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J. Biol. Chem., Vol. 277, Issue 20, 17804-17810, May 17, 2002

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Functional Classification of ADAMs Based on a Conserved Motif for Binding to Integrin ₉₁

IMPLICATIONS FOR SPERM-EGG BINDING AND OTHER CELL INTERACTIONS^*

Koji Eto, Clotilde Huet^§, Takehiko Tarui, Sergey Kupriyanov^§, Hai-Zhen Liu^§, Wilma Puzon-McLaughlin, Xi-Ping Zhang, Dean Sheppard^¶, Eva Engvall^§, and Yoshikazu Takada

From the  Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, ^§ The Burnham Institute, La Jolla, California 92037, and the ^¶ Lung Biology Center, Center for Occupational and Environmental Health, Cardiovascular Research Institute, and Department of Medicine, University of California, San Francisco, California 94143

Received for publication, January 4, 2002, and in revised form, February 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ADAMs (a disintegrin and metalloproteases) are members of the metzincin superfamily of metalloproteases. Among integrins binding to disintegrin domains of ADAMs are ₉₁ and _v3, and they bind in an RGD-independent and an RGD-dependent manner, respectively. Human ADAM15 is the only ADAM with the RGD motif in the disintegrin domain. Thus, both integrin ₉₁ and _v3 recognize the ADAM15 disintegrin domain. We determined how these integrins recognize the ADAM15 disintegrin domain by mutational analysis. We found that the Arg⁴⁸¹ and the Asp-Leu-Pro-Glu-Phe residues (residues 488-492) were critical for ₉₁ binding, but the RGD motif (residues 484-486) was not. In contrast, the RGD motif was critical for _v3 binding, but the other residues flanking the RGD motif were not. As the RX₆DLPEF ₉₁ recognition motif (residues 481-492) is conserved among ADAMs, except for ADAM10 and 17, we hypothesized that ₉₁ may recognize disintegrin domains in all ADAMs except ADAM10 and 17. Indeed we found that ₉₁ bound avidly to the disintegrin domains of ADAM1, 2, 3, and 9 but not to the disintegrin domains of ADAM10 and 17. As several ADAMs have been implicated in sperm-oocyte interaction, we tested whether the functional classification of ADAMs, based on specificity for integrin ₉₁, applies to sperm-egg binding. We found that the ADAM2 and 15 disintegrin domains bound to oocytes, but the ADAM17 disintegrin domain did not. Furthermore, the ADAM2 and 15 disintegrin domains effectively blocked binding of sperm to oocytes, but the ADAM17 disintegrin domain did not. These results suggest that oocytes and ₉₁ have similar binding specificities for ADAMs and that ₉₁, or a receptor with similar specificity, may be involved in sperm-egg interaction during fertilization. As ₉₁ is a receptor for many ADAM disintegrins and ₉₁ and ADAMs are widely expressed, ₉₁-ADAM interaction may be of a broad biological importance.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ADAMs¹ (a disintegrin and metalloproteases) or MDC (metalloprotease/disintegrin/cysteine-rich) proteins are a family of transmembrane glycoproteins of more than 30 members (see www.people.virginia.edu/~jag6n/Table_of_the_ADAMs.html and www.gene.ucl.ac.uk/nomenclature/genefamily/metallo. html). ADAMs have a prodomain, a metalloprotease domain, a disintegrin domain, a cysteine-rich region, an EGF-like domain, a transmembrane domain, and a cytoplasmic tail (1-3). Several ADAMs are involved in crucial biological processes such as fertilization (ADAM1, 2, and 3) (4-6) and muscle cell differentiation (meltrin-, ADAM12) (7, 8). Most, but not all, ADAMs have a catalytically active metalloprotease domain, which processes several biologically important cell surface proteins including tumor necrosis factor- (tumor necrosis factor converting enzyme (TACE), ADAM17) (for reviews, see Refs. 9 and 10), Alzheimer protein precursor (ADAM10 and 17) (11, 12), Delta (ADAM10) (9, 13, 14), and heparin-binding EGF (ADAM9) (15).

The ADAM disintegrin domains are homologous to snake venom disintegrins and are potential integrin ligands. Snake venom disintegrins bind to the platelet integrin _IIb3 in an RGD-dependent manner. Of all known ADAMs, ADAM15 is the only one that has the RGD motif in the disintegrin domain. Indeed, as we have reported, the ADAM15 disintegrin domain binds to integrin _v3 in an RGD-dependent manner (16). However, ADAM15 binds also to ₉₁ in an RGD-independent manner (17) and to integrin ₅₁ (18). Several laboratories, including ours, have reported that the non-RGD ADAM disintegrin domains interact with several integrins such as ADAM2 and 9 with 61 (5, 19), ADAM12 with ₉₁(17), ADAM23 with _v3 (20), and ADAM28 with ₄₁ (21).

At least three ADAMs have been shown to participate in fertilization (ADAM1, 2, and 3) (Refs. 5 and 22, and for review, see Ref. 23). The ADAMs on the sperm surface are processed and lack the pro- and metalloprotease domains; thus the disintegrin domains of ADAMs may be the most important in sperm-egg binding. Recent reports showed that several amino acid residues within the putative integrin-binding loop of the ADAM disintegrin domain are critical for sperm-egg interaction (24-26). The identity of the integrin(s) to which the disintegrin domains bind has not been fully established. As antibodies to integrin ₆ were shown to block sperm-egg binding, it has been proposed that integrin ₆₁ on the egg binds to ADAM2 and 3 on the sperm (26). However, oocytes from ₆-null mice can be fertilized in vitro (27), and anti-integrin ₆ subunit monoclonal antibody does not always block sperm binding (28). Thus, it is still unclear whether ₆₁ is the main or only integrin involved in sperm-egg interaction, and the specificity, occurrence, and significance of integrin-ADAM binding in other cells and tissues are not known (for review, see Ref. 23).

In the present study, we analyzed how integrins _v3 and ₉₁ recognize the ADAM15 disintegrin domain by mutating amino acid residues in the putative integrin-binding site of the disintegrin domain (Fig. 1a). We found that _v3 and ₉₁ recognize distinct motifs in the disintegrin domain, the RGD (residues 484-486) and the RX₆DLPEF (residues 481-492) motifs, respectively. The RX₆DLPEF ₉₁ recognition motif is conserved among ADAMs, except for ADAM10 and 17, and we provide evidence that ₉₁ recognizes several, perhaps all, ADAM disintegrins with this motif. We also found that oocytes and ₉₁ have similar binding specificities for ADAM disintegrin domains and propose that ₉₁, or a receptor with similar specificity, may be involved in sperm-oocyte interaction during fertilization. Considering that ₉₁ and ADAMs are widely expressed, ₉₁-ADAM interactions may have a broad significance in many biological and pathological processes such as fertilization, development, and tissue remodeling.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Production of Recombinant Disintegrin Domains as Glutathione S-Transferase (GST) Fusion Proteins-- Complementary DNA fragments encoding the disintegrin domain of ADAMs were amplified by polymerase chain reaction and cloned in a pGEX-2T vector (Amersham Biosciences) as described previously (16). The disintegrin domains were derived from mouse ADAM1 (Arg²⁴⁶-Gln³³⁴), mouse ADAM2 (Lys³⁸⁸-Pro⁴⁷⁹), mouse ADAM3 (Gly³⁹⁴-Glu⁴⁸⁶), human ADAM9 (Ser⁴¹³-Tyr⁵⁰³), mouse ADAM10 (Gly⁴⁵⁷-Thr⁵⁵⁴), human ADAM15 (Met⁴²⁰-Glu⁵¹⁰), mouse ADAM15 (Met⁴²¹-Glu⁵¹¹), and mouse ADAM17 (Ser⁴⁷⁴-Thr⁵⁶⁵). GST fusion proteins were produced and purified as described previously (16). Absorbance at 280 nm was measured to determine the concentration of purified proteins, and the amount of proteins was calculated as described previously (16). In some experiments, because GST binds to the egg plasma membrane (24), disintegrin domains used for bead coating (see below) were released from the GST by incubating with thrombin (1 unit/mg of protein) for 6 h at room temperature, and the free GST was removed by glutathione-agarose affinity chromatography.

Chinese Hamster Ovary (CHO) Cell Adhesion Assays-- CHO cells expressing _v3, human ₆, or human ₉ (designated ₃-, ₆-, or ₉-CHO cells, respectively) have been described elsewhere (17). Adhesion assays were performed as described previously (16). Briefly, wells of 96-well Immulon-2 microtiter plates (Dynatech Laboratories, Chantilly, VA) were coated with substrates in 100 µl of PBS (10 mM phosphate buffer, 0.15 M NaCl, pH 7.4) overnight at 4 °C. Remaining protein-binding sites were blocked by incubating with 1% bovine serum albumin (BSA) (Calbiochem) for 1 h at room temperature. After washing with PBS, CHO cells (10⁵ cells/well) in 100 µl of Dulbecco's modified Eagle's medium supplemented with 1% BSA were added to the wells and incubated at 37 °C for 1 h. After unbound cells were removed by rinsing the wells with Dulbecco's modified Eagle's medium, bound cells were quantified by measuring endogenous phosphatase activity (29).

Gamete Preparation for in Vitro Binding Assays-- Three-week-old B6129PF1/J female mice (Jackson Laboratory) were superovulated using standard hormonal treatment. Oocytes were collected 12-13 h after administration of human chorionic gonadotrophin, and cumulus cells were removed by incubation with hyaluronidase in Flushing and Holding medium (Specialty Media) for 5 min at 37 °C. The zona pellucida were softened in Flushing and Holding medium containing 10 µg/ml -chymotrypsin (Sigma) and then removed by passing the eggs through a narrow pipette as described previously (5). Zona pellucida-free eggs were allowed to recover in fertilization medium composed of Human Tubal Fluid medium (Irvine Scientific) supplemented with 5 mg/ml BSA fraction V (Sigma) for 1-2 h at 37 °C under mineral oil in a 5% CO₂ atmosphere.

Sperms were collected from 3-6-month-old B6129F1 male mice (Taconic Farms) by placing the cauda of the epididymis and the vas deferens in 1 ml of fertilization medium under mineral oil. Each tissue was slit open with the edge of an injection needle. Sperms were allowed to swim out for 20 min at 37 °C in an atmosphere of 5% CO₂. Tissues were removed from the medium, and spermatozoa were capacitated for 2-3 h under the same conditions. The sperm concentration was estimated with a hemocytometer.

Binding of Disintegrin-coated Beads to Eggs-- Fluorescent beads (0.2-µm yellow-green sulfate microspheres, Molecular Probes, Inc.) were coated overnight at 4 °C with purified GST-free disintegrin domains (0.3 mg/ml), washed with PBS, quenched for 1 h at 4 °C in fertilization medium containing 3% BSA, and resuspended by sonication with a water bath sonicator just before use. Zona pellucida-free eggs were incubated in 25-µl drops of 0.1% (v/v) coated beads in 3% BSA in fertilization medium under mineral oil for 1 h at 37 °C in a 5% CO₂ atmosphere. Eggs were then washed three times with fertilization medium, fixed in 0.5% glutaraldehyde, and analyzed by confocal microscopy (Bio-Rad, MRC 1024). To confirm that beads were coated with equal amounts of recombinant protein, aliquots of coated beads were boiled in Laemmli SDS sample buffer and subjected to SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue.

Sperm-Egg Binding Assay-- Zona pellucida-free eggs were preincubated with GST fusion proteins in 100-µl drops of fertilization medium under mineral oil for 30 min at 37 °C in a 5% CO₂ atmosphere. The concentration of GST elution buffer (100 mM Tris, 5 mM reduced glutathione) was adjusted to 10% (v/v) final concentration. Sperm was then added into the drops (2.5 × 10⁵ sperm/ml final concentration) and incubated for 1 h. Eggs were washed by removing medium from the drop and adding fresh Flushing and Holding medium three times. Finally eggs were fixed by adding glutaraldehyde to the drops to a final concentration of 0.5%. The number of spermatozoa bound to each egg was counted immediately under a microscope. Aliquots of the GST fusion proteins used were subjected to SDS-PAGE under reducing conditions, transferred onto nitrocellulose membrane, and stained with Ponceau S to confirm the amount and the quality of recombinant proteins at the end of the incubation. Control in vitro fertilization was routinely performed at ~50 × 10⁶ sperm/ml to ascertain the quality of gametes and to verify that more than 90% of the eggs could be fertilized.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Integrins _v3 and ₉₁ Bind to Distinct Motifs in the Disintegrin Domain of ADAM15-- Human ADAM15 is the only ADAM with the RGD motif within the putative integrin-binding sites of disintegrin domains (Fig. 1a). We have reported that _v3 and ₉₁ recognize the ADAM15 disintegrin domain in an RGD-dependent and an RGD-independent manner, respectively (17). To identify how different integrins recognize the ADAM15 disintegrin domain, we introduced mutations around the RGD motif. The mutant ADAM15 disintegrin domains were synthesized in bacteria as GST fusion proteins. The ability of the mutant disintegrin domains to bind to _v3 and ₉₁ was determined in cell adhesion assays with CHO cells expressing recombinant _v3 or ₉₁ (designated ₃-CHO cells and ₉-CHO cells, respectively). CHO cells transfected with expression vector (designated mock-CHO cells) were used as controls. CHO cells express endogenous _v1, _v5, and ₅₁ (30), but these integrins do not bind to ADAM15 (16).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Effect of point mutations on binding of cells expressing 91 (b-d) or v3 (b, e, and f) to the disintegrin domain of human ADAM15. a shows the position of the RGD motif in the putative integrin-binding region in the disintegrin domain of human ADAM15. Mutant disintegrin-GST fusion protein was generated in bacteria, and the disintegrin domain was cleaved and purified as described under "Materials and Methods." Adhesion of 9- and 3-CHO cells was tested as a function of the coating concentration of the disintegrin domain. TM, transmembrane.

Fig. 1b shows that mutating the RGD motif to SGA completely blocked the binding of _v3 but had no detectable effect on the binding of ₉₁ to the ADAM15 disintegrin domain, consistent with the previous report (17). We found that the R481A, C487A, D488A, L489A, E491, and F492A mutations negatively affected adhesion of ₉-CHO cells to the ADAM15 disintegrin domain (Fig. 1, c and d). In contrast, the same mutations flanking the RGD motif did not significantly affect adhesion of ₃-CHO cells to the ADAM15 disintegrin domain (Fig. 1, e and f). Mutating the Asp⁴⁸⁸ and Glu⁴⁹¹ simultaneously did not further affect the adhesion of ₉- or ₃-CHO cells to the ADAM15 disintegrin domain (Fig. 1, d and f). Altogether these results show that the _v3-ADAM15 interaction requires the RGD motif but not the surrounding residues. Thus, _v3 and ₉₁ recognize ADAM15 in distinct manners. Interestingly the residues critical for ₉₁ binding in the disintegrin domain, including Asp⁴⁸⁸, have also been identified as critical for sperm-egg binding in ADAM2 and 3 (24-26). The sperm-egg binding studies were performed without reference to any specific integrin, but the results are compatible with ₉₁ being the oocyte receptor.

The ₉₁ binding motif in ADAM15 is conserved in many ADAMs. The alignment of the sequences of the putative integrin-binding regions of several ADAM disintegrin domains is shown in Fig. 2a. The Arg⁴⁸¹, Asp⁴⁸⁸, Leu⁴⁸⁹, Pro⁴⁹⁰, Glu⁴⁹¹, and Phe⁴⁹² residues that are critical for ₉₁ binding in ADAM15 (designated the RX₆DLPEF motif) are conserved among the disintegrin domains of all mammalian ADAMs except for ADAM10, which has the sequence RX₅AREGI, and ADAM17, which has the sequence QX₇KGVSY, in this region. We thus hypothesized that ₉₁ is a common receptor for many, if not all, ADAM disintegrin domains with this motif. To address this hypothesis, we tested the disintegrin domains of several ADAMs for interaction with ₉₁ in cell attachment assays (Fig. 2, b and c). We found that ₉-CHO cells adhered to the disintegrin domains from ADAM1, 2, 3, and 9 as well as 15 used as a positive control. ₉-CHO cells do not spread very well on these disintegrins. The ₉-CHO cells did not significantly adhere to the disintegrin domains from ADAM10 or 17. Control mock-transfected CHO cells did not significantly adhere to any of the ADAMs tested. We further found that adhesion of ₉-CHO cells to the ADAMs was completely blocked by anti-₉ monoclonal antibody Y9A2 (data not shown), showing that the adhesion was ₉-specific. These results indicate that ₉₁ may be a common receptor for ADAM disintegrin domains with the RX₆DLPEF-like motif.

View larger version (53K): [in this window] [in a new window]   Fig. 2.   Interaction between 91 and ADAMs disintegrin domains. a, alignment of the putative integrin-binding sites of different ADAM disintegrin domains. Amino acid residues that are critical for 91 interaction are boxed. h, m, and rADAMs represent human, mouse, and rat ADAMs, respectively. ADAMs that have been identified in nonmammalian species (ADAM13, 14, and 16) are not shown. It should be noted that the critical residues are not conserved in ADAM10 and ADAM17. b and c, adhesion of 9-CHO cells to GST-ADAM disintegrin domains. GST-ADAM1, -ADAM2, -ADAM3, -ADAM9, and -ADAM15 disintegrin domains support adhesion of 9-CHO cells, but those of -ADAM10 and -ADAM17 do not.

Since ₆₁ has been implicated in ADAM2 and ADAM3 binding (26), we tested whether ₆₁ expressed in CHO cells bound to the ADAM2 and 3 disintegrin domains. We found that ₆₁ did not mediate adhesion of ₆-CHO cells to ADAM2 and 3 under the conditions in which ₉-CHO cells adhered to these ligands (data not shown).

Interaction of Oocytes with Disintegrin Domains of ADAMs-- Several residues in the putative integrin-binding region of ADAM1, 2, and 3 have been identified as critical for sperm-egg binding (24-26), but it has not been established which receptor(s) may be involved. Since ADAM1, 2, and 3 disintegrins bind avidly to ₉₁, we wanted to test whether the binding of sperm to the oocyte may have a specificity similar to disintegrin binding to ₉₁. To address this hypothesis, we first studied whether the recombinant ADAM disintegrin domains interact with oocytes. Although ADAM15 and 17 have not been shown to be present on sperm, they were used to test specificity.

We tested whether fluorescent beads coated with disintegrin domains of ADAM2, 15, and 17 bind to zona pellucida-free eggs. As GST alone binds to such eggs (24), the GST portion of the recombinant fusion proteins was cleaved off, and the recombinant GST-free disintegrin domains were purified prior to coating of the beads. Fig. 3 shows that the beads coated with the ADAM15 disintegrin domain bound efficiently to the oocytes, the binding being largely concentrated to the microvillar region of the egg (Fig. 3b, inset). ADAM2 disintegrin domain-coated beads also bound to the egg microvillar region (Fig. 3a) but to a lesser extent than ADAM15-coated beads. Beads coated with the ADAM17 disintegrin domain (Fig. 3c) and BSA-coated beads (not shown) did not bind to eggs.

View larger version (61K): [in this window] [in a new window]   Fig. 3.   Binding of disintegrin domains to the egg microvillar region. Fluorescent microspheres were coated with 0.3 mg/ml GST-free recombinant disintegrin domains of mouse ADAM2 (a), ADAM15 (b), and ADAM17 (c) and incubated with zona pellucida-free eggs. Bead binding was analyzed by confocal microscopy after washing. Left panel, phase contrast image. Right panel, projection of fluorescence images in a single plane. Pictures show results from one representative experiment of three. ADAM15- and to a lesser extent ADAM2-coated beads bound to the microvillar region (b, inset) of all eggs, while ADAM17-coated beads did not bind significantly. Scale bar, 100 µm.

We next tested whether recombinant disintegrin domains, used as GST fusion proteins, would inhibit the binding of sperm to oocytes. The disintegrin domains from ADAM2 and ADAM15 inhibited the binding of sperm to mouse eggs in a dose-dependent manner, whereas that from ADAM17 did not affect sperm-egg binding (Fig. 4). The ADAM15 disintegrin domain was a more potent inhibitor than the ADAM2 disintegrin domain, blocking sperm-egg binding completely at 3 µM. Consistent with previous reports, the ADAM2 disintegrin domain at 3 µM inhibited sperm-egg binding to about 50%. Altogether, these results indicate that the binding specificity of sperm for the oocyte is similar to that of ADAM disintegrin for integrin ₉₁.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   The disintegrin domain of ADAM15 inhibits the binding of sperm to the egg plasma membrane. The disintegrin domains produced as GST fusion proteins were tested for their ability to interfere with the in vitro binding of capacitated sperm to zona pellucida-free eggs. Data represent means ± S.E. (n = 18-21) from one representative experiment of three.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have shown here that the conserved RX₆DLPEF motif flanking the RGD motif in the ADAM15 disintegrin domain, but not the RGD motif itself, is important for binding of ADAM15 to ₉₁. The ADAM disintegrin domains with this motif, e.g. those of ADAM1, 2, 3, 9, 12, and 15, bind avidly to ₉₁, but those lacking this motif, ADAM10 and 17, do not. The RX₆DLPEF motif is conserved among all ADAM disintegrin domains except ADAM10 and 17 (Fig. 2a), suggesting that ₉₁ is a receptor for all ADAMs with this recognition motif and that binding of ADAM disintegrin domains to ₉₁ through this motif may have critical biological functions. ADAM10 and 17 represent a subfamily within the ADAM family with several distinct structural (and functional) features. The disintegrin domains of ADAM10 and 17 contain only 13 of the 15 cysteine residues characteristic of a typical type III snake venom disintegrin domain, indicating that the disintegrin function of ADAM10 and 17 may be different from those of ADAMs with the conserved RX₆DLPEF motif as well as from that of snake venom disintegrins and may not bind to any integrin at all.

The RGD motif is not important for ₉₁ binding to ADAM15. Thus, ₉₁ recognizes ADAM15 in a manner distinct from that of _v3. Human ADAM15 is the only ADAM with the RGD motif in the disintegrin domain, and the RGD motif is not conserved among ADAM disintegrin domains (even mouse ADAM15 does not have this motif), suggesting that RGD-dependent interaction of ADAM15 with integrins may have only limited importance. This is in stark contrast to many snake venom disintegrins in which the RGD motif is highly conserved. Snake venom disintegrins bind to _IIb3 or _v3 integrins and block thrombosis and hemostasis (for reviews, see Refs. 2 and 31).

The three-dimensional structures of the snake venom disintegrins echistatin (Protein Data Bank code 2ECH) and kistrin (1KST) are available. The "disintegrin loops" of echistatin and kistrin are protruding loops (approximately 15 Å long and 4 Å wide) with the RGD motif at the tip of the loops. The three-dimensional structure of the ADAM disintegrin is not available. We generated a molecular model of the "disintegrin loop region" of the ADAM15 disintegrin domain based on the echistatin structure (2ECH) using the SWISS-MODEL protein modeling server (Fig. 5) assuming that the ADAM disintegrin and snake venom disintegrins are similar in structure. In this model oppositely charged residues (Arg⁴⁸¹ and Glu⁴⁹¹, Arg⁴⁸⁴ and Asp⁴⁸⁸) and hydrophobic residues (Pro⁴⁸² and Pro⁴⁹⁰) are close to each other, stabilizing the loop. This model predicts that the Arg⁴⁸¹ and the DLPEF motif in the ₉₁ binding motif are located on the opposite side of the loop and that Arg⁴⁸¹ and Glu⁴⁹¹ are close to each other in space, although they are distant from each other in the primary structure. The RGD motif is present at the tip of the loop. It is likely that the entire loop sequence may be required for synthetic peptides of the ADAM disintegrins to be properly folded and effectively bind to ₉₁.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   A model of the integrin-binding site of ADAM15 disintegrin domain based on the echistatin structure (2ECH). This model was generated using the SWISS-MODEL protein modeling server (36-38). The alignment used for generating the model is shown. The amino acid residues that are critical for 91 binding are shown in bold.

₉₁ has been shown to mediate cell adhesion and migration but not cell spreading in vitro. The association of an adhesion receptor, such as ₉₁, with a metalloprotease, such as an ADAM, would be expected to facilitate cell migration in vivo in that the protease may digest and modify extracellular matrices and other tissue barriers in the immediate area of adhesion. The association between ₉₁ and ADAM would further be expected to occur between molecules on the same cell, i.e. in cis, as cell-cell interaction is not normally a feature of cell migration. In contrast, it has been proposed that shedding of growth factors occurs with the growth factor and ADAM expressed on different cells, i.e. in trans. ADAM9 is responsible for processing of heparin-binding EGF (15) and binds with high affinity to integrin ₉₁ (the present report). It will be interesting to know whether integrin ₉₁ is involved in the processing of heparin-binding EGF. Our identification of mutations that obliterate the binding of ₉₁ to ADAMs can now be used to test the role of ₉₁-ADAM binding on cell migration as well as on processing of heparin-binding EGF and other potential ADAM substrates. As the prominent sheddases ADAM10 and 17 do not bind to ₉₁, it is unlikely that ₉₁ is involved in the processing of the proteins they target, including tumor necrosis factor- and the Alzheimer precursor protein. Few ADAMs other than ADAM10 and 17 have been tested for capacity to process these proteins. It is possible that the specificities of the ADAM domains other than the metalloprotease domains and the cell type-specific expression of ADAM ligands, such as integrin ₉₁, will determine which proteins will be digested and when. The digestion may then occur either in cis or in trans depending on the situation. The metalloprotease domains themselves may be rather nonspecific. The relative nonspecificity of metalloproteases agrees with the lack of success in developing specific metalloprotease inhibitors despite a large effort in this area by the pharmaceutical companies.

As many ADAMs that have the RX₆DLPEF motif are present on sperm (5) and ADAMs have been implicated in sperm-egg binding during fertilization, we tested whether ₉₁-ADAM interaction may be involved in sperm-egg binding. Two recent studies demonstrate that the presence of acidic residues surrounding the cysteine in the disintegrin loop of ADAM2 (X(C/D)ECD) is essential for the binding of ADAM2 to the egg plasma membrane (24, 25). The underlined residues correspond to the RGD sequence in ADAM15. ADAM3, which also plays a role in sperm-egg binding, has the same characteristics in the sequence of its disintegrin loop (KSDCD). It has also been reported that synthetic peptides of the ADAM2 and 3 disintegrin domain putative integrin-binding site block sperm-egg interaction (5, 22, 24, 26). It is intriguing that the RX₆DLPEF ₉₁ recognition motif is overlapping with these peptide sequences. All studies to date show that the Asp residue is essential. This residue is not conserved in ADAM10 and 17.

We have shown here that the disintegrin domains of ADAM2 and 15, which effectively bind to integrin ₉₁, bound to murine oocytes and blocked sperm-egg binding, but the ADAM17 disintegrin domain, which does not bind to ₉₁, did not. Although ADAM15 has not been shown to be present on sperm, it shares the specificity with the sperm ADAM2; in fact, in our experiments, ADAM15 was more effective in egg binding and inhibition of sperm-egg binding that ADAM2, although these differences may be primarily technical. These binding studies suggest that the receptor for sperm on the oocytes is similar in specificity to ₉₁. These results are well explained if eggs express integrin ₉₁ on their surface. Although we confirmed the expression of ₉₁ in many mouse tissues (32-34), our efforts to identify ₉ on oocytes, using immunostaining in mouse ovary with anti-₉ cytoplasmic peptide antibody 1057 or in guinea pig ovary with either antibody 1057 or monoclonal antibody Y9A2, were not successful. The lack of detection of ₉ in the ovary suggests that either ₉ is not present or it is not accessible to antibodies. Another integrin(s) or other receptor with specificity similar to ₉₁ may exist in eggs.

It has been proposed that ₆₁ is the oocyte receptor for sperm (5). Recent findings that sperm-egg interaction is normal in ₆-null mice (27) and that the antibody to ₆, GoH3, does not always affect sperm-egg interaction (28, 35) have raised questions about the role of ₆₁ in sperm-egg binding and fusion. We thus tested the ability of ₆₁ to bind to the ADAM disintegrin domains. We did not detect adhesion of ₆-expressing CHO cells to the ADAM disintegrins under the conditions in which ₉-CHO cells adhere to the same ligands. However, it cannot be ruled out that ₆₁ may interact with the ADAM disintegrin domains under certain conditions in oocytes. Since we used CHO cells expressing human ₉ or ₆/hamster ₁ hybrid in this study, it also cannot be ruled out that human and hamster ₁ provide different ligand specificities.

The biological significance of the interaction between ADAMs and ₉₁ integrin is not known. ₉₁ is present in many developing and mature tissues including airway epithelia, the basal layer of squamous epithelia, smooth muscle, skeletal muscle, hepatocytes, neutrophils, and monocytes (32-34). Considering the wide distribution of both ADAMs and ₉₁, ADAM-₉₁ interactions may play a role in many physiological and pathological situations including muscle development and regeneration; vascular, cartilage, and other tissue remodeling; and perhaps fertilization. It remains a challenge to determine what this role may be.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants GM49899 (to Y. T.) and AR45446 (to E. E.).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.

To whom correspondence should be addressed: Dept. of Cell Biology, The Scripps Research Inst., VB-6, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-7636; Fax: 858-784-7645; E-mail: takada@scripps.edu.

Published, JBC Papers in Press, March 6, 2002, DOI 10.1074/jbc.M200086200

	ABBREVIATIONS

The abbreviations used are: ADAM, a disintegrin and metalloprotease; CHO, Chinese hamster ovary; GST, glutathione S-transferase; EGF, epidermal growth factor; BSA, bovine serum albumin.

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