THE PLOWLABORATORYCOLLABORATORSTatiana Byzova, Ph.D.Jun Qin, Ph.D.Olga Stenina, Ph.D.Tatiana Ugarova, Ph.D.RESEARCH ASSOCIATESElzbieta Pluskota, Ph.D.Valentin Yakubenko, Ph.D.POSTDOCTORAL FELLOWSBin Hu, Ph.D.Michelle Kish, Ph.D.Dmitry Solovjov, Ph.D.Carmen Swaisgood, Ph.D.Valentin Ustinov, Ph.D.PREDOCTORAL FELLOWNataly Podolnikova, M.S.TECHNICAL ASSOCIATESTimothy BurkeCarla DrummValeryi Lishko, Ph.D.Daniel TrepalShiying WangVISITING SCIENTISTSCzeslaw Cierniewski, Ph.D. 1Steve Busuttil, M.D. 21Univ. of Lodz, Poland2Case Western Reserve Univ.and Veterans Adm. Med. Ctr.,<strong>Cleveland</strong>, OHByzova, T.V., Goldman, C.K., Pampori, N., Thomas, K.A., Bett, A., Shattil, S.J., and E.F.Plow (2000) A mechanism for modulation of cellular responses to VEGF: activation of theintegrins. Mol. Cell 6:851-860.Plow, E.F., Haas, T.A., Zhang, L., Loftus, J., and J.W. Smith (2000) Ligand binding tointegrins. J. Biol. Chem. 275:21785-21788.Forsyth, C.B., Solovjov, D.A., Ugarova, T.P., and E.F. Plow (2001) Integrin α Mβ 2-mediatedcell migration to fibrinogen and its recognition peptides. J. Exp. Med. 193:1123-1134.Vinogradova, O., Velyvis, A., Velyviene, A., Hu, B., Haas, T., Plow, E., and J. Qin (2002) Astructural mechanism of integrin α IIbβ 3“inside-out” activation as regulated by its cytoplasmicface. Cell 110:587-597.Ustinov, V.A., and E.F. Plow (2002) Delineation of the key amino acids involved inneutrophil inhibitory factor binding to the I-domain supports a mosaic model for the capacityof integrin α Mβ 2to recognize multiple ligands. J. Biol. Chem. 277:18769-18776.Swaisgood, C.M., Schmitt, D., Eaton, D., and E.F. Plow (2002) In vivo regulation ofplasminogen function by plasma carboxypeptidase B. J. Clin. Invest. 110:1275-1282.128Edward F. Plow, Ph.D.Integrin and Plasminogen ReceptorsRegulate Cell Adhesion and MigrationCell adhesion and migration are essential for theformation, development and survival of allmulticellular organisms. Aberrations in theadhesive status of cells have major pathogeneticconsequences, including thrombosis, tumor growthand metastasis, and infection. <strong>Research</strong> within thislaboratory seeks to delineate molecular mechanismsthat regulate cell adhesion and migration. In particular,our efforts are directed toward analyzing thecontributions of two specific receptor systems to thesecellular responses: the integrin and plasminogenreceptor systems.The integrins are a large and broadly distributedfamily of adhesion receptors (>20 members, occurringon virtually every cell type). Each member is anoncovalent heterodimeric complex composed of anα and a β subunit. Key to many functions ofintegrins is their capacity to rapidly modulate theiraffinity for ligands, i.e., they can exist in ligandcompetentand in resting states, in which they do notbind ligands with high affinity. Also characteristic isthe capacity of each integrin to recognize multiple andstructurally unrelated ligands. Development of amolecular understanding of these two centralproperties of integrins is a general direction of thelaboratory.Integrin α IIbβ 3(GPIIb-IIIa) mediates plateletaggregation, an essential event in thrombus formation.We are attempting to understand the molecular basisfor ligand binding to this integrin. A combination ofmolecular biology, protein chemistry and biophysicalanalyses are being developed to identify the cation andligand contact sites within the polypeptide chains ofthe receptor. Our goal is to determine how cation andligand coordination sites cooperate to achieve receptorfunction. In parallel, we are seeking to understandhow the cytoplasmic tails of the α IIband β 3subunitstransmit signals to the extracellular domain to activatethe receptor’s ligand binding functions. Cell-permeablesynthetic peptides, corresponding to the cytoplasmictail of each subunit, have been shown to interact witheach other to form a functional cytoplasmic domain,and the structure of this cytoplasmic domain has beensolved by nuclear magnetic resonance spectroscopy.The Department of Molecular CardiologyThese studies are conducted collaboratively with thelaboratories of Dr. Jun Qin.Integrin α Mβ 2is expressed on leukocytes andplays a pivotal role in the transmigration of these cellsduring intravascular inflammatory responses, leading toatherosclerosis and restenosis, and extravascularinflammatory responses. The ligand repertoire of α Mβ 2is extremely broad. We are testing the hypothesis thatmany of the α Mβ 2ligands bind to overlapping but notidentical segments within a 200-amino-acid stretch, theI domain of the α Msubunit; and other regions of theα Mand β 2subunits influence subsequent responsessuch as adhesion and migration once ligand has beenengaged by the I domain. Since the crystal structure ofthe I domain is known, incisive mutagenesis strategiescan be implemented to test this hypothesis. Four α Mβ 2ligands of current interest are the fungal pathogenCandida albicans, neutrophil inhibitory factor (NIF),platelet membrane protein GPIb, and fibrinogen. Asoluble factor released from C. albicans, which supportsα Mβ 2-dependent cell adhesion and migration, is beingisolated and characterized. Studies of fibrinogenrecognition by α Mβ 2are conducted collaboratively withDr. Tatiana Ugarova, who is seeking the define the siteswithin fibrinogen recognized by the receptor as well asthe sites with α Mβ 2that recognize fibrinogen.In addition to its central role in fibrinolysis, theplasminogen system is also involved in cell migration.This latter function depends upon the interaction ofplasminogen with cell surface receptors. When bound,plasminogen is efficiently activated to plasmin, and thebound enzyme can cleave a variety of pericellularsubstrates to facilitate cell migration. Our currentefforts emphasize how plasminogen receptor expressionis modulated. We are investigating the capacity of celladhesion, proteolysis and apoptosis to modulateexpression of plasminogen receptors. Such modulationcan result in 5- to 10-fold changes in the expression ofnaturally occurring cells, such as neutrophils, to bindplasminogen. At the same time, we are also challengingthe hypothesis that plasminogen is crucial for cellmigration. These latter studies are being conducted inmice in which the gene for plasminogen has beeninactivated. These knockout mice are being used inmodels for the inflammatory response, atherosclerosisand restenosis, in which cell migration may contributeto pathogenesis. Recent studies have focused on therole of plasminogen in asthma and of a molecule,TAFI, which suppresses plasminogen binding to itscellular receptors.Recent large-scale genetic studies have identifiedparticular single nucleotide polymorphisms (SNPs)within members of the thrombospondin gene family asbeing associated with an increased risk of prematureatherosclerosis. The thrombospondins are large,extracellular matrix proteins that are reported to exert avariety of effects on blood and vascular cells. Incollaboration with Dr. Olga Stenina, we are determininghow SNPs in the thrombospondins alter structureand function as a potential means to define a mechanismunderlying atherosclerotic disease.
The Department of Molecular CardiologyACE: Biosynthesis and SecretionTHE I. SENLABORATORYAngiotensin-converting enzyme (ACE), adipeptidyl carboxypeptidase, is a keycomponent of the renin-angiotensinsystem that regulates blood pressure. Studieswith ACE knock-out mice have revealedadditional roles of ACE in renal physiology andmale fertility. Although ACE exists primarily as amembrane-bound cell-surfaceprotein, a soluble form ispresent under normalconditions in serum and otherbody fluids. Becauseinformation in the literaturesuggests that the specificphysiological function of cellboundACE may differ fromthose of ACE in circulation,production of soluble ACEfrom cell-bound ACE couldbe a significant point ofbiological regulation.We have been studyingthe process of ACE cleavagesecretionusing natural ACEproducingcells and cellstransfected with expressionvectors of ACE or itsmutants. Our studiesrevealed that the ectodomainof ACE is cleaved at aspecific site near the plasmamembrane by a membraneanchored metalloprotease, theACE-secretase. The cleavage specificity ismaintained not by amino acid sequence at oraround the cleavage site but by the presence ofthe distal ectodomain of the ACE protein thatactivates the ACE-secretase, suggesting that it isan atypical protease. The activity of themammalian secretase can be upregulated bytreatment of cells with phorbol esters,calmodulin inhibitors or protein tyrosinephosphatase inhibitors. Our research effort isdirected toward (1) identifying the sequences inectodomain of ACE thatactivates the secretase, (2)determining how thecleavage-secretion processis regulated, and (3)cloning and characterizationof ACE-secretase.These studies will providean understanding of howcell-bound ACE is releasedto the circulation in aregulated fashion.Our study is highlysignificant beyond theACE system as well.Although the cleavagesecretionprocess forproduction of solubleproteins from membraneboundforms is widelyused in biology, theresponsible secretases havenot been identified inmost systems. Thus,Indira Sen, Ph.D.identification andcharacterization of ACEsecretase will considerably advance our knowledgeof the secretases whose primary role is toselectively cleave and release many biologicallysignificant ectoproteins from the cell surface.POST DOCTORAL FELLOWSS. Sengupta, Ph.D.G. Karan, Ph.D.COLLABORATORSRoy A. Black, Ph.D. 1Janice G. Douglas, M.D. 2Ganes C.Sen, Ph.D. 31Immunex Corp., Seattle, WA2Dept. of Medicine, CaseWestern Reserve Univ.,<strong>Cleveland</strong>, OH3Dept. of Molecular Biology,CCFRamchandran, R., Kasturi, S., Douglas, J.G., and I. Sen (1996) Metalloprotease-mediated cleavage secretionof pulmonary ACE by vascular endothelial and kidney epithelial cells. Am. J. Physiol. 271:H744-H751.Sadhukhan, R., Sen, G.C., and I. Sen (1996) Synthesis and cleavage-secretion of enzymatically active angiotensin-convertingenzyme in Pichia pastoris. J. Biol. Chem. 271:18310-18313.Sadhukhan, R., Sen, G.C., Ramchandran, R., and I. Sen (1998) The distal ectodomain of angiotensin-convertingenzyme regulates its cleavage-secretion from the cell surface. Proc. Natl. Acad. Sci. USA 95:138-143.Sadhukhan, R., Santhamma, K.R., Reddy, P., Peschon, J.J. , Black, R.A., and I. Sen (1999) Unalteredcleavage and secretion of angiotensin-converting enzyme in tumor necrosis factor-α-converting enzyme-deficientmice. J. Biol. Chem. 274:10511-10516.Santhamma, K.R., and I. Sen (2000) Specific cellular proteins associate with angiotensin-converting enzymeand regulate its intracellular transport and cleavage-secretion. J. Biol. Chem. 275:23253-23258.129
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