship between cell adhesion and migration, on novel genetic approaches for the study of integrin function, and on the connections between integrins and cancer-causing genes have defined major new research directions.

Another senior investigator in the department, Martin Schwartz, pioneered the analysis of how cell adhesion controls the growth and survival of cells. More specifically, his studies have provided clues about the signals required for the survival of endothelial and epithelial cells. He has also discovered a connection between a cellular protooncogene, c-Abl, and the integrin family, and his recent analysis of the regulation of anchorage dependence of cell growth offers promising avenues for the development of new therapeutic approaches to cancer and heart disease.

Sanford Shattil, a world-renowned hematologist-oncologist who joined us in 1995, is another senior scientist in the department. His work on the mechanism of platelet adhesion and signal transduction has been on the cutting edge of this rapidly evolving field, and his observations have direct diagnostic and therapeutic implications for a variety of diseases that are complicated by arterial thrombosis. Dr. Shattil's strong clinical ties are bringing increased opportunities for directly translating to clinical practice fundamental work performed in the department.

The department provides a home for young scientists of outstanding potential. For example, Yoshikazu Takada is a leader in the analysis of the ligand-binding sites of integrins. His recent mutational studies of integrin subunits have begun to provide a comprehensive picture of how these adhesion receptors recognize their ligands. Timothy O'Toole is an outstanding young investigator who has played a major role in establishing the role of integrin cytoplasmic domains in integrin signaling. His recent accomplishments include elucidating common structural motifs in integrin cytoplasmic domains involved in activation.

Finally, recent work in my own laboratory has begun to identify new cell adhesion mechanisms. This new direction was prompted by the observation that plasminogen activator inhibitor-1, the primary inhibitor of the fibrinolytic system, not only binds avidly to the adhesive glycoprotein vitronectin but in so doing also inhibits the binding of cells to this matrix protein. These studies emphasize the existence of an integrin-independent mechanism of cell adhesion, and our goals are to delineate the relative roles and interactions between these integrin-dependent and integrin-independent systems.

The department has also provided a focus for investigators who share common interests in cell adhesion and cardiovascular disease but have primary appointments in other departments. Many of these scientists have joint appointments in the Department of Vascular Biology. For example, Tom Kunicki and Zavario Ruggeri of the Department of Molecular and Experimental Medicine are leading authorities on von Willebrand factor and its platelet receptors. Members of their laboratories have performed detailed structural and functional analyses on each of these proteins, resulting in the development of new antithrombotic strategies. Similarly, David Cheresh, whose primary appointment is in the Department of Immunology, is a leader in understanding the development of blood vessels and its therapeutic modification. His studies on synthetic and antibody inhibitors of integrin vß3 offer new promise for therapeutics for cancer and diabetic retinopathy.

Our program in cell adhesion has achieved an international reputation, as evidenced by the 15 articles published in Nature, Science, and Cell by investigators in this program. At the same time, international meetings such as the Cold Spring Harbor Symposia and a Nobel Symposium devoted to this topic have featured speakers from the department. Thus, the Department of Vascular Biology and TSRI offer a special environment for fundamental studies of cell adhesion in vascular biology and for translational research in cardiovascular, inflammatory, and neoplastic diseases in which the adhesive properties of cells have a contributory role.

In addition to research on cell adhesion, the department has a strong international presence in the field of plasminogen activation and will host the VI International Workshop on the Molecular and Cellular Biology of Plasminogen Activation later this year. The choice of the department as the host institution is significant because the workshop is considered by many to be the most important meeting in the field. The plasminogen activation system not only regulates formation and dissolution of fibrin but also plays a key role in a variety of normal and pathologic processes involving cell adhesion, migration, and invasion. In this regard, Ed Madison and Lindsey Miles continue to provide important insights into structure-function relationships in plasminogen and the plasminogen activators, and Ray Schleef and my group study the biosynthesis and mechanism of action of the plasminogen activator inhibitors. These studies have provided clues to the pathogenesis and treatment of cardiovascular disease, especially thrombosis and stroke.

In summary, the past year has been highly successful for the department in terms of scientific accomplishments, the maturation of ongoing projects, and the development of new research programs. Members of this department have presented plenary lectures at international meetings on cancer, thrombosis, and heart disease and at more fundamental venues such as the American Society for Cell Biology, the Federation of European Biochemical Societies, and the American Society for Molecular Biology and Biochemistry. All this progress has occurred with significantly increased funding from the National Institutes of Health, including a new program project grant (S. Shattil, principal investigator). We now have three major program project grants in the department. In addition, we have successfully competed for a large grant for equipment (M. Schwartz, principal investigator). Although we are still a relatively young department, our studies have provided a new understanding of the diseases of the vasculature, including atherosclerosis, thrombosis, stroke, hypertension, spontaneous bleeding, and tumor angiogenesis. I am confident that we will continue to build on this record of accomplishments in the forthcoming year.

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Investigators' Reports


Integrin Structure and Function

E. Baker, D. Calderwood, E. Tozer, C. Fenczik, P. Hughes, J. Lin, S. Liu, J. Ramos, M. Roesel, D. Rose, T. Sethi, R. Zent, M. Ginsberg

Glanzmann's thrombasthenia, an inherited bleeding disorder, can be caused by a defect or deficiency in platelet integrin IIbß3 (GP IIb-IIIa). Studies of thrombasthenia variants have facilitated determination of sites involved in the functions of IIbß3 and other integrins. Such sites include those that bind ligand and those that participate in the "activation" of IIbß3 required for high-affinity binding of ligands such as fibrinogen or a monoclonal antibody, PAC1. We isolated such variants, created in vitro with Chinese hamster ovary cells that express an activated form of IIbß3. These cells were exposed to the mutagen ethyl methane sulfonate, and variants that lost the capacity to bind PAC1 were isolated by fluorescence-activated cell sorting.

These variants were grouped into three phenotypic classes. One comprised integrin mutations that disrupt ligand binding; a second comprised mutations that interfere with the capacity of cells to activate the integrin. Most of these activation-defective mutations were in the integrin cytoplasmic domain, but, surprisingly, several were caused by mutations that affect three closely spaced residues in the ß3 extracellular domain. A third class of mutants had a defect in integrin activation not ascribable to changes in the integrin sequence. Thus, these three classes may represent mutated signaling molecules required for integrin activation. This unbiased genetic approach provides new insights into the structural basis of integrin function and may assist in determining the cellular events that regulate integrin function.

Rapid modulation of ligand-binding affinity (activation) is a central property of the integrin cell adhesion receptors. Using a screen for suppressors of integrin activation, we identified the small GTP-binding protein H-Ras and its effector kinase Raf-1 as negative regulators of integrin activation. H-Ras inhibited the activation of integrins with three distinct and ß subunit cytoplasmic domains. Suppression was not associated with integrin phosphorylation and was independent of both mRNA transcription and protein synthesis. Furthermore, suppression correlated with activation of the ERK MAP-kinase pathway. Thus, regulation of integrin affinity state is a novel, transcription-independent function of a Ras-linked MAP-kinase pathway that may mediate a negative feedback loop in integrin function.

PUBLICATIONS

Baker, E.K., Tozer, E.C., Pfaff, M., Shattil, S.J., Loftus, J.C., Ginsberg, M.H. A genetic analysis of integrin function: Glanzmann thrombasthenia in vitro. Proc. Natl. Acad. Sci. U.S.A. 94:1973, 1997.

Diaz-Gonzalez, F., Forsyth, J., Steiner, B., Ginsberg, M.H. Trans-dominant inhibition of integrin function. Mol. Biol. Cell 7:1939, 1996.

Du, X., Ginsberg, M.H. Integrin IIbß3 and platelet function. Thromb. Haemost., in press.

Eigenthaler, M., Hofferer, L., Shattil, S.J., Ginsberg, M.H. A conserved sequence motif in the integrin ß3 cytoplasmic domain is required for its specific interaction with ß3-endonexin. J. Biol. Chem. 272:7693, 1997.

Faull, R.J., Ginsberg, M.H. Inside-out signaling through integrins. J. Am. Soc. Nephrol. 7/8:1091, 1996.

Ginsberg, M.H., Diaz-Gonzalez, F. Cell adhesion molecules and endothelial cells in arthritis. In: Arthritis and Allied Conditions. Koopman, W.J. (Ed.). Williams & Wilkins, Baltimore, 1996, p. 479.

Ginsberg, M.H., Ruggeri, Z.M., Varki, A.P. Cell adhesion in vascular biology: Series introduction. J. Clin. Invest. 98:1505, 1996.

Hughes, P.E., Renshaw, M.W., Pfaff, M., Forsyth, J., Keivens, V.M., Schwartz, M.A., Ginsberg, M.H. Suppression of integrin activation: A novel function of a Ras/Raf-initiated MAP kinase pathway. Cell 88:521, 1997.

Huttenlocher, A., Ginsberg, M.H., Horwitz, A.F. Modulation of cell migration by integrin mediated cytoskeletal linkages and ligand binding affinity. J. Cell Biol. 134:1551, 1996.

Indig, F.E., Diaz-Gonzalez, F., Stuiver, I., Ginsberg, M.H. Analysis of the tetraspanin CD9-integrin IIbß3 (GPIIb-IIIa) complex in platelet membranes and transfected cells. Biochem. J., in press.

Kashiwagi, H., Schwartz, M.A., Eigenthaler, M., Davis, K.A., Ginsberg, M.H., Shattil, S.J. Affinity modulation of platelet integrin IIbß3 by ß3-endonexin, a selective binding partner of the ß3 integrin cytoplasmic tail. J. Cell Biol., in press.

Palecek, S.P., Loftus, J.C., Ginsberg, M.H., Horwitz, A.F., Lauffenburger, D.A. Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature 385:537, 1997.

Wu, C., Hughes, P.E., Ginsberg, M.H., McDonald, J.A. Identification of a new biological function for the integrin vß3: Initiation of fibronectin matrix assembly. Cell Adhes. Commun. 4:149, 1996.

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Local Expression of Hemostatic Genes

D. Loskutoff, G. Deng, V. de Waard, K. Fujisawa, H. Ihara, F. Samad, A. Stiko, G. Belogrudov, K. Yamamoto

Vascular hemostasis results from the exquisitely regulated interaction and balance between the coagulation and fibrinolytic systems, and imbalances in both systems promote vascular disease. However, little information is available about the cells that express hemostatic genes during normal physiologic conditions or how expression of these genes changes in various pathologic states. The primary hypotheses of our work are that expression of hemostatic genes is highly regulated and not restricted to the liver and that changes in the expression of these genes in tissues alters local hemostatic balance. We have been analyzing tissues from murine models of thrombotic disease to test these hypotheses. Although initial efforts focused on plasminogen activator inhibitor 1 (PAI-1), tissue factor, and vitronectin, we have now extended these studies to include von Willebrand factor, protein C, and transforming growth factor-ß (TGF-ß).

VON WILLEBRAND FACTOR

Although von Willebrand factor is often used as a biochemical marker for endothelial cells, little is known about its relative level of expression and regulation in endothelial cells in different vascular beds in vivo. Analysis by polymerase chain reaction revealed large differences in the concentration of von Willebrand factor mRNA in highly vascularized murine tissues. For example, the lung and brain contained 5--50 times higher concentrations of von Willebrand factor mRNA than did the kidney and liver, and endothelial cells in the lung and brain expressed abundant von Willebrand factor mRNA compared with similar cells in the liver and kidney. We often found signigicantly higher levels of von Willebrand factor mRNA and antigen in endothelial cells in larger vessels compared with microvessels and in venous endothelial cells compared with arterial endothelial cells. Intraperitoneal administration of endotoxin decreased the steady-state level of von Willebrand factor mRNA in all murine tissues examined except the heart and kidney. Thus, von Willebrand factor mRNA is differentially expressed in endothelial cells in different tissues and within the same vascular bed and is regulated by endotoxin in mice.

PROTEIN C

Activated protein C acts as an anticoagulant by inhibiting coagulation factors Va and VIIIa. Although the liver appears to be its primary site of synthesis, the finding that other components of this system are produced extrahepatically suggests that protein C itself may be synthesized in other tissues. Analysis of murine tissues for protein C mRNA revealed low but significant levels in the epididymis (1.7% of the level in liver), brain (1.1% of liver), and lung (0.8% of liver) and relatively high levels in the kidney (35% of liver) and testis (22% of liver). Protein C was detected in epithelial cells in the lung, kidney, and epididymis; in pyramidal neurons in the cerebrum; in Purkinje cells in the cerebellum; and in spermatogenic cells in the testis.

The expression of protein C mRNA in the kidney was significantly decreased in mice with renal disease (eg, in mice with autoimmune lupus nephritis or diabetic nephropathy and in endotoxin-treated mice with acute renal injury). The decreased renal expression of protein C may contribute to the increased procoagulant potential of the kidney during septic and inflammatory processes and to the progression of kidney disease associated with these conditions.

TGF-ß

TNF- is chronically elevated in adipose tissue in obese humans and mice and contributes to the insulin resistance, elevated PAI-1 levels, and cardiovascular complications associated with obesity and non--insulin-dependent diabetes mellitus. We observed that TGF-ß mRNA and protein also were higher in the adipose tissue of genetically obese mice than in the animals' lean counterparts. Administration of TNF- increased expression of TGF-ß mRNA in the adipose tissue of lean mice and stimulated production of TGF-ß by cultured adipocytes. Administration of TGF-ß increased concentrations of PAI-1 antigen in the plasma and PAI-1 mRNA in the adipocytes of lean mice and enhanced the rate of PAI-1 synthesis by adipocytes in vitro. These results suggest that TNF- contributes to the elevated expression of TGF-ß in the adipose tissue of obese mice and that TGF-ß may play a role in the vascular abnormalities associated with obesity and non--insulin-dependent diabetes mellitus.

OTHER GENES

A dramatic increase in PAI-1 activity in plasma and in PAI-1 mRNA in the kidneys was observed in mice in which autoimmune disease develops, and this increase appeared to correlate with the progression of lupus nephritis. The increase in PAI-1 mRNA was relatively specific for the kidney, although PAI-1 mRNA also was higher in the brains of diseased mice. Interestingly, mRNA for urokinase plasminogen activator decreased, and mRNA for tissue factor increased, and these changes also correlated with the development of lupus nephritis and fibrin deposition in the diseased kidneys. The induction of PAI-1 and tissue factor, and the decrease in urokinase plasminogen activator in the kidneys of mice prone to lupus nephritis may promote the formation of microthrombi and thus contribute to the progression of lupus nephritis in this model.

PUBLICATIONS

Deng, G., Curriden, S.A., Wang, S., Rosenberg, S., Loskutoff, D.J. Is plasminogen activator inhibitor-1 the molecular switch that governs urokinase receptor mediated cell adhesion and release? J. Cell Biol. 134:1563, 1996.

Estellés, A., Grancha, S., Gilabert, J., Thinnes, T., Chirivella, M., España, F., Aznar, J., Loskutoff, D.J. Abnormal expression of plasminogen activator inhibitors in patients with gestational trophoblastic disease. Am. J. Pathol. 149:1229, 1996.

Fearns, C., Loskutoff, D.J. Induction of plasminogen activator inhibitor 1 gene expression in murine liver by lipopolysaccharide: Cellular localization and role of endogenous tumor necrosis factor-. Am. J. Pathol. 150:579, 1997.

Lawrence, D.A., Loskutoff, D.J. Plasminogen activator inhibitor type-1. In: Human Protein Data. Haeberli, A. (Ed.). VCH, Weinheim, Germany, in press.

Luther, T., Flössel, C., Mackman, N., Bierhaus, A., Kasper, M., Albrecth, S., Sage, E.H., Iruela-Arispe, L., Grossmann, H., Ströhlein, A., Zhang, Y., Nawroth, P.P., Carmeliet, P., Loskutoff, D.J., Müller, M. Tissue factor expression during human and mouse development. Am. J. Pathol. 149:101, 1996.

Samad, F., Loskutoff, D. The fat mouse: A powerful genetic model to study elevated plasminogen activator inhibitor 1 in obesity/NIDDM. Thromb. Haemost., in press.

Samad, F., Loskutoff, D.J. Tissue distribution and insulin regulation of plasminogen activator inhibitor-1 in obese mice. Mol. Med. 2:568, 1996.

Samad, F., Schneiderman, J., Loskutoff, D. Expression of fibrinolytic genes in tissues from human atherosclerotic aneurysms and from obese mice. Proc. N.Y. Acad. Sci., in press.

Samad, F., Yamamoto, K., Pandey, M., Loskutoff, D.J. Elevated expression of transforming growth factor-ß in adipose tissue from obese mice: A potential role in the obesity-linked increase in plasminogen activator inhibitor-1? Mol. Med. 3:37, 1997.

Schneiderman, J., Adar, R., Engelberg, I., Bordin, G.M., Seiffert, D., Loskutoff, D.J., Dilley, R.B. Medical control of abdominal aortic aneurysm expansion rate. J. Vasc. Surg. 24:297, 1996.

Seiffert, D., Loskutoff, D.J. Type 1 plasminogen activator inhibitor induces multimerization of plasma vitronectin: A suggested mechanism for the generation of the tissue-form of vitronectin in vivo. J. Biol. Chem. 271:29644, 1996.

van Aken, B.E., Seiffert, D., Thinnes, T., Loskutoff, D.J. Localization of vitronectin in the normal and atherosclerotic human vessel wall. Histochem. Cell Biol., in press.

Yamamoto, K., Loskutoff, D.J. Fibrin deposition in tissues from endotoxin-treated mice correlates with decreases in the expression of urokinase-type but not tissue-type plasminogen activator. J. Clin. Invest. 97:2440, 1996.

Yamamoto, K., Loskutoff, D.J. The kidneys of mice with autoimmune disease acquire a hypofibrinolytic/procoagulant state that correlates with the development of glomerulonephritis and tissue microthrombosis. Am. J. Pathol., in press.

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Structure and Function of Tissue-Type Plasminogen Activator

L. Hervio, Y. Zhang, K. Tachias, E. Madison

The primary goal of our laboratory is to elucidate the relationships between structure and function in members of the chymotrypsin family of serine proteases, a gene family that contains many medically important members. We are especially interested in conformational changes and protein-protein interactions that regulate the activity of these key enzymes. To date, we have focused primarily on proteases of the fibrinolytic cascade, in particular, tissue-type plasminogen activator (tPA) and urokinase plasminogen activator (uPA), the two enzymes that catalyze the rate-limiting step in this cascade. The tight regulation of the activity of tPA in vivo, which involves conformational changes, interactions with at least six other proteins, and remarkably stringent substrate specificity, continues to provide a particularly fertile and intriguing model system.

Most recently, we have investigated regulation of the activity of tPA and uPA by zymogen activation, regulation of tPA by interaction with specific cofactors, evolution of substrate specificity by tPA and uPA, and coevolution of specificity between serine proteases and serpins (serine protease inhibitors). These studies have resulted in the design and characterization of variants of uPA that are resistant to inhibition by plasminogen activator inhibitor 1 and variants of tPA that (1) are resistant to inhibition by serpins, (2) have zymogen-like properties, (3) have a dramatically enhanced response and selectivity toward fibrin cofactors, and (4) have a significantly enhanced half-life in the circulation in vivo. These investigations have also produced variants of plasminogen activator inhibitor 1 that, unlike the wild-type protein, selectively inhibit tPA or uPA and a "revertant plasminogen activator inhibitor 1" that can rapidly inhibit one of our serpin-resistant variants of tPA.

During the past 2 years, we have developed several novel techniques. We recently described an improved protocol for using the polymerase chain reaction to accomplish site-directed mutagenesis. We have also reported a technique we call "protein loop grafting" that facilitates the targeting of a protein of interest to a desired, new receptor. We used this technique to construct a variant of tPA that bound to the platelet integrin IIbß3 with an apparent KD of 1 nM. In addition, we have developed a new technique that uses phage display to produce "substrate subtraction libraries" that can be used to elucidate subtle differences in specificity between closely related enzymes. These studies not only produced new insights into the evolution of specificity by serine proteases but also provided a rational basis for the design of highly selective, small-molecule inhibitors of tPA or uPA.

In addition to providing fundamental new insights into the molecular determinants of catalysis and specificity for the chymotrypsin family of enzymes, our studies on structure and function may aid in the development of improved therapeutic agents for the treatment of acute myocardial infarction and other thrombotic disorders. In current thrombolytic therapy, intravenous administration of tPA is used to lyse stable, occlusive thrombi. However, two mechanisms that normally regulate the activity of endogenous tPA, rapid inactivation of the enzyme by specific inhibitors present in human plasma and rapid removal of the enzyme from the circulation by hepatic receptors, have important adverse consequences for the therapeutic use of tPA. We have therefore constructed and are testing variants of tPA that are resistant to both of these constraints.

PUBLICATIONS

Fujise, K., Revelle, B.M., Stacy, L., Madison, E.L., Yeh, E.T.H., Willerson, J.T., Beck, P.J. A t-PA/P-selectin fusion protein is an effective thrombolytic agent. Circulation 95:715, 1997.

Ke, S., Coombs, G., Corey, D., Navre, M., Madison, E.L. Use of substrate subtraction libraries to distinguish closely related enzymes. J. Biol. Chem., in press.

Ke, S., Tachias, K., Lamba, D., Bode, W., Madison, E.L. Identification of a hydrophobic exosite on tissue-type plasminogen activator that modulates specificity for plasminogen. J. Biol. Chem. 272:1811, 1997.

Madison, E.L. Molecular determinants of the substrate and inhibitor specificity of tissue-type plasminogen activator. In: Proceedings of the International Symposium on the Chemistry and Biology of Serpins. Church, F. (Ed.). Plenum, New York, in press.

Tachias, K., Madison, E.L. Converting tissue-type plasminogen activator into a zymogen. J. Biol. Chem. 271:28749, 1996.

Tachias, K., Madison, E.L. Converting tissue-type plasminogen activator into a zymogen: Important role of lys 156. J. Biol. Chem. 272:28, 1997.

Tachias, K., Madison, E.L. Variants of tissue-type plasminogen activator that display extraordinary resistance to inhibition by the serpin plasminogen activator, type 1. J. Biol. Chem. 272:14580, 1997.

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Regulation of the Plasminogen Activation System

J. Felez,* Y. Gong, S. Hawley, R.J. Parmer,** S. Xue, L.A. Miles

* Institut de Recerca Oncologica, Barcelona, Spain
** University of California, San Diego, CA

Cell-surface receptors for plasminogen and the plasminogen activators, urokinase and tissue-type plasminogen activator, positively regulate the plasminogen activation system by enhancing activation of plasminogen and protecting plasmin from inactivation by protease inhibitors. These receptors also localize the proteolytic activity of plasmin on cell surfaces to promote degradation of the extracellular matrix, which is crucial for processes that require cell migration. We are investigating the structure, function, and regulation of these receptors.

We have identified an -enolase--related molecule (-ERM) as a plasminogen-binding protein present on the surfaces of peripheral blood monocytes, neutrophils, and monocytoid cell lines. We produced a monoclonal antibody to -ERM that recognized -ERM on the cell surface. By phage display of random fragments of human -enolase cDNA, we localized the epitope recognized by the antibody to a region within an external loop of -enolase. Other experiments showed that -ERM is not an integral membrane protein, suggesting that it may bind to a membrane protein. On different cell types, -ERM may be oriented differently on the cell surface: on some cell types, its carboxyl-terminal lysine (required for plasminogen binding and activation on cells) is available for interaction, whereas on other cell types, this key residue is less accessible. We are studying the mechanisms by which -ERM is bound to the cell surface.

In addition to -ERM, two major plasminogen-binding membrane proteins with Mr's similar to that of -enolase (but with distinct pIs) were detected. These proteins also expose carboxyl-terminal lysines on the cell surface. We are purifying these novel plasminogen-binding proteins.

The receptor for urokinase plasminogen activator (uPAR) positively regulates activation of plasminogen. Previous studies have shown that human urokinase does not bind to murine uPAR and that murine urokinase does not recognize human uPAR. However, we found that species specificity was not absolute: human urokinase bound with high affinity to hamster, rat, and bovine uPARs. We cloned hamster, rat, and bovine uPARs and compared the translated amino acid sequences with the published sequences for human and murine uPARs. When the five sequences were aligned, 18 amino acid residues were unique to the murine uPAR sequence. At 6 of these positions, all the other sequences shared the same amino acid residue. We are currently examining whether these differences may account for the inability of murine uPAR to recognize human urokinase.

The plasminogen activation system is negatively regulated by molecules that interfere with binding of plasminogen to its substrates and regulatory molecules. Lipoprotein(a), which is associated with atherosclerosis and with disease processes involving thrombosis, contains an apoprotein with a sequence highly homologous to the amino acid sequence of plasminogen. Hence, lipoprotein(a) binds directly to cells, fibrin, and the extracellular matrix and competes for the binding of plasminogen to these regulatory surfaces. These interactions may contribute to the proatherothrombogenic consequences of high levels of lipoprotein(a). We found that multiple domains within apoprotein(a) may modulate these interactions, and we are expressing these domains in both bacterial and mammalian systems to test their function directly.

The plasminogen activation system is also regulated by the synthesis and secretion of components of the system. Using several chromaffin cell sources, including the rat pheochromocytoma PC-12 chromaffin cell line, primary cultures of bovine adrenal chromaffin cells, and human pheochromocytoma cells, we found that tissue-type plasminogen activator enters the regulated secretory pathway and is packaged in and released directly from catecholamine storage vesicles. Therefore, these vesicles may be an important reservoir, and sympathoadrenal activation may be an important physiologic mechanism for the rapid release of this plasminogen activator.

PUBLICATIONS

Arza, B., Félez, J., Miles, L.A., Muñoz-Cánoves, P. Identification of an epitope of -enolase (a candidate plasminogen receptor) by phage display. Thromb. Haemost. 78:1097, 1997.

Félez, J., Miles, L.A., Fabregas, P., Jardi, M., Plow, E.F., Lijnen, H.R. Characterization of cellular receptors and interactive regions within reactants required for enhancement of plasminogen activation by tPA on the surface of leukocytic cells. Thromb. Haemost. 76:577, 1996.

Jardí, M., Inglés-Esteve, J., Burgal, M., Azqueta, C., Velasco, F., Miles, L.A., Félez, J. Distinct patterns of urokinase receptor (uPAR) expression by leukemic cells and peripheral blood cells. Thromb. Haemost. 76:1009, 1996.

Jenkins, G.R.E., Seiffert, D., Parmer, R.J., Miles, L.A. Regulation of plasminogen gene expression by interleukin-6 in hepatoma and murine liver cells. Blood 89:2394, 1997.

Kim, Sun-Ok, Plow, E.F., Miles, L.A. Regulation of plasminogen receptor expression on monocytoid cells by ß1-integrin-dependent cellular adherence to extracellular matrix proteins. J. Cell Biol. 271:23761, 1996.

Parmer, R.J., Mahata, M., Mahata, S., Sebald, M.T., O'Connor, D.T., Miles, L.A. Tissue plasminogen activator (t-PA) is targeted to the regulated secretory pathway: Catecholamine storage vesicles as a reservoir for the rapid release of t-PA. J. Biol. Chem. 272:1976, 1997.

Plow, E.F., Redlitz, A., Hawley, S.B., Xue, S., Herrin, T., Hoover-Plow, J.L., Miles, L.A. Assembly of the plasminogen system on cell surfaces. In: Handbook of Experimental Pharmacology: Fibrinolytics and Antifibrinolytics. Bachmann, F. (Ed.). Springer-Verlag, New York, in press.

Plow, E.F., Ugarova, T., Miles, L.A., Interaction of the fibrinolytic system with the vessel wall. Thromb. Hemorrhage, in press.

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Integrin Cytoplasmic Domains and Signaling

T.E. O'Toole, C. Buensuceso, D. Normand

Members of the integrin family of adhesion receptors are notable for their bidirectional signaling properties. In response to external agonists, cellular signals are transduced in an "inside out" fashion, inducing an extracellular conformational change of the integrin and allowing high-affinity binding of ligand. After binding, events are transduced in an "outside in" fashion, inducing cellular responses such as growth, differentiation, survival, and migration. Previous work has implicated the cytoplasmic domains of the and ß subunits of integrins in bidirectional signaling. Typical models propose that the interaction of these sequences with intracellular or membrane-bound proteins mediate these events.

We are using a number of systems to determine such protein interactions. With the yeast two-hybrid approach, we isolated ß3-endonexin, a cytosolic, protein that specifically binds integrin ß3, and we are studying its functional attributes. We are now adopting this strategy to screen for proteins that bind to other integrin tails. To complement the yeast two-hybrid system, we are also screening cDNA expression libraries with protein fragments or peptides. Targeted areas include an Asn-Pro-x-Tyr (NPXY) motif that is conserved in most of the integrin ß subunits, a conserved stretch of consecutive hydroxylated residues also in the ß cytoplasmic tail, and membrane proximal sequences in either the or ß tails. Other anticipated strategies for detecting interacting proteins include the use of affinity matrices or coimmunoprecipitation.

In a final area, we are examining the role of LIM domains in cell signaling. LIM domains are protein motifs characterized by a repetitive spacing of histidine and cysteine residues. As such, they can coordinate zinc ions and form a two-looped finger structure. These motifs have been implicated in protein-protein interactions. Data from other proteins suggest that integrin tails have the primary structure necessary for recognition by certain LIM domains. Also, a number of proteins that contain LIM domains reside in focal contacts, cellular clusters of integrins and other signaling molecules. Studies of the binding partners and signaling function of several LIM proteins are under way.

PUBLICATIONS

O'Toole, T.E. Integrin signaling: Building connections beyond the focal contact? Matrix Biol., in press.

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Intracellular Processing and Trafficking of Protease Inhibitors in Platelets

R.R. Schleef, J. Chang, I.M. Lang, L. Gombau, M. Riewald, C.F. Barbas*

* Department of Molecular Biology, TSRI

Platelets play a central role in cardiovascular diseases through the release and cell-surface expression of a variety of hemostatic proteins. During the formation of platelets within megakaryocytes in the bone marrow or after the release of platelets into the circulating blood, these molecules are deposited into storage organelles called -granules. Knowledge of the factors involved in the processing of proteins and their deposition into secretory granules will provide information on basic cellular activities and may reveal novel approaches for controlling the coagulation and fibrinolytic systems.

We have shown that coagulation and fibrinolytic protease inhibitors are packaged and stabilized within -granules in concert with a series of defined proteins in a calcium-dependent manner. To determine proteins that may participate in the targeting or storage of these potent inhibitors, we used filamentous bacteriophages to display proteins expressed by cells containing a regulated secretory pathway and enriched the proteins by using their affinity for different protease inhibitors. One novel cDNA clone that preferentially recognized solution-phase plasminogen activator inhibitor 1 and reacted positively with antibodies derived from a rabbit immunized with -granules was recombinately expressed, and the purified recombinant protein was used to generate polyclonal antibodies. These immunologic reagents were used to develop a purification protocol for a novel molecule that may be involved in the packaging of proteins into storage granules.

Because proteases are involved not only in the processing of proteins into storage granules but also in a number of basic cellular events, we have been analyzing the cytoplasmic molecules that control the activity of cell-associated proteases in the hematopoietic compartment. We have cloned a novel protease inhibitor that we call bomapin because its expression is restricted to hematopoietic cells within the bone marrow. To understand the expression of bomapin within the hematopoietic compartment, we examined RNA extracted from bone marrow or peripheral blood from healthy donors and from patients with leukemia. Bomapin mRNA was readily detected in normal bone marrow, at a level designated as medium. Bomapin expression in peripheral blood from healthy donors and from patients with chronic lymphocytic leukemia was low or undetectable. Blood from patients with chronic myeloid leukemia, chronic myelomonocytic leukemia, acute myeloid leukemia, and acute lymphocytic leukemia had low to medium levels of bomapin expression. In addition, one patient with acute monocytic leukemia had high levels of bomapin.

We extended these studies by analyzing the expression of bomapin in a series of tissue culture cell lines. Bomapin mRNA was detected in the monocytic THP-1 and AML-193 cell lines, and treatment of these cell lines with phorbol myristate acetate or TNF- reduced the bomapin mRNA levels over a 4-day period. Immunoblotting showed the presence of a 40-kD protein in the cytosol of THP-1 cells. Levels of bomapin antigen were correspondingly reduced after treatment with phorbol myristate acetate. Because phorbol myristate acetate and TNF- induce monocytic differentiation in THP-1 and AML-193 cells, these data suggest that bomapin may play a role in the regulation of protease activities, specifically during the early stages of cellular differentiation.

PUBLICATIONS

Lang, I.M., Barbas, C.F. III, Schleef, R.R. Recombinant rabbit Fab with binding activity to type 1 plasminogen activator inhibitor derived from a phage-display library against human -granules. Gene 172:295, 1996.

Lang, I.M., Chuang, T.L., Barbas, C.F. III, Schleef, R.R. Purification of storage granule protein-23: A novel protein identified by phage display technology and interaction with type 1 plasminogen activator inhibitor. J. Biol. Chem. 271:30125, 1996.

Lang, I.M., Moser, K.M., Schleef, R.R. Expression of Kunitz protease inhibitor containing forms of amyloid ß-protein precursor within vascular thrombi. Circulation 94:2728, 1996.

Salonen, E.-M., Gombau, L., Engvall, E., Schleef, R.R. Human glioma U-251 cells contain type 1 plasminogen activator inhibitor in a rapidly releasable form. FEBS Lett. 393:216, 1996.

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Signal Transduction by Integrins

M.A. Schwartz, S. Hess, J.M. Lewis, J.E. Meredith, L.S. Price, X.-D. Ren, M.W. Renshaw

In addition to its structural role in tissue organization and cell adhesion, the extracellular matrix regulates a variety of cell functions, including growth, survival, and gene expression. It appears that the ability of integrins to transduce signals across the plasma membrane accounts for many of these regulatory effects.

Much of our work is directed toward understanding why cells require both growth factors and integrin-mediated adhesion to proteins in the extracellular matrix to progress through the cell cycle. This fact implies that signals from growth factors and integrins must converge at some point to regulate key events in the cell cycle. Previously, we showed that activation of inositol lipid hydrolysis by growth factors required integrin-mediated adhesion, because adhesion controlled the levels of 4,5-phosphatidylinositol bisphosphate, the substrate for the phospholipase C that is activated by growth factors. Stimulation of inositol lipid synthesis by adhesion involved the small GTP-binding protein Rho. Thus, integrin- and Rho-dependent events act synergistically with a growth factor--dependent pathway to trigger production of second messengers that promote cell proliferation.

Malignant transformation of cells involves acquisition of both serum and anchorage independence; that is, cells can grow without growth factors or adhesion. Our model suggests that constitutive activation of a step on an integrin pathway should induce anchorage independence but not serum independence. To test this hypothesis and assess the role of Rho, we characterized the effects of two Rho nucleotide exchange factors, proteins that constitutively activate endogenous Rho. These proteins are oncogenic, but we found that they induce anchorage-independent growth without changing cells' requirement for serum. They also induce anchorage-independent activation of inositol lipid synthesis and enable cells in suspension to respond to growth factors as if the cells were adherent. These results support a model in which Rho mediates an early event on an integrin-dependent signaling pathway before convergence with growth factor pathways.

We have also studied the joint regulation of the MAP kinase pathway by integrins and growth factors. We found that serum or growth factors could activate the MAP kinase ERK2 in adherent cells but did so poorly in suspended cells. The point in the MAP kinase cascade that was sensitive to adhesion was between Raf and MEK1; steps before MEK1 were activated normally in suspended cells, but later steps were blocked. Thus, at least two important growth-control pathways (inositol lipids and MAP kinase) require both growth factors and cell adhesion.

We are also investigating the role of the Rho-family small GTPases cdc42 and Rac in integrin-mediated signal transduction. These proteins appear to be activated by cell adhesion and to mediate cell spreading, migration, and possibly other events. We are also studying the nonreceptor tyrosine kinase c-Abl. We showed that c-Abl transiently localizes to focal adhesions during cell spreading and that integrin-mediated adhesion regulates activity and localization of c-Abl kinase. More recently, we found that c-Abl interacts with the focal adhesion protein paxillin and participates in the integrin-induced activation of MAP kinase.

Finally, we are studying ß1c, an alternatively spliced variant of the integrin ß1 subunit. In ß1c, the C-terminal 20 residues of the ß1 cytoplasmic domain are replaced with 42 unique residues. The ß1c variant does not localize to focal adhesions and markedly inhibits progression of the cell cycle, leading to a block in late G1 near the restriction point. We are continuing to investigate the mechanism by which ß1c inhibits cell growth.

PUBLICATIONS

Brooks, P.C., Klemke, R.L., Schön, S., Lewis, J.M., Schwartz, M.A., Cheresh, D.A. Insulin-like growth factor receptor cooperates with integrin vß5 to promote tumor cell dissemination in vivo. J. Clin. Invest. 99:1390, 1997.

Hughes, P.E., Renshaw, M.W., Pfaff, M., Forsyth, J., Keivens, V.M., Schwartz, M.A., Ginsberg, M.H. Suppression of integrin activation: A novel function of a Ras/Raf-initiated MAP kinase pathway. Cell 88:521, 1997.

Kashiwagi, H., Schwartz, M.A., Eigenthaler, M., Davis, K.A., Ginsberg, M.H., Shattil, S.J. Affinity modulation of platelet integrin IIbß3 by ß3-endonexin, a selective binding partner of the ß3 integrin cytoplasmic tail. J. Cell Biol. 137:1433, 1997.

Kreisberg, J.I., Radnik, R.A., Schwartz, M.A. Involvement of Rho and myosin phosphorylation in cAMP-induced disassembly of actin stress fibers. Am. J. Physiol. 273:F283, 1997.

Lewis, J.M., Baskaran, R., Taagepera, S., Schwartz, M., Wang, J.Y.-J. Integrin regulation of c-Abl tyrosine kinase activity and cytoplasmic-nuclear transport. Proc. Natl. Acad. Sci. U.S.A. 93:15174, 1996.

Lewis, J.M., Cheresh, D.A., Schwartz, M.A. Protein kinase C regulates vß5-dependent cytoskeletal associations and FAK phosphorylation. J. Cell Biol. 134:1323, 1996.

Meredith, J.E., Schwartz, M.A. Integrins, adhesion and apoptosis. Trends Cell Biol. 7:146, 1997.

Renshaw, M.W., Toksoz, D., Schwartz, M.A. Involvement of the small GTPase Rho in integrin-mediated activation of MAP kinase. J. Biol. Chem. 271:21691, 1996.

Schwartz, M.A., Toksoz, D., Khosravi-Far, R. Transformation by Rho exchange factor oncogenes is mediated by activation of an integrin dependent pathway. EMBO J. 15:6525, 1996.

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Regulation of the Adhesive and Signaling Functions of Platelet Integrins

S.J. Shattil, J. Gao, M.H. Ginsberg, H. Kashiwagi, L. Leng, N. Pampori

The research areas of cell adhesion and signaling, originally considered separate, have begun to converge because of discoveries that cell adhesion receptors are often signaling receptors: the receptors can transmit biochemical and mechanical information in both directions across the plasma membrane. A good example is the integrin superfamily of ß heterodimeric receptors.

The adhesive function of integrins is required for interactions between cells and between cells and the extracellular matrix during embryonic development and pathophysiologic responses to tissue injury and disease. The signaling function of integrins is needed to optimize the adhesion response, because the affinity and avidity of these receptors for ligands are usually tightly regulated by the cell. Moreover, the binding of adhesive ligands to integrins triggers tyrosine phosphorylation and other biochemical reactions required for anchorage-dependent cell growth, differentiation, and survival. Bidirectional signaling requires specific interactions of integrin ß subunits with intracellular signaling proteins on the one hand and with extracellular adhesive ligands on the other. Efforts in this laboratory are centered on understanding the molecular bases of these interactions.

One focus is to characterize direct interactions of the cytoplasmic tails of integrin IIbß3 with specific intracellular signaling molecules in platelets. This integrin is necessary for fibrinogen-dependent platelet aggregation and hemostasis and is also involved in pathologic formation of thrombus in arteriosclerosis and other vascular diseases. Furthermore, specific mutations or deletions within the ß3 cytoplasmic tail, although rare, are responsible for heritable bleeding disorders due to defective integrin signaling. Currently, we are investigating functional if not physical interactions between IIbß3 and nonreceptor tyrosine kinases and phosphatases, phosphatidylinositol 3-kinases, and small GTPases of the Rho family. In addition, genetic screening techniques are being used to discover proteins that interact directly with the cytoplasmic tails of IIb or ß3. Once these proteins are determined, their roles in integrin signaling are evaluated. A case in point is a novel integrin tail--binding protein called ß3-endonexin.

ß3-Endonexin is a 111 amino acid polypeptide that was detected on the basis of its ability to interact with the cytoplasmic tail of the ß3 integrin subunit. ß3-Endonexin is widely expressed in human tissues, including platelets. The interaction with the cytoplasmic tail is selective for the ß3 tail, because ß3-endonexin does not bind to other integrin or ß tails. Mutagenesis studies of integrin tails expressed in yeast and in mammalian cells showed that this selectivity of ß3-endonexin is accounted for by a linear sequence of amino acids at the carboxyl-terminus of the ß3 cytoplasmic tail. This sequence is different from that of any other integrin tail, but it is conserved in ß3 subunits across species. Recent studies in other laboratories have detected additional intracellular proteins, structurally distinct from ß3-endonexin, that interact selectively with the ß1 or ß2 cytoplasmic tails. Thus, endonexin may be a member of a class of integrin tail--binding proteins that are specific for and influence the function of only a single type of integrin subunit.

To explore the function of ß3-endonexin, we fused its cDNA with that of green fluorescent protein (GFP) and transiently transfected the fusion protein into Chinese hamster ovary cells that stably express IIbß3. GFP/ß3-endonexin was found in both the cytoplasm and the nucleus. PAC1, a fibrinogen-mimetic monoclonal antibody, was used to monitor the affinity of IIbß3 in transfectant cells. Cells transfected with GFP alone bound little or no PAC1, indicating that IIbß3 was in a low-affinity state. In contrast, GFP/ß3-endonexin switched IIbß3 into an energy-dependent high-affinity state that was capable of binding PAC1 and supporting fibrinogen-dependent aggregation of the cells. Studies with ß3 cytoplasmic tail mutants indicated that this functional effect of ß3-endonexin tracked with its ability to bind to the ß3 tail. Thus, ß3-endonexin can modulate the affinity of IIbß3 in a manner that is structurally specific and subject to metabolic regulation.

By analogy, the adhesive function of platelets may be regulated by similar interactions between proteins. Furthermore, most cells contain several different integrins but must respond to environmental cues in a specific and coordinated manner. Modulation of integrin function by integrin tail--binding proteins such as ß3-endonexin may help determine the specificity of cellular responses to adhesive ligands.

PUBLICATIONS

Abrams, C., Shattil, S.J. The platelet integrin, GP IIb-IIIa (IIbß3). In: The Platelet. Lapetina, E. (Ed.). JAI Press, Greenwich, CT, 1997, p. 67. Part of the series Advances in Molecular and Cell Biology.

Baker, E.K., Tozer, E.C., Pfaff, M., Shattil, S.J., Loftus, J.L., Ginsberg, M.H. A genetic analysis of integrin function: Glanzmann thrombasthenia in vitro. Proc. Natl. Acad. Sci. U.S.A. 94:1973, 1997.

Brugge, J.S., Clark, E.A., Shattil, S.J. Platelet tyrosine phosphorylation. In: The Platelet. Lapetina, E. (Ed.). JAI Press, Greenwich, CT, 1997, p. 335. Part of the series Advances in Molecular and Cell Biology.

Eigenthaler, M., Hofferer, L., Shattil, S.J., Ginsberg, M.H. A conserved sequence motif in the integrin ß3 cytoplasmic domain is required for its specific interaction with ß3-endonexin. J. Biol. Chem. 272:7693, 1997.

Eigenthaler, M., Shattil, S.J. ß3-Endonexin. In: Guidebook to the Cytoskeletal and Motor Proteins. Kreis, T., Vale, R. (Eds.). Oxford University Press, New York, in press.

Eigenthaler, M., Shattil, S.J. Integrin signaling and the platelet cytoskeleton. Curr. Top. Membr. 43:265, 1996.

Kashiwagi, H., Eigenthaler, E., Schwartz, M.A., Davis, K., Ginsberg, M.H., Shattil, S.J. Affinity modulation of platelet integrin IIbß3 by ß3-endonexin, a selective binding partner of the ß3 integrin cytoplasmic tail. J. Cell Biol. 137:1433, 1997.

Kunicki, T.J., Annis, D.S., Shattil, S.J. A molecular basis for affinity modulation of Fab ligand binding to integrin IIbß3. J. Biol. Chem. 271:20315, 1996.

McCrae, K.R., Shattil, S.J. Disorders of platelet function. In: Textbook of Internal Medicine, 3rd ed. Kelley, W.N. (Ed.). Lippincott, Philadelphia, 1997, p. 1419.

Phillips, D.R., Teng, W., Arfsten, A., Nanizzi-Alaimo, L., White, M.M., Longhurst, C., Shattil, S.J., Randolph, A., Jakubowski, J.A., Jennings, L.K., Scarborough, R.M. Effect of Ca2+ on integrilin-GP IIb-IIIa interactions: Enhanced GP IIb-IIIa binding and inhibition of platelet aggregation by reductions in the concentration of ionized calcium in plasma anticoagulated with citrate. Circulation, in press.

Ruggeri, Z.M., FitzGerald, G.A., Shattil, S.J. Platelet thrombus formation and anti-platelet therapy. In: Molecular Basis of Heart Disease. Chien, K.R., et al. (Eds.). Saunders, Philadelphia, in press.

Shattil, S.J., Gao, J., Kashiwagi, H. Not just another pretty face: Regulation of platelet function at the cytoplasmic face of integrin IIbß3. Thromb. Haemost. 77:220, 1997.

Shattil, S.J., Ginsberg, M.H. Integrin signaling in vascular biology. J. Clin. Invest. 100:1, 1997.

Wiedmer, T., Sims, P., Shattil, S.J. Use of flow cytometry in the analysis of activated platelets and platelet-derived microparticles. In: Flow Cytometry of the Megakaryocyte-Platelet System. Scharf, R., Clemetson, K. (Eds.). Elsevier/North-Holland Biomedical Press, New York, in press.

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Structure and Functions of Integrins

Y. Takada, T. Kamata, A. Irie, W. Puzon-McLaughlin, X. Zhang

Cell adhesion plays crucial roles in wound healing, development, immune responses, and metastasis. Integrins, heterodimers that act as receptors during cell adhesion, mediate interactions between cells and between cells and the extracellular matrix. The integrin 4ß1 recognizes vascular cell adhesion molecule-1 and the alternatively spliced IIICS part of fibronectin (connecting segment-1). Vascular cell adhesion molecule-1 is expressed on activated endothelial cells and constitutively on bone marrow stromal cells. Mounting evidence indicates that 4ß1 plays a central role in leukocyte recruitment. This integrin also initiates lymphocyte contact ("tethering") in vitro under shear and in the absence of a selectin contribution.

Monoclonal antibodies to 4ß1 have therapeutic effects in numerous animal models of disease, such as experimental allergic encephalomyelitis, contact hypersensitivity, nonobese diabetes, allergic lung inflammation, and inflammatory bowel disease. Therefore, interactions between 4ß1 and its ligand are a therapeutic target for many diseases. Understanding the mechanism of ligand binding and detecting ligand-binding sites are important for designing inhibitors that modulate these interactions.

The N-terminal part of integrin subunits (approximately 440 amino acids) contains seven sequence repeats. Recently, we localized the putative ligand-binding sites of 4 (residues 108--268 of 4), which span repeats 2--5 of the seven N-terminal repeats of 4, by mapping epitopes of antibodies to 4 that block its function. By introducing multiple mutations into the putative ligand-binding sites, we determined that Tyr-187 and Gly-190, which are clustered in repeat 3 of 4, are critical residues for ligand binding to Y187 and G190.

We localized additional regions critical for ligand binding by using another strategy: swapping the predicted loop structures within or close to the putative ligand-binding sites of 4 with the corresponding regions of 5. Interestingly, swapping residues 112--131 in repeat 2 and residues 237--247 in repeat 4 completely blocked cell adhesion to immobilized ligands. The reduced affinity to ligand of these swapped mutants was not restored by activation with manganese. However, swapping residues 40--52 in repeat 1, residues 151--164 in repeat 3, or residues 282--288 (which contain a putative cation-binding motif) in repeat 5 did not affect or only slightly reduced adhesion to these ligands. The binding of several function-blocking antibodies was blocked by swapping residues 112--131, 151--164, and 186--191 (which contain Y187 and G190, residues critical for ligand binding). These results suggest that these predicted loops in repeats 2--4 are likely to be directly involved in interactions between 4ß1 and its ligand.

Recently, it was proposed that these seven N-terminal sequence repeats fold into a ß-propeller domain. The proposed domain contains seven four-stranded ß-sheets arranged in a torus around a pseudosymmetry axis. Integrin ligands and a putative magnesium ion are predicted to bind to the upper face of the ß-propeller. The calcium-binding motifs in the integrin subunit are thought to be on the lower face of the ß-propeller. Our mutagenesis data are consistent with this ß-propeller model, in which the regions of 4 critical for binding to vascular cell adhesion molecule-1 and fibronectin are adjacent to each other, although they are not adjacent in the primary structure, and are located in the upper face of the ß-propeller model, the predicted ligand-binding site.

PUBLICATIONS

Faull, R.J., Wang, J., Leavesley, D.I., Puzon, W., Russ, G.R., Vestweber, D., Takada, Y. A novel activating anti-ß1 integrin monoclonal antibody binds to the cystein-rich repeats in the ß1 chain. J. Biol. Chem. 271:25099, 1996.

Huang, S., Kamata, T., Takada, Y., Ruggeri, Z.M., Nemerow, G.R. Adenovirus interaction with distinct integrins mediates separate events in cell entry and gene delivery to hematopoietic cells. J. Virol. 70:4502, 1996.

Irie, A., Kamata, T., Takada, Y. Multiple loop structures critical for ligand binding of the integrin 4 subunit in the upper face of the ß-propeller model. Proc. Natl. Acad. Sci. U.S.A. 94:7198, 1997.

Isobe, T., Hisaoka, T., Shimizu, A., Okuno, M., Aimoto, S., Takada, Y., Saito, Y., Takagi, J. Propolypeptide of von Willebrand factor is a novel ligand for very late antigen-4 integrin. J. Biol. Chem. 272:8447, 1997.

King, S.L., Kamata, T., Cunningham, J.A., Emsley, J., Liddington, R.C., Takada, Y., Bergelson, J.M. Echovirus 1 interaction with the human VLA-2 I domain: Indentification of two independent virus contact sites distinct from the MIDAS residues. J. Biol. Chem., in press.

Mould, A.P., Askari, J.A., Aota, S.I., Yamada, K.M., Irie, A., Takada, Y., Mardon, H.J., Humphries, M.J. Defining the topology of integrin 5ß1-fibronectin interactions using inhibitory anti-5 and anti-ß1 monoclonal antibodies: Evidence that the synergy sequence of fibronectin is recognized by the N-terminal repeats of the 5 subunit. J. Biol. Chem. 272:17283, 1997.

Puzon-McLaughlin, W., Takada, Y. Critical residues for ligand binding in an I domain-like structure of the integrin ß1 subunit. J. Biol. Chem. 271:20438, 1996.

Takada, Y., Kamata, T., Irie, A., Puzon-McLaughlin, W., Zhang, X.-P. Structural basis of integrin-mediated signal transduction (a review). Matrix Biol., in press.

Takagi, J., Isobe, T., Takada, Y., Saito, Y. Structural interlock between ligand-binding site and stalk-like region of ß1 integrin revealed by a monoclonal antibody recognizing conformation-dependent epitope. J. Biochem. 121:914, 1997.

Takagi, J., Kamata, T., Meredith, J., Puzon-McLaughlin, W., Takada, Y. Changing ligand specificities of vß1 and vß3 integrins by swapping a short diverse sequence of the ß subunit. J. Biol. Chem. 272:19794, 1997.

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