SDS-PAGE analysis of purified recombinant ADAM 23 produced in E. coli. Five-microliter aliquots of bacterial extracts (pGEX-3X and pGEX-3X ADAM 23) as well as 1 μl of purified ADAM 23 were analyzed by SDS-PAGE. The sizes in kilodaltons of the molecular size markers (MWM) are indicated at the right.

Analysis of ADAM 23 interaction with cell lines expressing specific integrins. (A) Microtiter plates were coated with ADAM 23-GST (10 μg/ml) and seeded with parental CHO cells or with CHO cells transfected with the indicated integrins. (B) Effect of a function-blocking anti-αvβ3 antibody (LM 609) on β3-CHO and NB100 neuroblastoma cell adhesion mediated by ADAM 23. Control, control sample (without addition of antibody); LIA 1/2, samples analyzed with this blocking anti-β1 antibody.

Analysis of ADAM 23 expression in human tissues and cell lines. (A) Approximately 2 μg of polyadenylated RNA from the indicated tissues were analyzed by hybridization with the full-length cDNA isolated for human ADAM 23. The positions of RNA size markers are shown. Filters were subsequently hybridized with a human actin probe to ascertain the differences in RNA loading among the different samples. (B) Reverse transcription-PCR analysis of ADAM 23 expression in human neuroblastoma NB100 and SH-S_y5_y cells. N1 and N2 indicate negative controls for each cell line. M, molecular size markers.

Comparison of the domain structure of ADAM 23 and human ADAMs. The amino acid sequences of the regions around the consensus sequence for metalloproteinases (HEXXHXXGXXH) and the putative integrin-binding loops of ADAMs are shown. Sequences for human fertilin α (ADAM 1) and cyritestin 1 (ADAM 3) are not included, because their respective genes are not functional (Jury et al., 1997; Frayne and Hall, 1998). The disintegrin domain of decysin is truncated and lacks the region compared in the alignment of the remaining family members. All amino acid sequences were extracted from the SwissProt database. The modular structure of members of the recently described ADAM-TS subfamily containing thrombospondin-1 repeats is shown by Hurskainen et al. (1999).

Delineation of the ADAM 23 region mediating the interaction with αvβ3. (A) Effect of a point mutation (E→A) in the sequence of ADAM 23 on its adhesive-promoting properties represented as percentage of adhesion using neuroblastoma cell lines. ADAM 23 wild-type and experiments using mutant ADAM 23 are shown as white and black bars, respectively. Inset, SDS-PAGE gel with both ADAM 23 wild-type (lane 1) and E→A mutant (lane 2). M, molecular weight markers. (B) Effect of a synthetic peptide (AVNECDIT, pep 330) on the adhesion of NB100 cells. Pep 331 indicates the assay carried out with a scrambled peptide (DCVTNIAE). Two different concentrations of peptides were used: 20 μg/ml (black bars) and 40 μg/ml (gray bars). White bars indicate control using NB100 cells grown on ADAM 23.

Interaction between human αvβ3 and the disintegrin domain of ADAM 23. (A) Purified αvβ3 integrin and Sepharose beads containing recombinant ADAM 23-GST were incubated in a Mn-containing buffer. After repeated washings, the presence of bound αvβ3 was analyzed by SDS-PAGE of proteins solubilized from Sepharose beads after treatment with Laemmli denaturing buffer. Staining was carried out with silver nitrate. (B) Western blot analysis of the solubilized material from Sepharose beads using antibodies anti-αv, anti-β3, and anti-β1. M, molecular weight markers (indicated at the left). Lane 1, 300 ng of αvβ3, α1β1, or α5β1; lane 2, beads incubated with the indicated integrins; lane 3, beads containing recombinant ADAM 23-GST (500 ng) incubated with the integrins; lane 4, beads containing GST incubated with the indicated integrins.

Interaction between human αvβ3 and HeLa cells transfected with full-length cDNA for ADAM 23. (A) HeLa cells were transfected with the expression vector pcDNA3-ADAM 23-HA containing the full-length cDNA for ADAM 23 linked to a 24-bp oligonucleotide coding for the HA epitope. Transfected cells were added to microtiter plates coated with 10 μg/ml purified αvβ3. Cells were allowed to adhere for 2 h at 37°C. Nonbound cells were removed by washing, and adhesion was observed at the light microscopic level using a 20× high-power objective. (B) Immunofluorescence analysis of HeLa cells transfected with plasmid pcDNA3-ADAM 23-HA. Top, transfected cells were incubated with a monoclonal anti-HA antibody (12CA5) diluted 1:2500 followed by another incubation with goat anti-mouse fluoresceinated antibody (diluted 1:50). Fluorescence was observed under a confocal laser microscope and localized to the surface of the ADAM 23-HA transfected cells. Bottom, negative control showing HeLa cells transfected with pcDNA3.

Dose- and cation-dependent analysis of the effect of ADAM 23 on NB100 adhesion. (A) Culture dishes were coated with the indicated concentrations of ADAM 23, BSA, and GST and blocked for nonspecific binding with BSA (2.5%). Cells were allowed to adhere for 2 h at 37°C. Nonbound cells were removed by washing, and adhesion was determined by counting bound cells per unit area using a 20× high-power objective and an ocular grid. (B) Analysis of the effect of divalent cations on NB100 adhesion mediated by ADAM 23. The experiments were performed as above with the exception that cells were washed three times in PBS and resuspended in the same buffer supplemented either with 1 mM MgCl₂, 50 μM MnCl₂, and 1 mM CaCl₂ or 1 mM MgCl₂ plus 5 mM EDTA. C, control cells, corresponding to NB100 cells plated on wells coated with BSA in the absence of divalent cations. The results presented are the mean of three independent experiments.

Analysis of the role of ADAM 23 in neuroblastoma cell adhesion. Adhesion of NB 100 cells to dishes coated with 10 μg/ml fibronectin (A) or with 10 μg/ml recombinant ADAM 23 (B) is shown. Several differences in cell morphology are found at the light microscopic level. Scanning electron microscopy proved that differences in cell morphology between NB 100 cells adherent to fibronectin (C) and those adherent to ADAM 23 (D) are mainly due to variations in the number and length of surface protrusions. Cells adherent to fibronectin (C) have a relatively low number of long asymmetric filopodial extensions, whereas those attached to ADAM 23 are devoid of long surface extensions but show many short protrusions resembling microspikes, most of which appeared to be firmly attached to the glass coverslip. The effect of ADAM 23 on F-actin and vinculin distribution is shown in E–H. Confocal optical sections of NB100 cells adherent to fibronectin (E and G) and ADAM 23 (F and H) were double labeled for F-actin (rhodamine-phalloidine; red) and vinculin (anti-vinculin, visualized with FITC-labeled goat anti-rabbit; green). Neuroblastoma cells adherent to fibronectin (E) show relatively little F-actin in the central region of the cell and numerous parallel bundles at the axis of the filopodial protrusions. In cells adherent to ADAM 23 (F), the assembly of actin filaments is lower, but they are mainly located at specific cortical regions and display a moderate polarization. Vinculin in neuroblastoma cells adherent to fibronectin (G) is mainly located at the sites of filopodial protrusion. Vinculin-positive patches in cells adherent to ADAM 23 (H) show a lower degree of aggregation, but they are also concentrated at specific cortical regions.