Integrin Activation by Dithiothreitol or Mn2+ Induces a Ligand-occupied Conformation and Exposure of a Novel NH2-terminal Regulatory Site on the β1Integrin Chain
1998; Elsevier BV; Volume: 273; Issue: 14 Linguagem: Inglês
10.1074/jbc.273.14.7981
ISSN1083-351X
AutoresHeyu Ni, Anli Li, Neil Simonsen, John A. Wilkins,
Tópico(s)Signaling Pathways in Disease
ResumoIntegrins can be expressed in at least three functional states (i.e. latent, active, and ligand-occupied). However, the molecular bases for the transitions between these states are unknown. In the present study, changes in the accessibility of several β1 epitopes (e.g.N29, B44, and B3B11) were used to probe activation-related conformational changes. Dithiothreitol or Mn2+ activation of integrin-mediated adhesion in the human B cell line, IM9, resulted in a marked increase in the exposure of the B44 epitope, while N29 expression levels were most sensitive to dithiothreitol treatment. These results contrasted with the epitope expression patterns of spontaneously adherent K562 cells, where N29 was almost fully accessible and B44 was low. Addition of a soluble ligand resulted in a marked increase in B44 levels, suggesting that this antibody detected a ligand-induced binding site. The N29 epitope was mapped to a cysteine-rich region near the NH2 terminus of the integrin chain, thus defining a novel regulatory site.These studies indicate that the activation of integrin function by different stimuli may involve related but nonidentical conformations. Both Mn2+ and dithiothreitol appear to induce localized conformational changes that mimic a ligand-occupied receptor. This differs from the “physiologically” activated integrins on K562 cells that display a marked increase in overall epitope accessibility without exposure of the ligand-induced binding site epitopes. The increased exposure of the N29 site on K562 cells may indicate a role for this region in the regulation of integrin function. Integrins can be expressed in at least three functional states (i.e. latent, active, and ligand-occupied). However, the molecular bases for the transitions between these states are unknown. In the present study, changes in the accessibility of several β1 epitopes (e.g.N29, B44, and B3B11) were used to probe activation-related conformational changes. Dithiothreitol or Mn2+ activation of integrin-mediated adhesion in the human B cell line, IM9, resulted in a marked increase in the exposure of the B44 epitope, while N29 expression levels were most sensitive to dithiothreitol treatment. These results contrasted with the epitope expression patterns of spontaneously adherent K562 cells, where N29 was almost fully accessible and B44 was low. Addition of a soluble ligand resulted in a marked increase in B44 levels, suggesting that this antibody detected a ligand-induced binding site. The N29 epitope was mapped to a cysteine-rich region near the NH2 terminus of the integrin chain, thus defining a novel regulatory site. These studies indicate that the activation of integrin function by different stimuli may involve related but nonidentical conformations. Both Mn2+ and dithiothreitol appear to induce localized conformational changes that mimic a ligand-occupied receptor. This differs from the “physiologically” activated integrins on K562 cells that display a marked increase in overall epitope accessibility without exposure of the ligand-induced binding site epitopes. The increased exposure of the N29 site on K562 cells may indicate a role for this region in the regulation of integrin function. Members of the integrin family mediate cellular interactions with elements of their microenvironment (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8966) Google Scholar, 2Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2809) Google Scholar, 3Schwartz M.A. Schaller M.D. Ginsberg M.H. Ann. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1461) Google Scholar). These contacts can lead to cellular adhesion, migration, and activation (4Sjaastad M.D. Nelson W.J. Bioessays. 1997; 19: 47-55Crossref PubMed Scopus (133) Google Scholar, 5Haazenberger D. Klominek J. Holgersson J. Bergstrom S.-E. Sundqvist K.-G. J. Immunol. 1997; 158: 76-84PubMed Google Scholar, 6Pytela R. Pierschbacher M.D. Ruoslahti E. Cell. 1985; 40: 191-198Abstract Full Text PDF PubMed Scopus (686) Google Scholar). In a number of cell types, such as platelets and leukocytes, the activities of integrins are tightly regulated such that host cell activation is required before cell binding can proceed (7Marguerie G.A. Plow E.F. Biochemistry. 1981; 20: 1074-1080Crossref PubMed Scopus (37) Google Scholar, 8Wright S.D. Meyer B.C. J. Immunol. 1986; 136: 1759-1764PubMed Google Scholar, 9Shimizu Y. Van Seventer G.A. Morgan K.J. Shaw S. Nature. 1990; 345: 250-253Crossref PubMed Scopus (536) Google Scholar). This prerequisite ensures that integrin function is operative only at the appropriate anatomical or pathological sites.Although the structural basis for the underlying changes associated with the acquisition of integrin functionality is unknown, data from a number of different biochemical and immunological approaches clearly demonstrate activation-associated alterations in integrin conformation (10Calvette J.J. Thromb. Haemostasis. 1994; 72: 1-15Crossref PubMed Scopus (177) Google Scholar, 11Calvette J.J. Mann K. Schafer W. Fernandez-Lafuente R. Guisan J.M. Biochem. J. 1994; 298: 1-7Crossref PubMed Scopus (24) Google Scholar, 12Sims P.J. Ginsberg M.H. Plow E.F. Shattil S.J. J. Biol. Chem. 1991; 266: 7345-7352Abstract Full Text PDF PubMed Google Scholar). Antibody-binding studies and protease-susceptibility studies have shown that there are activation-associated changes in the accessibility of regions of the complex (10Calvette J.J. Thromb. Haemostasis. 1994; 72: 1-15Crossref PubMed Scopus (177) Google Scholar, 11Calvette J.J. Mann K. Schafer W. Fernandez-Lafuente R. Guisan J.M. Biochem. J. 1994; 298: 1-7Crossref PubMed Scopus (24) Google Scholar). Fluorescent energy transfer studies on αIIbβ3 have also demonstrated that there are alterations in the spacing and interaction of αIIb and β3 in the activated integrin structure (12Sims P.J. Ginsberg M.H. Plow E.F. Shattil S.J. J. Biol. Chem. 1991; 266: 7345-7352Abstract Full Text PDF PubMed Google Scholar). Changes in epitope expression are also observed following receptor occupancy (13Frelinger III, A.L. Du X. Plow E.F. Ginsberg M.H. J. Biol. Chem. 1991; 266: 17106-17111Abstract Full Text PDF PubMed Google Scholar, 14Mould A.P. Garratt A.N. Askari J.A. Akiyama S.K. Humphries M.J. FEBS Lett. 1995; 363: 118-122Crossref PubMed Scopus (122) Google Scholar, 15Faull R.J. Kovach N.L. Harlan J.M. Ginsberg M.H. J. Cell Biol. 1993; 121: 155-162Crossref PubMed Scopus (207) Google Scholar, 16Bazzoni G. Shih D.-T Buck C.A. Hemler M.E. J. Biol. Chem. 1995; 270: 25570-25577Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Collectively the data suggest that the activated integrin complex acquires a more open conformation than is observed in the latent structure.Recently models of integrin activation have been proposed that involve allosteric mechanisms for the acquisition of an adhesion-competent conformation (17Mould A.P. J. Cell Sci. 1996; 109: 2613-2618Crossref PubMed Google Scholar, 18Lee O.-J Rieu P. Arnaout M.A. Liddington R. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). Support for such a model derives from the observations that the binding of ligand to purified integrin inhibits the binding of an inhibitory antibody to the β1 chain (19Mould A.P. Akiyama S.K. Humphries M.J. J. Biol. Chem. 1996; 271: 20365-20374Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The pattern of inhibition displays characteristics that are most compatible with an allosteric mechanism. However, as pointed out by Mould (17Mould A.P. J. Cell Sci. 1996; 109: 2613-2618Crossref PubMed Google Scholar), the situation with the integrins is more complex than a classical allosteric mechanism, as the “active” integrin does not necessarily acquire a conformation that approximates the ligand-bound receptor. Thus the existence of multiple intermediate conformations have been suggested.Activation of integrin function can be achieved by a variety of stimuli (20Wilkins J.A. Stupack D.G. Stewart S. Caixia S. Eur. J. Immunol. 1991; 21: 517-522Crossref PubMed Scopus (103) Google Scholar, 21Chan B.M. Wong J.G. Rao A. Hemler M.E. J. Immunol. 1991; 147: 398-404PubMed Google Scholar, 22Dransfield I. Cabanas C. Craig A. Hogg N. J. Cell Biol. 1992; 116: 219-226Crossref PubMed Scopus (399) Google Scholar, 23Elices M.J. Urry L.A. Hemler M.E. J. Cell Biol. 1991; 112: 169-181Crossref PubMed Scopus (344) Google Scholar, 24Davis G.E. Camarillo C.W. J. Immunol. 1993; 151: 7138-7156PubMed Google Scholar, 25Edwards B.S. Curry M.S. Southon E.A. Chong A.S.-F. Graf L.H. Blood. 1995; 86: 2288-2301Crossref PubMed Google Scholar, 26Peerschke E.I. Thromb. Haemostasis. 1995; 73: 862-867Crossref PubMed Scopus (49) Google Scholar). Mn2+ and the bifunctional reducing agent, DTT, 1The abbreviation used is: DTT, dithiothreitol. 1The abbreviation used is: DTT, dithiothreitol. have been shown to activate integrin binding in a number of systems (22Dransfield I. Cabanas C. Craig A. Hogg N. J. Cell Biol. 1992; 116: 219-226Crossref PubMed Scopus (399) Google Scholar, 23Elices M.J. Urry L.A. Hemler M.E. J. Cell Biol. 1991; 112: 169-181Crossref PubMed Scopus (344) Google Scholar, 24Davis G.E. Camarillo C.W. J. Immunol. 1993; 151: 7138-7156PubMed Google Scholar, 25Edwards B.S. Curry M.S. Southon E.A. Chong A.S.-F. Graf L.H. Blood. 1995; 86: 2288-2301Crossref PubMed Google Scholar, 26Peerschke E.I. Thromb. Haemostasis. 1995; 73: 862-867Crossref PubMed Scopus (49) Google Scholar). Since both of these agents activate purified integrins, it would appear that their effects on adhesion might be directly on the receptor complex (19Mould A.P. Akiyama S.K. Humphries M.J. J. Biol. Chem. 1996; 271: 20365-20374Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 24Davis G.E. Camarillo C.W. J. Immunol. 1993; 151: 7138-7156PubMed Google Scholar). These agents may provide useful probes for the analysis of the changes associated with integrin activation and ligand binding.We have previously described a panel of regulatory antibodies to the human β1 integrin chain and localized their continuous epitopes (27Stupack D.G. Shen C. Wilkins J.A. Cell. Immunol. 1994; 155: 237-245Crossref PubMed Scopus (14) Google Scholar, 28Wilkins J.A. Li A. Ni H. Stupack D.G. Shen C. J. Biol. Chem. 1996; 271: 3046-3051Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 29Ni H. Wilkins J.A. Cell Adhes. Commun. 1997; (in press)Google Scholar). Three noncompeting groups of antibodies were identified, and one set of antibodies was shown to react with the membrane proximal β1 region (28Wilkins J.A. Li A. Ni H. Stupack D.G. Shen C. J. Biol. Chem. 1996; 271: 3046-3051Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The present study localizes a novel stimulatory region to the cysteine-rich amino-terminal portion of the β1 chain. Furthermore, it is demonstrated that it is possible to generate functionally “activated” integrins with overlapping but nonidentical conformations.DISCUSSIONThe present studies provide several new pieces of data relevant to integrin activation. 1) The stimulatory antibody, N29, recognizes a new regulatory region located near the NH2 terminus of the β1 molecule. 2) The stimulatory antibody, B44, identifies an epitope, which is exposed on ligand binding. 3) Mn2+ and DTT induce changes in β1 epitope accessibility, which resemble those observed in the ligand occupied receptor. 4) The overall accessibility of epitopes in physiologically active integrins is increased relative to those on nonadherent cells or on Mn2+- and DTT-activated cells.The initial assumption that N29 might identify an activation epitope does not appear to be fully supported by the results of this study. In the case of DTT-treated cells, there was an almost total exposure of the N29 epitope associated with activation of adhesion. However, the N29 levels on spontaneously adhesive cells such as K562 and Jurkat 2H. Ni and J. A. Wilkins, unpublished results. or following treatment with Mn2+ were elevated 2–4-fold, such that 20–30% of the integrins displayed this epitope. There were also low but detectable levels of N29 exposure on nonadherent cells. Thus the correlation between integrin functional status and N29 accessibility appeared to be semiquantitative rather than a qualitative one.The antibody B44 identifies an epitope, which under normal conditions appears to be of very limited accessibility. Thus the expression levels of this epitope on adhesion competent cells such as Jurkat and K562 are significantly lower than the total integrin levels. However, occupancy of integrin by ligand or by an RGD-containing antagonist results in a marked increase in B44 expression. The B44 epitope is reduction resistant under SDS-polyacrylamide gel electrophoresis conditions, implying that the antibody detects a continuous peptide sequence. It appears that ligand binding exposes the cryptic epitope to the solvent and renders it antibody accessible. However, it is unlikely that this epitope represents a ligand contact site as B44 binding has been shown to induce adherence in Jurkat cells (28Wilkins J.A. Li A. Ni H. Stupack D.G. Shen C. J. Biol. Chem. 1996; 271: 3046-3051Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The properties of B44 most resemble those of two other antibodies, 15/7 (35Puzon-McLaughlin W. Yednock T.A. Takada Y. J. Biol. Chem. 1996; 271: 16580-16585Crossref PubMed Scopus (48) Google Scholar) and HUTS-21 (36Luque A. Gomez M. Puzon W. Takada Y. Sanchez-Madrid F. Cabanas C. J. Biol. Chem. 1996; 271: 11067-11075Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar), which detect integrins in a ligand-occupied or high affinity state. These antibodies have been shown to react with epitopes that are located in the cysteine rich region of the β1 (residues 355–425). However, to date it has not been possible to determine the location of the B44 epitope.Treatment of IM9 cells with Mn2+ induces B44 epitope expression. The implication is that the Mn2+ induces alterations that resemble those caused by ligand binding to a competent integrin. It has been suggested that Mn2+ may stimulate adhesion by forming a co-ordination complex with residues in the cation binding domains of the integrin and the aspartate residue of the ligand (37Bergelson J.M. Hemler M.E. Curr. Biol. 1995; 5: 615-617Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 38D'Souza S.E. Haas T.A. Piotrowicz R.S. Byers-Ward V. McGrath D.E. Soule H.R. Cierniewski C. Plow E.F. Smith J.W. Cell. 1994; 79: 659-667Abstract Full Text PDF PubMed Scopus (204) Google Scholar), or by facilitating the ligand entry to the binding site via an exchange mechanism (18Lee O.-J Rieu P. Arnaout M.A. Liddington R. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). Recently it has been proposed that Mn2+ may induce a conformation resembling the ligand occupied receptor thus permitting ligand access to the binding region of the integrin (17Mould A.P. J. Cell Sci. 1996; 109: 2613-2618Crossref PubMed Google Scholar). The binding pattern of B44 is compatible with the latter explanation of Mn2+ action. However, it does not address the issue of the relative contributions of Mn2+ to cation-facilitated exchange and ligand co-ordination.Activation of adhesion by reducing agents has also been described in several systems. Edwards et al. (25Edwards B.S. Curry M.S. Southon E.A. Chong A.S.-F. Graf L.H. Blood. 1995; 86: 2288-2301Crossref PubMed Google Scholar) noted that there was an obligate requirement for a bifunctional thiol with a minimal spacing of four carbons between the two –SH groups. Early studies on the activation of platelet adhesion by DTT indicated that there were changes in αIIbβ3 electrophoretic mobility associated with activation by this agent (26Peerschke E.I. Thromb. Haemostasis. 1995; 73: 862-867Crossref PubMed Scopus (49) Google Scholar). The DTT-dependent activation of mutant αIIbβ3 in platelets from a patient with Glanzmann's thrombasthenia by DTT was shown to be associated with the appearance of activation epitopes (39Kouns W.C. Steiner B. Kunicki T.J. Moog S. Jutzi J. Jennings L.K. Cazenave J.-P. Lanza F. Blood. 1994; 84: 1108-1115Crossref PubMed Google Scholar). However, DTT-induced activation of αLβ2 mediated adhesion of natural killer cells to intercellular adhesion molecule 1-expressing target cells failed to reveal conformational changes using two reporter antibodies (25Edwards B.S. Curry M.S. Southon E.A. Chong A.S.-F. Graf L.H. Blood. 1995; 86: 2288-2301Crossref PubMed Google Scholar). Furthermore, these authors could not demonstrate the appearance of free thiol groups in the αLβ2 complex, implying that the integrin chains were not directly modified by DTT treatment (25Edwards B.S. Curry M.S. Southon E.A. Chong A.S.-F. Graf L.H. Blood. 1995; 86: 2288-2301Crossref PubMed Google Scholar). The present data clearly indicate that significant conformational changes are induced by DTT as access to the B44 and the N29 epitopes are markedly increased.The increased B3B11 and N29 expression on K562 cells implies that physiologically activated integrins undergo changes that allow an increased accessibility to the membrane proximal and NH2-terminal regions of the molecule. Although the integrins on these cells are in an adhesion competent state, ligand binding is required for B44 epitope expression. These results would seem to suggest that there is an intermediate conformation in which the integrin is adhesion-competent but unoccupied. The fact that agents such as Mn2+ and DTT induce conformations that resemble the ligand-occupied state suggests that they stimulate adhesion competence by generating integrin intermediates that are distinct from the native active forms observed in K562. Although different functional forms of integrins have been described or postulated (16Bazzoni G. Shih D.-T Buck C.A. Hemler M.E. J. Biol. Chem. 1995; 270: 25570-25577Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 17Mould A.P. J. Cell Sci. 1996; 109: 2613-2618Crossref PubMed Google Scholar, 40Yednock T.A. Cannon C. Vandevert C. Goldbach E.G. Shaw G. Ellis D.K. Liau C. Fritz L.C. Tanner L.I. J. Biol. Chem. 1995; 270: 28740-28750Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), it is unclear at this point whether active forms such as those induced by DTT or Mn2+ are representative of physiological integrin intermediates. These observations suggest that caution should be exhibited when attempting to correlate competent states induced by these agents with those found in physiologically activated integrins.The localization of the N29 epitope between residues 15 and 54 places it in a highly conserved cysteine-rich region (41Calvette J.J. Henschen A. Gonzalez-Rodriguez J. Biochem. J. 1991; 274: 63-71Crossref PubMed Scopus (158) Google Scholar). This area has not previously been identified as a regulatory site, although it is adjacent to region that has been shown to be a cation and ligand sensitive in the β3 chain (34Honda S. Tomiyama Y. Pelletier A.J. Annis D. Honda Y. Orchekowski R. Ruggeri Z. Kunicki T.J. J. Biol. Chem. 1995; 270: 11947-11954Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Unlike the β3 situation, the binding of N29 is relatively insensitive to the cationic composition of the extracellular milieu. Thus if a homologous region exists in the β1 chain it would appear that it is not located in the N29 reactive 15–54 sequence of the molecule.The antibodies N29, B44, and B3B11/JB1B were originally identified because of their abilities to stimulate Jurkat adherence to collagen and fibronectin (28Wilkins J.A. Li A. Ni H. Stupack D.G. Shen C. J. Biol. Chem. 1996; 271: 3046-3051Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). It is noteworthy that in those cases where their corresponding epitopes have been identified (28Wilkins J.A. Li A. Ni H. Stupack D.G. Shen C. J. Biol. Chem. 1996; 271: 3046-3051Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 29Ni H. Wilkins J.A. Cell Adhes. Commun. 1997; (in press)Google Scholar), the stimulatory epitopes map to regions that are in close proximity to residues that are predicted to be involved in disulfide bonds between sequentially distant cysteines (i.e. Cys7–Cys415and Cys444–Cys671). The present results extend those of others employing interspecies β1 chimeras (16Bazzoni G. Shih D.-T Buck C.A. Hemler M.E. J. Biol. Chem. 1995; 270: 25570-25577Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar,17Mould A.P. J. Cell Sci. 1996; 109: 2613-2618Crossref PubMed Google Scholar, 42Takada Y. Puzon W. J. Biol. Chem. 1993; 268: 17597-17601Abstract Full Text PDF PubMed Google Scholar, 43Faull R.J. Wang J. Leavesley D.I. Puzon W. Russ G.R. Vestweber D. Takada Y. J. Biol. Chem. 1996; 271: 25099-25106Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 44Shih D.-T Edelman J.M. Horowitz A.F. Grunwald G, B. Buck C.A. J. Cell Biol. 1993; 122: 1361-1371Crossref PubMed Scopus (45) Google Scholar) and expands the locations of regulatory sites to include both the membrane proximal and the distal regions of the β1 (Fig. 8).It might be speculated that the NH2-terminal region of the β1 chain is involved in the normal control of integrin function. The increased N29 expression on K562 could reflect a situation in which physiologically activated integrins undergo a conformational change to expose this site. The exposure may indicate accessibility to the ligand-binding site. However, it seems unlikely that N29 contact is required for binding as Mn2+ induces adhesion competence with minimal effects on N29 exposure. Subsequent to ligand binding, the B44 epitope is expressed, and this presumably reflects a secondary change in the integrin conformation, perhaps as a consequence of ligand displacement of previously buried residues. It is important to bear in mind that, although the results of the antibody studies indicate changes in the accessibility of β1integrin epitopes following activation, the basis for these changes are unknown. They could relate to integrin conformational changes, to alterations in the patterns of integrin-associated proteins, or to both of these mechanisms. Studies with purified integrin may permit the differentiation of these possibilities. Members of the integrin family mediate cellular interactions with elements of their microenvironment (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8966) Google Scholar, 2Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2809) Google Scholar, 3Schwartz M.A. Schaller M.D. Ginsberg M.H. Ann. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1461) Google Scholar). These contacts can lead to cellular adhesion, migration, and activation (4Sjaastad M.D. Nelson W.J. Bioessays. 1997; 19: 47-55Crossref PubMed Scopus (133) Google Scholar, 5Haazenberger D. Klominek J. Holgersson J. Bergstrom S.-E. Sundqvist K.-G. J. Immunol. 1997; 158: 76-84PubMed Google Scholar, 6Pytela R. Pierschbacher M.D. Ruoslahti E. Cell. 1985; 40: 191-198Abstract Full Text PDF PubMed Scopus (686) Google Scholar). In a number of cell types, such as platelets and leukocytes, the activities of integrins are tightly regulated such that host cell activation is required before cell binding can proceed (7Marguerie G.A. Plow E.F. Biochemistry. 1981; 20: 1074-1080Crossref PubMed Scopus (37) Google Scholar, 8Wright S.D. Meyer B.C. J. Immunol. 1986; 136: 1759-1764PubMed Google Scholar, 9Shimizu Y. Van Seventer G.A. Morgan K.J. Shaw S. Nature. 1990; 345: 250-253Crossref PubMed Scopus (536) Google Scholar). This prerequisite ensures that integrin function is operative only at the appropriate anatomical or pathological sites. Although the structural basis for the underlying changes associated with the acquisition of integrin functionality is unknown, data from a number of different biochemical and immunological approaches clearly demonstrate activation-associated alterations in integrin conformation (10Calvette J.J. Thromb. Haemostasis. 1994; 72: 1-15Crossref PubMed Scopus (177) Google Scholar, 11Calvette J.J. Mann K. Schafer W. Fernandez-Lafuente R. Guisan J.M. Biochem. J. 1994; 298: 1-7Crossref PubMed Scopus (24) Google Scholar, 12Sims P.J. Ginsberg M.H. Plow E.F. Shattil S.J. J. Biol. Chem. 1991; 266: 7345-7352Abstract Full Text PDF PubMed Google Scholar). Antibody-binding studies and protease-susceptibility studies have shown that there are activation-associated changes in the accessibility of regions of the complex (10Calvette J.J. Thromb. Haemostasis. 1994; 72: 1-15Crossref PubMed Scopus (177) Google Scholar, 11Calvette J.J. Mann K. Schafer W. Fernandez-Lafuente R. Guisan J.M. Biochem. J. 1994; 298: 1-7Crossref PubMed Scopus (24) Google Scholar). Fluorescent energy transfer studies on αIIbβ3 have also demonstrated that there are alterations in the spacing and interaction of αIIb and β3 in the activated integrin structure (12Sims P.J. Ginsberg M.H. Plow E.F. Shattil S.J. J. Biol. Chem. 1991; 266: 7345-7352Abstract Full Text PDF PubMed Google Scholar). Changes in epitope expression are also observed following receptor occupancy (13Frelinger III, A.L. Du X. Plow E.F. Ginsberg M.H. J. Biol. Chem. 1991; 266: 17106-17111Abstract Full Text PDF PubMed Google Scholar, 14Mould A.P. Garratt A.N. Askari J.A. Akiyama S.K. Humphries M.J. FEBS Lett. 1995; 363: 118-122Crossref PubMed Scopus (122) Google Scholar, 15Faull R.J. Kovach N.L. Harlan J.M. Ginsberg M.H. J. Cell Biol. 1993; 121: 155-162Crossref PubMed Scopus (207) Google Scholar, 16Bazzoni G. Shih D.-T Buck C.A. Hemler M.E. J. Biol. Chem. 1995; 270: 25570-25577Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Collectively the data suggest that the activated integrin complex acquires a more open conformation than is observed in the latent structure. Recently models of integrin activation have been proposed that involve allosteric mechanisms for the acquisition of an adhesion-competent conformation (17Mould A.P. J. Cell Sci. 1996; 109: 2613-2618Crossref PubMed Google Scholar, 18Lee O.-J Rieu P. Arnaout M.A. Liddington R. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). Support for such a model derives from the observations that the binding of ligand to purified integrin inhibits the binding of an inhibitory antibody to the β1 chain (19Mould A.P. Akiyama S.K. Humphries M.J. J. Biol. Chem. 1996; 271: 20365-20374Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The pattern of inhibition displays characteristics that are most compatible with an allosteric mechanism. However, as pointed out by Mould (17Mould A.P. J. Cell Sci. 1996; 109: 2613-2618Crossref PubMed Google Scholar), the situation with the integrins is more complex than a classical allosteric mechanism, as the “active” integrin does not necessarily acquire a conformation that approximates the ligand-bound receptor. Thus the existence of multiple intermediate conformations have been suggested. Activation of integrin function can be achieved by a variety of stimuli (20Wilkins J.A. Stupack D.G. Stewart S. Caixia S. Eur. J. Immunol. 1991; 21: 517-522Crossref PubMed Scopus (103) Google Scholar, 21Chan B.M. Wong J.G. Rao A. Hemler M.E. J. Immunol. 1991; 147: 398-404PubMed Google Scholar, 22Dransfield I. Cabanas C. Craig A. Hogg N. J. Cell Biol. 1992; 116: 219-226Crossref PubMed Scopus (399) Google Scholar, 23Elices M.J. Urry L.A. Hemler M.E. J. Cell Biol. 1991; 112: 169-181Crossref PubMed Scopus (344) Google Scholar, 24Davis G.E. Camarillo C.W. J. Immunol. 1993; 151: 7138-7156PubMed Google Scholar, 25Edwards B.S. Curry M.S. Southon E.A. Chong A.S.-F. Graf L.H. Blood. 1995; 86: 2288-2301Crossref PubMed Google Scholar, 26Peerschke E.I. Thromb. Haemostasis. 1995; 73: 862-867Crossref PubMed Scopus (49) Google Scholar). Mn2+ and the bifunctional reducing agent, DTT, 1The abbreviation used is: DTT, dithiothreitol. 1The abbreviation used is: DTT, dithiothreitol. have been shown to activate integrin binding in a number of systems (22Dransfield I. Cabanas C. Craig A. Hogg N. J. Cell Biol. 1992; 116: 219-226Crossref PubMed Scopus (399) Google Scholar, 23Elices M.J. Urry L.A. Hemler M.E. J. Cell Biol. 1991; 112: 169-181Crossref PubMed Scopus (344) Google Scholar, 24Davis G.E. Camarillo C.W. J. Immunol. 1993; 151: 7138-7156PubMed Google Scholar, 25Edwards B.S. Curry M.S. Southon E.A. Chong A.S.-F. Graf L.H. Blood. 1995; 86: 2288-2301Crossref PubMed Google Scholar, 26Peerschke E.I. Thromb. Haemostasis. 1995; 73: 862-867Crossref PubMed Scopus (49) Google Scholar). Since both of these agents activate purified integrins, it would appear that their effects on adhesion might be directly on the receptor complex (19Mould A.P. Akiyama S.K. Humphries M.J. J. Biol. Chem. 1996; 271: 20365-20374Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 24Davis G.E. Camarillo C.W. J. Immunol. 1993; 151: 7138-7156PubMed Google Scholar). These agents may provide useful probes for the analysis of the changes associated with integrin activation and ligand binding. We have previously described a panel of regulatory antibodies to the human β1 integrin chain and localized their continuous epitopes (27Stupack D.G. Shen C. Wilkins J.A. Cell. Immunol. 1994; 155: 237-245Crossref PubMed Scopus (14) Google Scholar, 28Wilkins J.A. Li A. Ni H. Stupack D.G. Shen C. J. Biol. Chem. 1996; 271: 3046-3051Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 29Ni H. Wilkins J.A. Cell Adhes. Commun. 1997; (in press)Google Scholar). Three noncompeting groups of antibodies were identified, and one set of antibodies was shown
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