The Integrin alpha 1 A-domain Is a Ligand Binding Site for Collagens and Laminin*

(Received for publication, January 6, 1997, and in revised form, February 24, 1997)

David A. Calderwood Dagger , Danny S. Tuckwell , Johannes Eble §, Klaus Kühn and Martin J. Humphries par

From the Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom, the § Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, and the  Max-Planck-Institut für Biochemie, 82152 Martinsreid, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The integrin alpha 1beta 1 is a cell surface receptor for collagens and laminin. The alpha 1 subunit contains an A-domain, and the A-domains of other integrins are known to mediate ligand binding. To determine the role of the alpha 1 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 alpha 1 A-domain and tested its ability to bind collagens and laminin. In solid phase assays, the A-domain from alpha 1 was found to bind to collagen I, collagen IV, and laminin in a largely cation-dependent manner. The alpha 2 A-domain, from the alpha 2beta 1 integrin, also bound to these ligands, but the binding hierarchy differed from that seen for alpha 1. 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. alpha 1 A-domain bound to all three fragments, while the alpha 2 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 alpha 1beta 1 and alpha 2beta 1 integrins.


INTRODUCTION

Integrins are a family of alpha beta 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 (1). The integrin family contains at least 16 alpha  subunits, seven of which contain an ~200 amino acid inserted domain in their N-terminal region (I or A-domain) (2, 3). 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 (2, 4).

Mapping studies that have localized the epitopes of anti-functional monoclonal antibodies to the A-domains of the alpha 1, alpha 2, alpha L, alpha M, and alpha X integrins initially suggested a role for A-domains in ligand binding (5-10). More recently, it has also been demonstrated that isolated recombinant A-domains from alpha 2, alpha L, and alpha M are capable of binding ligands (11-15).

The integrin alpha 1beta 1 is a receptor for collagen I, collagen IV, and laminin (16). These interactions, like all integrin-ligand binding events, are cation-dependent and require Mg2+ or Mn2+; Ca2+, however, does not support binding (17). A similar pattern of ligand binding is found for the closely related integrin alpha 2beta 1, which also interacts with collagen I, collagen IV, and laminin (16). alpha 1beta 1 and alpha 2beta 1 have, however, been shown to differ in their relative affinities for ligands, with alpha 1beta 1 binding collagen IV and laminin with higher affinity (18). Integrins alpha 1beta 1 and alpha 2beta 1 also bind to a proteolytic fragment of collagen IV, CB3 (19), but are believed to have distinct binding sites within this fragment (18).

To investigate the role of the alpha 1 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 alpha 1, alpha 2, and alpha M integrin A-domains. We find that integrin alpha 1 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-alpha 1 monoclonal antibody (mAb).1 alpha 2 A-domain also binds these ligands, while alpha M A-domain does not. The alpha M A-domain does, however, bind to fibrinogen, while the alpha 1 and alpha 2 A-domains do not. This binding is largely cation-dependent, and real time binding studies using surface plasmon resonance (SPR) analysis suggests that cation, A-domain, and ligand may form a ternary complex. By using proteolytic fragments of collagen IV, the ligand specificity of the closely related alpha 1 and alpha 2 A-domains has been compared. The results indicate that recombinant A-domains retain the specificity reported for intact receptors.


EXPERIMENTAL PROCEDURES

Materials

Oligonucleotides were synthesized on an Applied Biosystems 391 DNA synthesizer. Antibodies were obtained from the following sources: monoclonal mouse anti-human integrin alpha 1, 5E8D9 (Upstate Biotechnology Inc., New York); monoclonal mouse anti-human integrin alpha 2, Gi9 (The Binding Site, Birmingham, United Kingdom.) and 16B4 (previously reported as 1C11) raised as described by Mould et al. (20); monoclonal mouse anti-human integrin alpha M, 44 (Autogen Bioclear, Devizes, UK); anti-glutathione S-transferase (GST) rabbit polyclonal antibody (Autogen Bioclear). Rat tail tendon type I collagen, human placental type IV collagen, and human fibrinogen were obtained from Sigma, Poole, Dorset, UK. Rat laminin was obtained from Life Technologies Ltd., Paisley, UK. The collagen IV fragments CB3, F1, and F4 were produced as described previously (18, 19).

Production of Recombinant Integrin A-domains

The production of recombinant alpha 2 A-domain has already been described (12). The alpha 1 and alpha M A-domains were produced in a similar manner. DNA coding for the A-domains from integrins alpha 1 and alpha M was produced by reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA prepared from the A375 human melanoma cell line (for alpha 1) or human buffy coat lymphocytes (for alpha M) was a gift of L. J. Green (University of Manchester, UK). First strand cDNA was generated using a 3' primer spanning the predicted end of the integrin A-domains and incorporating a SalI site (alpha 1, 5'-TTTGTCGACTCAGGCTTCCAGG GCAAATATTCTTTCTCC-3'; alpha M, 5'-TTTGTCGACTCAACCCTCGATCGCAAAGATCTTCTCCCG-3'). Polymerase chain reaction (PCR) amplification of this cDNA was then carried out using the thermostable proof-reading DNA polymerase Pfu (Stratagene Ltd., Cambridge, UK) with the above 3' primer and a 5' primer designed to produce DNA coding for 17 amino acids preceding the predicted start of the integrin A-domain (21) and including a BamHI site (alpha 1, 5'-TTTGGATCCGTCAGCCCCACATTTCAAGTCGTGAATTCC-3'; alpha M, 5'-TTTGGATCCAACCTACGGCAGCAGCCCCAG-3'). 50 cycles, each consisting of 1 min at 94 °C, 1 min at 55 °C, and 2.5 min at 72 °C, were carried out. PCR products, of the correct Mr, were then excised from a 1% agarose gel. After digestion with BamHI and SalI, the products were ligated into pUC119 and used to transform Escherichia coli strain DH5alpha F'. A-domain sequences from transformants were sequenced by the dideoxy chain termination method of Sanger (22) and compared with the published sequences (alpha 1 and alpha M, see Refs. 23 and 24, respectively). The sequenced DNA was then subcloned into the expression vector pGEX-4T3 (25) (Pharmacia, Milton Keynes, UK). Transformants were screened, and protein was produced as described for alpha 2 (12) except that dithiothreitol was not required for GST removal from alpha 1 and alpha M A-domains. For experiments using fusion protein, a single glutathione affinity step was performed and thrombin digestion was not carried out. The recombinant A-domains produced were 214 (alpha 1), 224 (alpha 2), and 211 (alpha M) amino acids long. N-terminal amino acid sequencing of thrombin-cleaved alpha 1 and alpha M A-domains confirmed the predicted starting sequence.

Biotinylation of Proteins

A-domain-GST fusion proteins, collagen I, and fibrinogen were biotinylated as follows. Protein was diluted to 1 mg/ml in 150 mM NaCl, 10 mM Tris-HCl, pH 7.5 (TBS), and dry sulfo-N-hydroxysuccinimido-biotin (Pierce, Chester, UK) was added to give a ratio of 1:1 (w/w) protein:biotin. The mixture was incubated for 1 h at room temperature and then dialyzed against TBS (or 0.1 M acetic acid for collagen I) to remove unincorporated biotin.

Ligand Binding Assays

Binding of soluble ligand to immobilized A-domain was measured using assays adapted from Tuckwell et al. (12). A-domain fusion proteins (10 µg/ml in PBS without divalent cations, PBS-) were coated to 96-well microtitre plates (Immulon 4, Dynatech, Billingshurst, UK) overnight at 4 °C. Wells were then blocked with 50 mg/ml BSA in TBS for 1 h at room temperature and, washed twice with TBS, and biotinylated ligand in TBS, 1 mg/ml BSA, plus cation, was added for 3 h at 37 °C. Plates were washed three times with TBS, 1 mM MnCl2, and ExtrAvidin peroxidase (Sigma; 10 µg/ml in TBS, 1 mM MnCl2) was added for 15 min. After washing three times with TBS, 1 mM MnCl2, bound ligand was visualized with 0.1 M sodium acetate, 0.05 M NaH2PO4, 2 mM 2'2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 0.03% H2O2. Wells were read at 405 nm on a plate reader.

A-domain Binding Assays

Assays measuring binding of soluble A-domains to immobilized ligand were carried out essentially as above, but plates were coated with ligand (usually at 30 µg/ml) instead of fusion protein and binding of biotinylated A-domain fusion protein was measured. For antibody detection of unbiotinylated A-domain fusion protein binding to ligand, the assay was performed as above up to the streptavidin step where 5 µg/ml anti-GST antibody in TBS, 1 mg/ml BSA, 1 mM MnCl2 was added for 1 h instead. Wells were washed three times with TBS, 1 mM MnCl2, and 1:500 peroxidase-linked goat anti-rabbit antibody (Sigma) in TBS, 1 mg/ml BSA, 1 mM MnCl2 was added for 45 min. Wells were washed three times with TBS, 1 mM MnCl2, and binding was detected with ABTS as above.

Surface Plasmon Resonance

The kinetic parameters (apparent association and dissociation rate constants ka and kd, respectively) and the apparent equilibrium constant (KD) for A-domain binding to collagen I and IV were measured using SPR on a BIAcoreTM (Biacore, St. Albans, UK). This biosensor device was used in accordance with the manufacturer instructions. Briefly, collagen I or IV was covalently coupled via primary amine groups to the dextran matrix of a CM5 sensor chip. A-domain solution was flowed over the chip at 5 µl/min, and binding was measured as a function of time. TBS, 1 mM MnCl2 was used as running buffer throughout, and injections of TBS, 10 mM EDTA were used to remove bound A-domain, regenerating the surface for further binding experiments. The curve fitting software, BIAevaluation, was used to fit these results to the simple first order interaction model A + B iff  AB. This produced values for ka and kd, allowing the apparent equilibrium constant KD to be derived from kd/ka. To measure binding in the presence of divalent cations other than Mn2+, a 10-min injection of TBS, 10 mM EDTA was passed over the chip (to remove any residual Mn2+) immediately prior to injection of A-domain in the specified cation.


RESULTS

Generation of Integrin alpha 1 and alpha M A-domain cDNA

The reverse transcriptase-polymerase chain reaction was used to generate integrin alpha 1 A-domain cDNA from A375 human melanoma cell line RNA and integrin alpha M A-domain cDNA from human buffy coat lymphocyte RNA. After cloning into pUC119, sequencing revealed that the alpha M A-domain cDNA matched the published sequence (24). Sequencing of alpha 1 A-domain cDNA revealed three differences from the published sequence (23): 1) an inserted Thr at position 502; 2) a deleted Ala at position 511, which put the sequence back in frame after the earlier insertion, and 3) a Cys to Thr mutation at position 674. These differences result in two changes in the predicted amino acid sequence of the protein, a lysine to glutamate at position 170 and a threonine to isoleucine at 228, numbered from the start of the mature polypeptide. Repeating the RT-PCR using two different A375 RNA samples produced the same sequence, and RT-PCR using human smooth muscle RNA also gave the same sequence. As all PCR reactions used a proof-reading DNA polymerase and produced the same sequence from different RNA samples, we believe that it represents an accurate human alpha 1 integrin sequence.

Expression of Integrin alpha 1 and alpha M A-domains in E. coli

Integrin A-domain cDNAs were cloned into the pGEX-2T3 expression vector and used to transform E. coli. After induction, transformants expressed GST-A-domain fusion proteins of ~50 kDa as reported for the alpha 2 A-domain (12). Fusion proteins were purified on glutathione-agarose columns and were either used directly or cleaved with thrombin and passed through a second glutathione-agarose column to produce purified 25 kDa A-domain. N-terminal sequencing of purified A-domains showed that cleavage had occurred at the expected site. SDS-PAGE showed the recombinant protein was at least 90% pure (data not shown).

All A-domains reacted specifically with previously characterized mAbs: the alpha 2 A-domain bound a number of anti-alpha 2 integrin mAbs, including Gi9, an anti-functional mAb. The alpha 1 A-domain was recognized by the anti-functional anti-alpha 1 integrin mAb 5E8D9 in ELISA, and alpha M A-domain bound the anti-functional anti-alpha M mAb 44 (data not shown).

Collagen I Binds to alpha 1 and alpha 2 A-domains but Not to alpha M A-domain

The integrins alpha 1beta 1 and alpha 2beta 1 are reported to be collagen receptors, while integrin alpha Mbeta 2 binds non-collagenous ligands. To investigate the role of A-domains in binding collagen, the binding of biotinylated collagen I to A-domain fusion proteins was measured. Fig. 1 shows that the A-domain fusion proteins from alpha 1 and alpha 2 integrins support dose-dependent, saturable binding of collagen I, while the alpha M A-domain exhibits very little binding. Collagen I shows a higher maximal binding to the alpha 1 A-domain than to the alpha 2 A-domain. The observed differences in binding were not due to different coating efficiencies of the GST-fusion proteins as the coating concentration chosen (10 µg/ml) gave very similar, almost maximal, coating of all three fusion proteins to the plate as measured by anti-GST antibody in ELISA (data not shown). The differences between alpha 1 and alpha 2 A-domains may instead reflect differences in the amount of correctly folded A-domain in the different samples rather than a difference in the number of binding sites per A-domain. Binding of collagen I was dependent on the conformation of the triple helix, as heat denaturation of the collagen I at 50 °C for 30 min inhibited its binding to alpha 1 and alpha 2 A-domains (data not shown).


Fig. 1. Collagen I binding to immobilized A-domains in ligand binding assays. Binding to alpha 1 A-domain fusion protein (bullet ), alpha 2 A-domain fusion protein (black-down-triangle ), alpha M A-domain fusion protein (black-square), and BSA only (open circle ) in 1 mM MnCl2 was measured. Data are mean ± S.E. and n >=  9 from three experiments normalized for 30 µg/ml collagen binding to alpha 1 A-domain.
[View Larger Version of this Image (17K GIF file)]

Fibrinogen Binds the alpha M A-domain Specifically in a Cation-dependent Manner

To confirm the specificity of the A-domains for their ligands, we investigated binding of the known alpha Mbeta 2 ligand fibrinogen to all three A-domains. Biotinylated fibrinogen bound to the alpha M A-domain in the presence of 1 mM Mn2+ but did not bind to alpha 1 or alpha 2 A-domains, above the GST control (Fig. 2). Interestingly the binding to GST was higher than to BSA. Fibrinogen binding to alpha M A-domain was reduced to GST levels by 5 mM EDTA indicating that the binding was cation-dependent. The binding of biotinylated fibrinogen to alpha M A-domain was also reduced to the levels of the GST control by the anti-functional anti-alpha M mAb 44 (data not shown).


Fig. 2. Fibrinogen binding to immobilized A-domains in ligand binding assays. Binding to alpha 1 A-domain fusion protein (bullet ), alpha 2 A-domain fusion protein (black-down-triangle ), alpha M A-domain fusion protein (black-square), GST (black-triangle), and BSA (open circle ) in 1 mM MnCl2 and to alpha M A-domain in 5 mM EDTA (square ) was measured. Data are mean ± S.E. and n >=  12 from five experiments normalized for 10 µg/ml fibrinogen binding to alpha M A-domain.
[View Larger Version of this Image (18K GIF file)]

Cation-dependence of Collagen I Binding to A-domains

Having shown that the alpha M A-domain interaction with fibrinogen required divalent cations, we investigated the effect of different divalent cations on biotinylated collagen I binding to alpha 1 and alpha 2 A-domains. Fig. 3A shows that collagen binding to alpha 1 A-domain was completely inhibited by EDTA and, therefore, requires divalent cations. However, the nature of the divalent cation had little effect as Ca2+, Mg2+, and Mn2+ all supported similar levels of binding. Of the three cations, Mn2+ supported the highest levels of binding. This cation profile does not match that reported for the whole integrin, where Mg2+ was required for ligand binding and Ca2+ did not support binding (17). Collagen binding to alpha 2 A-domain was inhibited by EDTA (Fig. 3B); however, in this case Ca2+ supported only very little binding, and Mg2+ and Mn2+ produced identical levels of collagen binding. This is in agreement with previously published data (12). Thus, while the A-domains from integrins alpha 1 and alpha 2 both bind collagen I, they differ in their cation specificities.


Fig. 3. The effect of cations on collagen I binding to immobilized alpha 1 (A) and alpha 2 (B) A-domain fusion protein was measured in the presence of 1 mM MnCl2 (bullet ), 1 mM MgCl2 (black-square), 1 mM CaCl2 (black-down-triangle ), and 5 mM EDTA (black-triangle). Binding to GST (square ) and BSA (open circle ) was measured in 1 mM MnCl2. Data are mean ± S.E. and n >=  9 from three experiments normalized for 10 µg/ml collagen binding to A-domain in the presence of 1 mM MnCl2.
[View Larger Version of this Image (14K GIF file)]

The binding of soluble A-domain to immobilized collagen was also investigated. This was carried out using biotinylated A-domain fusion proteins or by antibody detection of the GST moiety on the A-domain fusion protein. Both methods demonstrated concentration-dependent saturable binding of alpha 1 A-domain to collagen I; however, EDTA only partially inhibited this interaction (Fig. 4A). Inhibition was similar in both biotinylation and antibody detection assays, with slightly more inhibition seen for the antibody detection assays (data not shown). In both assays, inhibition was maximal at lower A-domain protein concentrations. Similar results were obtained for alpha 2 A-domain binding to collagen I (data not shown).


Fig. 4. alpha 1 A-domain binding to immobilized collagen I. A, microtitre plates were coated with substrate at 30 µg/ml and blocked with BSA, and GST-A-domain fusion protein was added. After incubating for 3 h at 37 °C, unbound fusion protein was washed off. Binding was detected using anti-GST antibody, a peroxidase-conjugated goat anti-rabbit IgG secondary antibody, and ABTS. Binding of alpha 1 A-domain fusion protein to collagen I in 1 mM MnCl2 (bullet ) and 5 mM EDTA (black-square) and to BSA in 1 mM MnCl2 (open circle ). Shown are the binding of GST in 1 mM MnCl2 to collagen I (black-triangle) and BSA (black-down-triangle ). Data are mean ± S.E. and n >=  9 from four experiments normalized for 30 µg/ml alpha 1 A-domain binding to collagen I. B, alternatively biotinlyated A-domain fusion protein was used, and binding was detected using ExtrAvidin-peroxidase and ABTS. The effect of heat denaturation of collagen I (hdCI), or alpha 1 A-domain (hd A-dom.) by heating to 50 °C for 30 min, on ligand binding was investigated. Results are mean ± S.E. and n = 9 from three experiments. Background binding to BSA has been subtracted and results normalized for native alpha 1 A-domain binding to native collagen I in the presence of 1 mM MnCl2.
[View Larger Version of this Image (17K GIF file)]

The discrepancy between cation-dependence of collagen binding to immobilized A-domain and A-domain binding to immobilized collagen is difficult to explain. The interaction appeared to be specific as, under these conditions, binding was still dependent on the triple-helical conformation of collagen and the native A-domain structure, as heat denaturation of either component strongly inhibited binding (Fig. 4B, and data not shown).

To address the apparent discrepancies in solid phase assay results, the cation dependence of the A-domain-collagen interaction was further investigated using SPR measurements on a BIAcore. Collagen I was covalently immobilized onto the sensor chip, and binding of A-domain was measured. These measurements were performed for both alpha 1 and alpha 2 A-domain fusion proteins at a range of concentrations (3-100 µg/ml). Fig. 5 shows a typical sensorgram of alpha 1 A-domain binding to collagen I in the presence of 1 mM Mn2+. Association and dissociation phases are marked and removal of bound A-domain with 10 mM EDTA is shown. EDTA injection removed 86 ± 2% of the bound alpha 1 A-domain (mean ± S.E.; n = 33) and 82 ± 8% of the bound alpha 2 A-domain (mean ± S.E.; n = 10), indicating that the presence of divalent cation is required for maintenance of the bound complex, not simply for A-domain-collagen binding. GST failed to bind collagen in these assays. Binding of fusion proteins in the presence of different cations was also investigated, and, as already seen for biotinylated collagen binding to A-domains, 1 mM Mn2+, Mg2+, and Ca2+ all supported alpha 1 A-domain binding while only Mn2+ and Mg2+ supported alpha 2 A-domain binding. No binding occurred without addition of divalent cations (data not shown). This supports the ligand-binding assay results that demonstrated a cation-dependent interaction.


Fig. 5. Sensorgram of alpha 1 A-domain fusion protein binding to collagen I immobilized on a BIAcore CM5 sensor chip. The sensorgram is annotated to indicate different stages in the experiment. Running buffer was TBS, 1 mM MnCl2. B indicates that only running buffer was passing over the chip surface. Assoc shows binding to the chip during injection of 10 µg/ml alpha 1 A-domain fusion protein. During the dissociation phase (Dis), only running buffer was again passing over the chip. Bound A-domain was removed during the regeneration phases (Reg 1 and Reg 2). This involved injection of TBS, 10 mM EDTA. Following the initial regeneration, most of the binding was removed, and further injections of EDTA did not remove the residual binding. The large change in response during regeneration is due to differences in the refractive index of the buffers.
[View Larger Version of this Image (19K GIF file)]

alpha 1 A-domain Binds to Collagen IV and Laminin

In addition to collagen I, alpha 1beta 1 is also reported to bind collagen IV and laminin (16). The binding of collagen IV could not be studied using immobilized A-domains because collagen IV is a poor biotinylation substrate.2 Antibody detection of alpha 1 A-domain fusion protein binding to immobilized collagen I, collagen IV, and laminin showed that the alpha 1 A-domain bound all these ligands in a concentration-dependent, saturable manner (Fig. 6A). The alpha 1 A-domain exhibited very similar levels of binding to collagen I and collagen IV, with laminin bound to a lesser extent. In addition, CB3, an integrin-binding proteolytic fragment of collagen IV, supported a similar level of binding to intact collagen IV although binding was less at lower A-domain concentrations. To test the specificity of the A-domain binding, we investigated cation dependence and antibody inhibition at low A-domain concentrations. Fig. 6B shows that 0.2 µg/ml of biotinylated alpha 1 A-domain bound collagen I, collagen IV, laminin, and CB3 and that binding was inhibited by EDTA and the anti-alpha 1 integrin mAb 5E8D9.


Fig. 6. alpha 1 A-domain binding to immobilized collagen IV and laminin. A, binding of alpha 1 A-domain fusion protein to collagen I (bullet ), collagen IV (black-triangle), laminin (black-square), CB3 (black-down-triangle ), and BSA (open circle ) was measured in antibody detection assays. Ligands were coated at 10 µg/ml. Data are mean ± S.E. and n >=  6 from four experiments normalized for 30 µg/ml alpha 1 A-domain binding to collagen I. B, A-domain binding assays were used to measure binding of biotinylated fusion protein (0.2 µg/ml) to immobilized ligands coated at 10 µg/ml in the presence of 1 mM MnCl2, 1 mM MnCl2 with 10 µg/ml 5E8D9, 1 mM MnCl2 with 10 µg/ml 16B4, or 5 mM EDTA. Data are mean ± S.E. and n >=  9 from three experiments. Background binding to BSA has been subtracted from each column, and results are normalized for binding to collagen I in the presence of 1 mM MnCl2. CI, collagen I; CIV, collagen IV; Lam, laminin.
[View Larger Version of this Image (17K GIF file)]

Collagen IV and Laminin Are Ligands for the alpha 2 A-domain

The integrin alpha 2beta 1 is also a receptor for laminin and collagen IV, so we investigated the binding of alpha 2 A-domain to these ligands and compared it with alpha 1 A-domain. Fig. 7 shows that alpha 2 A-domain bound to collagen IV and laminin; however, in this case, collagen I was a better ligand than collagen IV and laminin. Furthermore, CB3 supported much less binding than collagen IV, unlike the case with alpha 1. Thus, while alpha 1 and alpha 2 A-domains bound the same range of ligands, comparison of Figs. 6A and 7 indicate that the relative binding to these ligands differed between the two A-domains. GST showed only background levels of binding to laminin, collagen I, and collagen IV (data not shown).


Fig. 7. A-domain binding to different ligands. Binding of alpha 2 A-domain fusion protein in 1 mM MnCl2 to collagen I (bullet ), collagen IV (black-triangle), laminin (black-square), CB3 (black-down-triangle ), and BSA (open circle ) was measured in antibody detection assays. Ligands were coated at 10 µg/ml. Data are mean ± S.E. and n >=  6 from four experiments normalized for 30 µg/ml alpha 2 A-domain binding to collagen I.
[View Larger Version of this Image (18K GIF file)]

alpha 1 and alpha 2 A-domain Binding to Collagen IV Fragments

Proteolytic fragments of collagen IV were used to further investigate the differences between alpha 1 and alpha 2 A-domains and the integrin binding sites in collagen IV. As described above, both alpha 1 and alpha 2 A-domains bound to CB3, which is consistent with results obtained for whole integrins in solid phase binding assays and cell attachment assays (12, 18, 19). Further proteolysis of this fragment produces four smaller fragments, F1-F4, and investigation of integrin binding sites in these fragments demonstrated that both alpha 1beta 1 and alpha 2beta 1 were able to bind to F1 but only alpha 1beta 1 bound to F4 (18). F4 lacks the N- and C-terminal regions present in CB3 and F1, which are thought to contain the alpha 2beta 1 recognition site. Antibody detection of fusion protein binding demonstrated that alpha 1 A-domain bound both F1 and F4 in a concentration-dependent saturable manner, while alpha 2 A-domain bound only poorly, if at all, to F1 and not at all to F4. This binding was inhibited by 5 mM EDTA (data not shown).

Measurement of Apparent Binding Constants for A-domain Collagen Interactions

Binding of alpha 1 and alpha 2 A-domain fusion proteins to collagen I and IV was investigated on a BIAcore. BIAevaluation, the kinetic analysis curve-fitting software supplied with the BIAcore, was used to determine the ka and kd values. Prior to fitting the association and dissociation phases of the curve, the binding of A-domain to a blank sensor chip was measured and subtracted from the binding curve. Binding to the uncoated surface was negligible. Binding of a range of fusion protein concentrations (3-100 µg/ml) to collagen I and IV-coated chips was measured. Covalent coupling of collagen I to the chip was much more efficient than for collagen IV, and consequently, signals obtained for binding to collagen I were higher than those to collagen IV. Binding of alpha 2 A-domain to collagen IV could be detected using the BIAcore; however, signals were too low to allow accurate curve fitting. Values obtained are shown in Table I. The BIAevaluation software provides a number of statistical parameters to judge the fitting of the binding model to the experimental data. These indicated that a simple association model provided a good approximation to the experimental data; however, residual plots, comparing the fitted data with experimental results, indicated that a more complex interaction may occur. That the kinetic constants produced provide a good measure of the molecular interactions involved was shown by comparison of a simulated binding curve, produced using the calculated constants, with the experimental results (Fig. 8). This demonstrated that both curves were very similar. The binding curves obtained for A-domain binding to immobilized collagens in solid phase assays can also be fitted to produce apparent KD values for the interaction (26, 27). These values are shown in Table I and exhibit generally good agreement with those obtained using the BIAcore.

Table I. Kinetic constants for alpha 1 and alpha 2 A-domain interaction with collagens

The ka and kd values were generated using the BIAevaluation analysis program, on data recorded for a range of fusion protein concentrations (60-2000 nM) binding to collagen-coated sensor chips. Binding and dissociation was performed in TBS, 1 mM MnCl2. Results are mean ± S.E. ND, not determined; constants for alpha 2 A-domain interaction with collagen IV could not be measured using SPR due to low levels of binding. KD values were calculated as kd/ka from SPR data or from curve fitting analysis of solid phase data, pooled from at least three independent experiments. The ka and kd values were generated using the BIAevaluation analysis program, on data recorded for a range of fusion protein concentrations (60-2000 nM) binding to collagen-coated sensor chips. Binding and dissociation was performed in TBS, 1 mM MnCl2. Results are mean ± S.E. ND, not determined; constants for alpha 2 A-domain interaction with collagen IV could not be measured using SPR due to low levels of binding. KD values were calculated as kd/ka from SPR data or from curve fitting analysis of solid phase data, pooled from at least three independent experiments.
A-domain Ligand ka (× 103 M-1 s-1) kdis (× 10-5 s-1) KD (nM) (SPR) n (SPR) KD (nM), solid phase

 alpha 1 Collagen I 4.1  ± 0.39 8.2  ± 0.61 24  ± 3.4 18 23  ± 2.3
 alpha 1 Collagen IV 12  ± 1.3 7.3  ± 2.0 6.0  ± 1.2 9 34  ± 10
 alpha 2 Collagen I 67  ± 0.85 120  ± 13 180  ± 25 6 54  ± 6
 alpha 2 Collagen IV ND ND ND ND 115  ± 2


Fig. 8. Comparison of experimental results for alpha 1 A-domain fusion protein binding to collagen I with a simulated results curve generated using calculated ka and kd values. The experimental sensorgram (solid line) was recorded using a flow rate of 5 µl/ml and a 10-min injection of 0.6 µM alpha 1 A-domain. The simulated sensorgram (dotted line) was modelled using the experimental parameters and a ka value of 4 × 103 M-1 s-1 and kd value of 8 × 10-5 s-1. To allow comparison of the curves, the signal prior to injection was adjusted to zero, and injection was considered to start at time 0.
[View Larger Version of this Image (16K GIF file)]


DISCUSSION

We have produced a recombinant A-domain from the alpha 1 integrin and compared its ligand binding characteristics with recombinant A-domains from the alpha 2 and alpha M integrins. Our key findings are that (i) the alpha 1 integrin A-domain is a largely cation-dependent ligand binding domain, (ii) the alpha 1 and alpha 2 A-domains show similar but distinct ligand binding specificities, and (iii) measurement of binding affinities for A-domain-collagen interactions produces values comparable with those reported for integrin-collagen interactions, suggesting that A-domains are the major collagen binding sites in integrins.

Recombinant integrin A-domains and solid phase ligand-binding assays have been used to investigate A-domain-ligand interactions. The results showed that recombinant A-domains from alpha 1 and alpha 2 integrins bind collagen I, collagen IV, laminin, and the collagen IV fragment CB3, but not the alpha Mbeta 2 ligand fibrinogen. This is the first direct demonstration that the alpha 1 A-domain is a ligand-binding module, and the first observation of laminin binding by A-domains. Interestingly, while the alpha 1 and alpha 2 A-domains were found qualitatively to bind the same ligands, their binding profiles were quantitatively different, and their binding sites within collagen IV appeared to be separate. The specificity of these interactions was confirmed by demonstrating the requirement for native structure of both ligand and A-domain and through antibody inhibition studies.

Published reports of the ligand specificity of alpha 1beta 1 and alpha 2beta 1 match these findings with isolated A-domains. Thus, cell binding, antibody blocking, affinity chromatography, and solid phase assays have variously been employed to demonstrate alpha 1beta 1 binding to collagen types I, III, IV, and VI and to laminin (5, 17, 18, 28-31). In addition, the CB3 fragment of collagen IV and fragments F1 and F4 of CB3 have been shown to contain the alpha 1beta 1 binding site (18, 19). The alpha 2beta 1 integrin binds a similar range of ligands (16); however, the affinity of interaction with collagens is different from that of integrin alpha 1beta 1, and alpha 1beta 1 appears to bind laminin better than alpha 2beta 1 (18). The binding site for alpha 2beta 1 in collagen IV has also been localized to the CB3 fragment (19); however, the alpha 1beta 1 and alpha 2beta 1 binding sites are apparently separate (18). The A-domains thus mimic almost all of the ligand binding activity of the intact alpha 1beta 1 and alpha 2beta 1 integrins and appear to be key ligand-recognition modules within the intact heterodimer.

To characterize the A-domain-collagen interactions further and address the relative contribution of the A-domain to integrin-ligand binding affinity, the kinetics of binding were measured using SPR. These results showed that the alpha 1 A-domain has a higher affinity for collagens than does the alpha 2 A-domain. Comparison of apparent binding affinities produced from analysis of solid phase binding data indicated a similar pattern of binding; however, the differences between alpha 1 and alpha 2 binding were less notable. The apparent binding constants for alpha 1beta 1 binding to collagen IV have been measured using both BIAcore and solid phase binding and inhibition assays.3 These techniques produced KD values in the range of 1-5 nM, which is in good agreement with the figures obtained for alpha 1 A-domain binding to collagen IV (6-34 nM; Table I). Kern et al. (18) have obtained similar values for alpha 1beta 1 binding to CB3; however, this varied from 1 to 30 nM depending on the divalent cations present. alpha 2beta 1 binding to CB3 showed similar affinity but was more sensitive to cations (1-110 nM) (18). We measured the affinity of alpha 2 A-domain for collagen IV as 115 nM. Taken together, these findings indicate that the binding affinity of alpha 1 A-domain and alpha 1beta 1 binding to collagen IV are similar, suggesting that the A-domain is the major ligand binding site in the integrin. The variation in affinity reported for the intact receptor is dependent on divalent cation and suggests that the binding site can be regulated by different cations. As discussed below, this may suggest allosteric regulation of the A-domain by cation-binding regions lying outside the A-domain. As the recombinant A-domain is free of this regulation, it may account for the variation between KD values measured in the presence of Mn2+ for the A-domains and those reported for whole integrins. With this in mind, it should be noted that while the simple association model closely approximates the BIAcore data, some non-random variation from the model suggests that a more complex interaction may occur; this may be due to conformational changes in the A-domain.

Integrins are known to require divalent cations for ligand binding, and a number of regions have been proposed to act as cation-binding sites. The crystal structures of the alpha M and alpha L A-domains have now been solved and show a single divalent cation-binding site at one end of the domain (4, 32). While it is accepted that intact integrins require cations for ligand binding, cation dependence, independence, and partial dependence have all been reported for isolated A-domain binding to ligands (11, 12, 33, 34). Results reported here show largely cation-dependent binding; however, some variation in cation-dependence was observed. Binding of soluble A-domain to immobilized collagen is only partially cation-dependent, while binding of biotinylated collagen to immobilized A-domain is completely cation-dependent. BIAcore analysis of A-domain-collagen binding showed an absolute requirement for divalent cations, and binding that had already taken place could be reversed with EDTA, suggesting that there is a requirement for cation to remain bound during the integrin-ligand interaction. This suggests that divalent cations are normally required for A-domain ligand binding; however, some cation-independent binding may be seen due to non-native conformations of the recombinant A-domains.

The exact role of divalent cations in ligand binding remains unclear as it is difficult to determine whether the cation binding produces a ligand binding conformation in the A-domain or is itself required as a bridge between ligand and A-domains. Data from mutagenesis and peptide binding studies (14, 11) suggest that cation is not absolutely required for A-domain-ligand binding but that it normally regulates integrin-ligand binding. Qu and Leahy (35) have recently shown that the crystal structures of the recombinant alpha L A-domain in the presence and absence of divalent cation are very similar, suggesting that the cation-dependence of ligand binding is not due to stabilization of a ligand binding conformation. The presence or absence of divalent cation does, however, have a profound effect on the charge distribution on the cation binding face of the A-domain. As epitope mapping and mutagenesis studies suggest that the ligand binding and cation binding sites are located on the same face of the molecule, surface charge might account for the cation requirement of ligand binding. Our finding that cation was required to maintain ligand binding is consistent with both this and a cation bridge model. In some recombinant A-domains, non-native structures may mean that even in the absence of divalent cation the conformation and charge distribution are such that ligand binding can occur.

In the intact integrin, the situation is further complicated by the presence of other cation-binding sites in the alpha  and beta subunits. Cation regulation of the whole integrin does not always match that seen in the isolated A-domain. For example, it is reported that Ca2+ will not support collagen binding by alpha 2beta 1, or alpha 1beta 1; indeed, Ca2+ may actually inhibit Mg2+-induced collagen binding (18, 17). We have shown that, while Ca2+ will support only low levels of collagen binding to alpha 2 A-domain, it will allow binding to alpha 1 A-domain. It is possible that, while the A-domain in the intact alpha 1beta 1 will bind Ca2+, potentially permitting ligand binding, Ca2+ binding at other sites on the integrin normally precludes binding.

In conclusion, we have demonstrated that the A-domain from alpha 1 integrin can bind ligands and that laminin is a ligand for A-domains. We demonstrate that distinctions between alpha 1beta 1 and alpha 2beta 1 binding to collagen IV are also observed with isolated A-domains and that the affinity of A-domain binding to collagen is similar to that reported for whole integrin binding. Finally, we show that A-domain-ligand binding is largely divalent cation-dependent and suggest that cation, ligand, and A-domain form a ternary complex.


FOOTNOTES

*   This work was supported by grants from the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Present address: Dept. Vascular Biology, The Scripps Research Institute, La Jolla, CA 92037.
par    To whom correspondence should be addressed: Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, 2.205 Stopford Bldg., Oxford Rd., Manchester, M13 9PT, UK. Tel.: 44-161-275-5071; Fax: 44-161-275-5082.
1   The 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).
2   D. A. Calderwood, unpublished observation.
3   J. Eble, unpublished data.

ACKNOWLEDGEMENTS

We are grateful to Dr. Linda Green for the gifts of RNA and to Dr. Sylvie Cot from Biacore for critical review of BIAcore results.


REFERENCES

  1. Hynes, R. O. (1992) Cell 69, 11-25 [Medline] [Order article via Infotrieve]
  2. Colombatti, A., Bonaldo, P., and Doliana, R. (1993) Matrix 13, 297-306 [Medline] [Order article via Infotrieve]
  3. Shaw, S. K., Cepek, K. L., Murphy, E. A., Russell, G. J., Brenner, M. B., and Parker, C. M. (1994) J. Biol. Chem. 269, 6016-6025 [Abstract/Free Full Text]
  4. Lee, Jie-O., Rieu, P., Arnaout, M. A., and Liddington, R. (1995) Cell 80, 631-638 [Medline] [Order article via Infotrieve]
  5. Kern, A., Briesewitz, R., Bank, I., and Marcantonio, E. E. (1994) J. Biol. Chem. 269, 22811-22816 [Abstract/Free Full Text]
  6. Kamata, T., Puzon, W., and Takada, Y. (1994) J. Biol. Chem. 269, 9659-9663 [Abstract/Free Full Text]
  7. Diamond, M. S., Garcia-Aguilar, J., Bickford, J. K., Corbi, A. L., and Springer, T. A. (1993) J. Cell Biol. 120, 1031-1043 [Abstract]
  8. Landis, R. C., Bennet, R., and Hogg, N. (1993) J. Cell Biol. 120, 1519-1527 [Abstract]
  9. Landis, R. C., McDowall, A., Holness, C. L. L., Littler, A. J., Simmons, D. L., and Hogg, N. (1994) J. Cell Biol. 126, 529-537 [Abstract]
  10. Bilsland, C. A. G., Diamond, M. S., and Springer, T. A. (1994) J. Immunology 152, 4582-4589 [Abstract/Free Full Text]
  11. Kamata, T., and Takada, Y. (1994) J. Biol. Chem. 269, 26006-26010 [Abstract/Free Full Text]
  12. Tuckwell, D. S., Calderwood, D. A., Green, L. J., and Humphries, M. J. (1995) J. Cell Sci. 107, 1629-1637
  13. Randi, A. M., and Hogg, N. (1994) J. Biol. Chem. 269, 12395-12398 [Abstract/Free Full Text]
  14. Ueda, T., Rieu, P., Brayer, J., and Arnaout, M. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10680-10684 [Abstract/Free Full Text]
  15. Zhou, L., Lee, D. H. S., Plescia, J., Lau, C. Y., and Altieri, D. C. (1994) J. Biol. Chem. 269, 17075-17079 [Abstract/Free Full Text]
  16. Tuckwell, D. S., and Humphries, M. J. (1993) Crit. Rev. Oncol. Hematol. 15, 149-171 [Medline] [Order article via Infotrieve]
  17. Luque, A., Sanchez-Madrid, F., and Cabanas, C. (1994) FEBS Letts. 346, 278-284 [CrossRef][Medline] [Order article via Infotrieve]
  18. Kern, A, Eble, J., Golbik, R., and Kühn, K. (1993) Eur. J. Biochem. 215, 151-159 [Abstract]
  19. Vandenberg, P., Kern, A., Ries, A., Luckenbill-Edds, L., Mann, K., and Kühn, K. (1991) J. Cell Biol. 113, 1475-1483 [Abstract]
  20. Mould, A. P., Askari, J. A., Akiyama, S. K., Yamada, K., and Humphries, M. J. (1991) Biochem. Soc. Trans. 19, 361S [Medline] [Order article via Infotrieve]
  21. Michishita, M., Videm, V., and Arnaout, M. A. (1993) Cell 72, 857-867 [Medline] [Order article via Infotrieve]
  22. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463 [Abstract]
  23. Briesewitz, R., Epstein, M. R., and Marcantonio, E. E. (1993) J. Biol. Chem. 268, 2989-2996 [Abstract/Free Full Text]
  24. Corbi, A. L., Kishimoto, T. K., Miller, L. J., and Springer, T. A. (1988) J. Biol. Chem. 263, 12403-12411 [Abstract/Free Full Text]
  25. Smith, J. W., and Johnston, K. S. (1988) Gene (Amst.) 67, 31-40 [CrossRef][Medline] [Order article via Infotrieve]
  26. Mould, A. P., Askari, J. A., Craig, S. E., Garratt, A. N., Clements, J., and Humphries, M. J. (1994) J. Biol. Chem. 269, 27224-27230 [Abstract/Free Full Text]
  27. Woska, J. R., Morelock, M. M., Jeanfavre, D. D., and Bormann, B.-J. (1996) J. Immunol. 156, 4680-4685 [Abstract/Free Full Text]
  28. Kramer, R. H., and Marks, N. (1989) J. Biol. Chem. 264, 4684-4688 [Abstract/Free Full Text]
  29. Tomaselli, K. J., Hall, D. E., Flier, L. A., Gehlsen, K. R., Turner, D. C., Carbonetto, S., and Reichard, L. F. (1990) Neuron 5, 651-662 [Medline] [Order article via Infotrieve]
  30. Gullberg, D., Gehlsen, K. R., Turner, D. C., Åhlen, K., Zijenah, L. S., Barnes, M. J., and Rubin, K. (1993) EMBO J. 11, 3865-3873 [Abstract]
  31. Rossino, P., Defilippi, P., Silengo, L., and Tarone, G. (1991) Cell Regul. 2, 1021-1033 [Medline] [Order article via Infotrieve]
  32. Qu, A., and Leahy, D. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10277-10281 [Abstract]
  33. Rieu, P., Ueda, T., Haruta, I., Sharma, C. P., and Arnaout, M. A. (1994) J. Cell Biol. 127, 2081-2091 [Abstract]
  34. Muchowski, P. J., Zhang, L., Chang, E. R., Soule, H. R., Plow, E. F., and Moyle, M. (1994) J. Biol. Chem. 269, 26419-26423 [Abstract/Free Full Text]
  35. Qu, A., and Leahy, D. L. (1996) Structure (Lond.) 4, 931-942 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.