The Integrin α1 A-domain Is a Ligand Binding Site for Collagens and Laminin

The integrin α1β1 is a cell surface receptor for collagens and laminin. The α1 subunit contains an A-domain, and the A-domains of other integrins are known to mediate ligand binding. To determine the role of the α1 A-domain in ligand binding and the extent to which it reproduced the ligand binding activity and specificity of the parent molecule, we produced recombinant α1 A-domain and tested its ability to bind collagens and laminin. In solid phase assays, the A-domain from α1 was found to bind to collagen I, collagen IV, and laminin in a largely cation-dependent manner. The α2 A-domain, from the α2β1 integrin, also bound to these ligands, but the binding hierarchy differed from that seen for α1. This is the first demonstration of laminin binding by A-domains. Specificity of A-domain-ligand binding was further investigated using the triple-helical proteolytic fragment of collagen IV, CB3, and its subfragments, F1 and F4. α1 A-domain bound to all three fragments, while the α2 A-domain bound CB3 less well and exhibited little binding to F1 and no binding to F4. These differences mirror previous reports of distinct integrin binding sites in collagen IV and for the first time identify a limited proteolytic fragment of a ligand that contains integrin A-domain binding activity. To gain insight into the contribution that the A-domain makes to ligand binding within the whole integrin heterodimer, we measured binding constants for A-domain-collagen interactions using surface plasmon resonance biosensor technology. The values obtained were similar to those reported for intact integrin binding, suggesting that the A-domain is the major collagen binding site in the α1β1 and α2β1 integrins. The integrin α1β1 is a cell surface receptor for collagens and laminin. The α1 subunit contains an A-domain, and the A-domains of other integrins are known to mediate ligand binding. To determine the role of the α1 A-domain in ligand binding and the extent to which it reproduced the ligand binding activity and specificity of the parent molecule, we produced recombinant α1 A-domain and tested its ability to bind collagens and laminin. In solid phase assays, the A-domain from α1 was found to bind to collagen I, collagen IV, and laminin in a largely cation-dependent manner. The α2 A-domain, from the α2β1 integrin, also bound to these ligands, but the binding hierarchy differed from that seen for α1. This is the first demonstration of laminin binding by A-domains. Specificity of A-domain-ligand binding was further investigated using the triple-helical proteolytic fragment of collagen IV, CB3, and its subfragments, F1 and F4. α1 A-domain bound to all three fragments, while the α2 A-domain bound CB3 less well and exhibited little binding to F1 and no binding to F4. These differences mirror previous reports of distinct integrin binding sites in collagen IV and for the first time identify a limited proteolytic fragment of a ligand that contains integrin A-domain binding activity. To gain insight into the contribution that the A-domain makes to ligand binding within the whole integrin heterodimer, we measured binding constants for A-domain-collagen interactions using surface plasmon resonance biosensor technology. The values obtained were similar to those reported for intact integrin binding, suggesting that the A-domain is the major collagen binding site in the α1β1 and α2β1 integrins. Integrins are a family of αβ heterodimeric cell surface receptors, responsible for cell-cell and cell-extracellular matrix interactions. The specificity and regulation of these interactions is critical to many biological processes, including embryonic cell migration, wound healing, and the immune response (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9212) Google Scholar). The integrin family contains at least 16 α subunits, seven of which contain an ∼200 amino acid inserted domain in their N-terminal region (I or A-domain) (2Colombatti A. Bonaldo P. Doliana R. Matrix. 1993; 13: 297-306Crossref PubMed Scopus (156) Google Scholar, 3Shaw S.K. Cepek K.L. Murphy E.A. Russell G.J. Brenner M.B. Parker C.M. J. Biol. Chem. 1994; 269: 6016-6025Abstract Full Text PDF PubMed Google Scholar). This domain is homologous to the von Willebrand factor A-domain, a module also found in a number of other membrane, plasma, and matrix proteins (2Colombatti A. Bonaldo P. Doliana R. Matrix. 1993; 13: 297-306Crossref PubMed Scopus (156) Google Scholar, 4Lee Jie-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (812) Google Scholar). Mapping studies that have localized the epitopes of anti-functional monoclonal antibodies to the A-domains of the α1, α2, αL, αM, and αX integrins initially suggested a role for A-domains in ligand binding (5Kern A. Briesewitz R. Bank I. Marcantonio E.E. J. Biol. Chem. 1994; 269: 22811-22816Abstract Full Text PDF PubMed Google Scholar, 6Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar, 7Diamond M.S. Garcia-Aguilar J. Bickford J.K. Corbi A.L. Springer T.A. J. Cell Biol. 1993; 120: 1031-1043Crossref PubMed Scopus (480) Google Scholar, 8Landis R.C. Bennet R. Hogg N. J. Cell Biol. 1993; 120: 1519-1527Crossref PubMed Scopus (113) Google Scholar, 9Landis R.C. McDowall A. Holness C.L.L. Littler A.J. Simmons D.L. Hogg N. J. Cell Biol. 1994; 126: 529-537Crossref PubMed Scopus (89) Google Scholar, 10Bilsland C.A.G. Diamond M.S. Springer T.A. J. Immunology. 1994; 152: 4582-4589PubMed Google Scholar). More recently, it has also been demonstrated that isolated recombinant A-domains from α2, αL, and αM are capable of binding ligands (11Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar, 12Tuckwell D.S. Calderwood D.A. Green L.J. Humphries M.J. J. Cell Sci. 1995; 107: 1629-1637Google Scholar, 13Randi A.M. Hogg N. J. Biol. Chem. 1994; 269: 12395-12398Abstract Full Text PDF PubMed Google Scholar, 14Ueda T. Rieu P. Brayer J. Arnaout M.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10680-10684Crossref PubMed Scopus (164) Google Scholar, 15Zhou L. Lee D.H.S. Plescia J. Lau C.Y. Altieri D.C. J. Biol. Chem. 1994; 269: 17075-17079Abstract Full Text PDF PubMed Google Scholar). The integrin α1β1 is a receptor for collagen I, collagen IV, and laminin (16Tuckwell D.S. Humphries M.J. Crit. Rev. Oncol. Hematol. 1993; 15: 149-171Crossref PubMed Scopus (32) Google Scholar). These interactions, like all integrin-ligand binding events, are cation-dependent and require Mg2+or Mn2+; Ca2+, however, does not support binding (17Luque A. Sanchez-Madrid F. Cabanas C. FEBS Letts. 1994; 346: 278-284Crossref PubMed Scopus (33) Google Scholar). A similar pattern of ligand binding is found for the closely related integrin α2β1, which also interacts with collagen I, collagen IV, and laminin (16Tuckwell D.S. Humphries M.J. Crit. Rev. Oncol. Hematol. 1993; 15: 149-171Crossref PubMed Scopus (32) Google Scholar). α1β1 and α2β1 have, however, been shown to differ in their relative affinities for ligands, with α1β1 binding collagen IV and laminin with higher affinity (18Kern A Eble J. Golbik R. Kühn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (183) Google Scholar). Integrins α1β1 and α2β1 also bind to a proteolytic fragment of collagen IV, CB3 (19Vandenberg P. Kern A. Ries A. Luckenbill-Edds L. Mann K. Kühn K. J. Cell Biol. 1991; 113: 1475-1483Crossref PubMed Scopus (209) Google Scholar), but are believed to have distinct binding sites within this fragment (18Kern A Eble J. Golbik R. Kühn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (183) Google Scholar). To investigate the role of the α1 A-domain in ligand binding, we have generated recombinant A-domain in a bacterial expression system and tested its binding to a range of integrin ligands. The specificity of A-domain ligand binding has also been examined by comparing recombinant α1, α2, and αM integrin A-domains. We find that integrin α1 A-domain binds to collagen I, collagen IV, and laminin in a saturable, concentration-dependent manner and that the binding can be inhibited by an anti-functional anti-α1 monoclonal antibody (mAb). 1The abbreviations used are: mAb, monoclonal antibody; RT-PCR, reverse transcriptase-polymerase chain reaction; GST, glutathione S-transferase; TBS, tris-buffered saline; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; SPR, surface plasmon resonance; ABTS, 2′2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). α2 A-domain also binds these ligands, while αM A-domain does not. The αM A-domain does, however, bind to while the α1 and α2 A-domains not. This binding is largely and time binding studies using surface plasmon resonance that A-domain, and ligand a using proteolytic of collagen IV, the ligand specificity of the closely related α1 and α2 A-domains has been The that recombinant A-domains the specificity reported for intact were an were obtained from the monoclonal integrin α1, monoclonal integrin α2, and reported by S.K. K. Humphries M.J. Biochem. 1991; PubMed Scopus Google monoclonal integrin αM, antibody IV and were obtained from laminin was obtained from The collagen IV CB3, and were produced (18Kern A Eble J. Golbik R. Kühn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (183) Google Scholar, P. Kern A. Ries A. Luckenbill-Edds L. Mann K. Kühn K. J. Cell Biol. 1991; 113: 1475-1483Crossref PubMed Scopus (209) Google Scholar). The of recombinant α2 A-domain has been D.S. Calderwood D.A. Green L.J. Humphries M.J. J. Cell Sci. 1995; 107: 1629-1637Google Scholar). The α1 and αM A-domains were produced in a similar manner. for the A-domains from integrins α1 and αM was produced by reverse transcriptase-polymerase chain from the cell or was a of L. J. Green of was generated using a the of the integrin A-domains and a site αM, chain of this was using the with the and a to for amino the of the integrin A-domain Arnaout M.A. Cell. 1993; Full Text PDF PubMed Scopus Google and including a site αM, of at at and at were of the were from a and the were into and used to A-domain from were by the chain of F. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and with the and αM, R. Marcantonio E.E. J. Biol. Chem. 1993; Full Text PDF PubMed Google and A.L. L.J. Springer T.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, The was into the expression PubMed Scopus Google were and was produced for α2 D.S. Calderwood D.A. Green L.J. Humphries M.J. J. Cell Sci. 1995; 107: 1629-1637Google that was not for from α1 and αM A-domains. using a glutathione affinity was and was not The recombinant A-domains produced were and amino N-terminal amino acid of α1 and αM A-domains the collagen I, and were was to in and was to a of The was for at and acid for collagen to of ligand to A-domain was measured using from D.S. Calderwood D.A. Green L.J. Humphries M.J. J. Cell Sci. 1995; 107: 1629-1637Google Scholar). A-domain proteins in were to at were with in for at with TBS, and ligand in TBS, BSA, was for at were three with TBS, and in TBS, was for three with TBS, bound ligand was with were at a binding of A-domains to ligand were but were with ligand at of and binding of A-domain was antibody of A-domain binding to the was to the antibody in TBS, BSA, was for were three with TBS, and antibody in TBS, BSA, was for were three with TBS, and binding was with The and constants and the for A-domain binding to collagen and IV were measured using a This biosensor was used in with the collagen or IV was to the matrix of a A-domain was the at and binding was measured a of TBS, was used and of TBS, were used to bound A-domain, the surface for further binding The was used to these to the first A This produced values for and the to be To binding in the of other a of TBS, was the to of A-domain in the The reverse transcriptase-polymerase chain was used to integrin α1 A-domain from cell and integrin αM A-domain from into that the αM A-domain the A.L. L.J. Springer T.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of α1 A-domain three differences from the R. Marcantonio E.E. J. Biol. Chem. 1993; Full Text PDF PubMed Google an inserted at a at which the in the and a to at These differences in in the amino acid of the a to at and a to at from the of the the using produced the and using also the all used a and produced the from we that it an α1 integrin A-domain were into the expression and used to proteins of reported for the α2 A-domain D.S. Calderwood D.A. Green L.J. Humphries M.J. J. Cell Sci. 1995; 107: 1629-1637Google Scholar). proteins were and were used or with and a to N-terminal of A-domains that at the the recombinant was at least not A-domains with the α2 A-domain bound a number of integrin including an anti-functional The α1 A-domain was by the anti-functional anti-α1 integrin in ELISA, and αM A-domain bound the anti-functional not The integrins α1β1 and α2β1 are reported to be collagen receptors, while integrin binds ligands. To investigate the role of A-domains in binding the binding of collagen to A-domain proteins was that the A-domain proteins from α1 and α2 integrins support binding of collagen I, while the αM A-domain little binding. a higher binding to the α1 A-domain to the α2 The differences in binding were not to of the proteins the of all three proteins to the measured by antibody in not The differences α1 and α2 A-domains differences in the of A-domain in the a in the number of binding sites of collagen was the of the of the collagen at for inhibited its binding to α1 and α2 A-domains not To the specificity of the A-domains for their ligands, we investigated binding of the known ligand to all three A-domains. bound to the αM A-domain in the of but not bind to α1 or α2 the the binding to was higher to binding to αM A-domain was to by that the binding was The binding of to αM A-domain was also to the of the by the anti-functional not shown that the αM A-domain with we investigated the of collagen binding to α1 and α2 A-domains. A that collagen binding to α1 A-domain was inhibited by the of the little Ca2+, and all similar of binding. the three the of binding. This does not that reported for the whole integrin, was for ligand binding and not support binding (17Luque A. Sanchez-Madrid F. Cabanas C. FEBS Letts. 1994; 346: 278-284Crossref PubMed Scopus (33) Google Scholar). binding to α2 A-domain was inhibited by however, in this little binding, and and produced of collagen binding. This is in with D.S. Calderwood D.A. Green L.J. Humphries M.J. J. Cell Sci. 1995; 107: 1629-1637Google Scholar). while the A-domains from integrins α1 and α2 bind collagen I, differ in their The binding of A-domain to collagen was also This was using A-domain proteins or by antibody of the the A-domain demonstrated concentration-dependent binding of α1 A-domain to collagen however, inhibited this was similar in and antibody assays, with seen for the antibody not In assays, was at A-domain were obtained for α2 A-domain binding to collagen not The of collagen binding to A-domain and A-domain binding to collagen is to The to be these binding was the triple-helical of collagen and the A-domain of inhibited binding and not To the in solid phase the of the A-domain-collagen was further investigated using a was the and binding of A-domain was These were for α1 and α2 A-domain proteins at a range of a of α1 A-domain binding to collagen in the of and are and of bound A-domain with is of the bound α1 A-domain and of the bound α2 A-domain that the of is for of the bound not for A-domain-collagen binding. to bind collagen in these of proteins in the of was also seen for collagen binding to and all α1 A-domain binding while and α2 A-domain binding. binding of not This the that demonstrated a cation-dependent In to collagen I, α1β1 is also reported to bind collagen IV and laminin (16Tuckwell D.S. Humphries M.J. Crit. Rev. Oncol. Hematol. 1993; 15: 149-171Crossref PubMed Scopus (32) Google Scholar). The binding of collagen IV not be using A-domains collagen IV is a A. of α1 A-domain binding to collagen I, collagen IV, and laminin that the α1 A-domain bound all these ligands in a manner The α1 A-domain exhibited similar of binding to collagen and collagen IV, with laminin bound to a In CB3, an proteolytic fragment of collagen IV, a similar of binding to intact collagen IV binding was less at A-domain To the specificity of the A-domain binding, we investigated and antibody at A-domain that of α1 A-domain bound collagen I, collagen IV, and CB3 and that binding was inhibited by and the anti-α1 integrin The integrin α2β1 is also a receptor for laminin and collagen IV, we investigated the binding of α2 A-domain to these ligands and it with α1 that α2 A-domain bound to collagen IV and however, in this collagen was a ligand collagen IV and laminin. CB3 less binding collagen IV, the with α1. while α1 and α2 A-domains bound the range of ligands, of A and that the relative binding to these ligands differed the A-domains. of binding to collagen I, and collagen IV not of collagen IV were used to further investigate the differences α1 and α2 A-domains and the integrin binding sites in collagen α1 and α2 A-domains bound to CB3, which is with obtained for whole integrins in solid phase binding and cell D.S. Calderwood D.A. Green L.J. Humphries M.J. J. Cell Sci. 1995; 107: 1629-1637Google Scholar, A Eble J. Golbik R. Kühn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (183) Google Scholar, P. Kern A. Ries A. Luckenbill-Edds L. Mann K. Kühn K. J. Cell Biol. 1991; 113: 1475-1483Crossref PubMed Scopus (209) Google Scholar). of this fragment fragments, and of integrin binding sites in these demonstrated that α1β1 and α2β1 were to bind to F1 but α1β1 bound to (18Kern A Eble J. Golbik R. Kühn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (183) Google Scholar). the and in CB3 and which are to contain the α2β1 of binding demonstrated that α1 A-domain bound F1 and in a concentration-dependent while α2 A-domain bound at to F1 and not at all to F4. This binding was inhibited by not of α1 and α2 A-domain proteins to collagen and IV was investigated a the with the was used to determine the and to the and of the the binding of A-domain to a was measured and from the binding to the surface was of a range of to collagen and was of collagen to the was for collagen IV, and obtained for binding to collagen were higher those to collagen of α2 A-domain to collagen IV be using the however, were to obtained are shown in I. The a number of to the of the binding to the These that a a to the however, comparing the with that a the constants produced a of the interactions was shown by of a binding produced using the with the This demonstrated that were The binding obtained for A-domain binding to collagens in solid phase can also be to values for the J. Humphries M.J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google J. Google Scholar). These values are shown in and with those obtained using the constants for α1 and α2 A-domain with solid and values were generated using the for a range of binding to and was in TBS, are not constants for α2 A-domain with collagen IV not be measured using to of binding. values were from or from of solid phase from at least three in a The and values were generated using the for a range of binding to and was in TBS, are not constants for α2 A-domain with collagen IV not be measured using to of binding. values were from or from of solid phase from at least three We have produced a recombinant A-domain from the α1 integrin and its ligand binding with recombinant A-domains from the α2 and αM integrins. are that the α1 integrin A-domain is a largely cation-dependent ligand binding the α1 and α2 A-domains similar but distinct ligand binding and of binding affinities for A-domain-collagen interactions values with those reported for interactions, suggesting that A-domains are the major collagen binding sites in integrins. integrin A-domains and solid phase have been used to investigate A-domain-ligand interactions. The that recombinant A-domains from α1 and α2 integrins bind collagen I, collagen IV, and the collagen IV fragment CB3, but not the ligand This is the first demonstration that the α1 A-domain is a and the first of laminin binding by A-domains. while the α1 and α2 A-domains were found to bind the ligands, their binding were and their binding sites within collagen IV to be The specificity of these interactions was by the for of ligand and A-domain and antibody reports of the ligand specificity of α1β1 and α2β1 these with isolated A-domains. cell binding, antibody affinity and solid phase have been to α1β1 binding to collagen I, IV, and and to laminin (5Kern A. Briesewitz R. Bank I. Marcantonio E.E. J. Biol. Chem. 1994; 269: 22811-22816Abstract Full Text PDF PubMed Google Scholar, A. Sanchez-Madrid F. Cabanas C. FEBS Letts. 1994; 346: 278-284Crossref PubMed Scopus (33) Google Scholar, A Eble J. Golbik R. Kühn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (183) Google Scholar, N. J. Biol. Chem. Full Text PDF PubMed Google Scholar, D.C. S. Full Text PDF PubMed Scopus Google Scholar, D.C. K. M.J. K. J. 1993; Scopus (209) Google Scholar, P. P. L. Cell 1991; PubMed Scopus (89) Google Scholar). In the CB3 fragment of collagen IV and F1 and of CB3 have been shown to contain the α1β1 binding site (18Kern A Eble J. Golbik R. Kühn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (183) Google Scholar, P. Kern A. Ries A. Luckenbill-Edds L. Mann K. Kühn K. J. Cell Biol. 1991; 113: 1475-1483Crossref PubMed Scopus (209) Google Scholar). The α2β1 integrin binds a similar range of ligands (16Tuckwell D.S. Humphries M.J. Crit. Rev. Oncol. Hematol. 1993; 15: 149-171Crossref PubMed Scopus (32) Google however, the affinity of with collagens is from that of integrin and α1β1 to bind laminin α2β1 (18Kern A Eble J. Golbik R. Kühn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (183) Google Scholar). The binding site for α2β1 in collagen IV has also been localized to the CB3 fragment (19Vandenberg P. Kern A. Ries A. Luckenbill-Edds L. Mann K. Kühn K. J. Cell Biol. 1991; 113: 1475-1483Crossref PubMed Scopus (209) Google however, the α1β1 and α2β1 binding sites are (18Kern A Eble J. Golbik R. Kühn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (183) Google Scholar). The A-domains all of the ligand binding activity of the intact α1β1 and α2β1 integrins and to be within the intact To the A-domain-collagen interactions further and the relative contribution of the A-domain to integrin-ligand binding the of binding were measured using These that the α1 A-domain has a higher affinity for collagens does the α2 of binding affinities produced from of solid phase binding a similar pattern of however, the differences α1 and α2 binding were less The binding constants for α1β1 binding to collagen IV have been measured using and solid phase binding and These values in the range of which is in with the obtained for α1 A-domain binding to collagen IV Kern (18Kern A Eble J. Golbik R. Kühn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (183) Google have obtained similar values for α1β1 binding to however, this from to the α2β1 binding to CB3 similar affinity but was to (18Kern A Eble J. Golbik R. Kühn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (183) Google Scholar). We measured the affinity of α2 A-domain for collagen IV these that the binding affinity of α1 A-domain and α1β1 binding to collagen IV are suggesting that the A-domain is the major ligand binding site in the The in affinity reported for the intact receptor is and that the binding site can be by this regulation of the A-domain by the the recombinant A-domain is of this it for the values measured in the of for the A-domains and those reported for whole integrins. this in it be that while the closely the from the that a this be to in the Integrins are known to require for ligand binding, and a number of have been to The of the αM and A-domains have been and a site at of the domain Jie-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (812) Google Scholar, A. D.L. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). it is that intact integrins require for ligand binding, and have all been reported for isolated A-domain binding to ligands (11Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar, 12Tuckwell D.S. Calderwood D.A. Green L.J. Humphries M.J. J. Cell Sci. 1995; 107: 1629-1637Google Scholar, P. T. I. Arnaout M.A. J. Cell Biol. 1994; PubMed Scopus Google Scholar, L. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). reported largely cation-dependent however, in was of A-domain to collagen is while binding of collagen to A-domain is of A-domain-collagen binding an for and binding that be with suggesting that is a for to bound the integrin-ligand This that are for A-domain ligand however, binding be seen to of the recombinant A-domains. The role of in ligand binding it is to determine the binding a ligand binding in the A-domain or is a ligand and A-domains. from and binding studies T. Rieu P. Brayer J. Arnaout M.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10680-10684Crossref PubMed Scopus (164) Google Scholar, T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google that is not for A-domain-ligand binding but that it integrin-ligand binding. and A. D.L. Full Text Full Text PDF PubMed Scopus Google have shown that the of the recombinant A-domain in the and of are suggesting that the of ligand binding is not to of a ligand binding The or of does, however, have a the the binding of the and studies that the ligand binding and binding sites are the of the molecule, surface for the of ligand binding. that was to ligand binding is with this and a In recombinant that in the of the and are that ligand binding can In the intact integrin, the is further by the of other sites in the α and regulation of the whole integrin does not that seen in the isolated it is reported that not support collagen binding by α2β1, or collagen binding (18Kern A Eble J. Golbik R. Kühn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (183) Google Scholar, A. Sanchez-Madrid F. Cabanas C. FEBS Letts. 1994; 346: 278-284Crossref PubMed Scopus (33) Google Scholar). We have shown while support of collagen binding to α2 A-domain, it binding to α1 is while the A-domain in the intact α1β1 bind Ca2+, ligand binding, binding at other sites the integrin binding. In we have demonstrated that the A-domain from α1 integrin can bind ligands and that laminin is a ligand for A-domains. We that α1β1 and α2β1 binding to collagen IV are also with isolated A-domains and that the affinity of A-domain binding to collagen is similar to that reported for whole integrin binding. we that A-domain-ligand binding is largely cation-dependent and that and A-domain a We are to Green for the of and to from for critical of