Receptor-ligand interaction between CD44 and osteopontin (Eta-1)

Science

Volumn 271, Issue 5248, 1996, Pages 509-512

  Weber, Georg F ^b   Ashkar, Samy ^b   Glimcher, Melvin J ^b   Cantor, Harvey ^b  

^a HARVARD MEDICAL SCHOOL   (United States)

^b NONE

Author keywords

[No Author keywords available]

Indexed keywords

CELL SURFACE RECEPTOR; HERMES ANTIGEN; HYALURONIC ACID; OSTEOPONTIN;

ANIMAL CELL; ARTICLE; CELL ADHESION; CELL FUNCTION; CELL GROWTH; CELL MOTION; CHEMOTAXIS; CONTROLLED STUDY; LIGAND BINDING; METASTASIS; MOUSE; NONHUMAN; PRIORITY JOURNAL; RECEPTOR BINDING; TUMOR CELL;

ANIMALIA;

EID: 0030064334     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5248.509     Document Type: Article

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28. 2-terminal sequence analysis
29. 2-terminal sequence analysis
30. 6 WEHI-3B cells (about half the saturating concentration), incubated at room temperature in the presence or absence of antibody to CD44 (anti-CD44) or control antibody [mAb Pgp-1, clone IM7 from Pharmingen; mAb KM 81, clone TIB241 from the American Type Culture Collection (ATCC), and rat IgG from Sigma] at 3 μg or a 200-fold excess of unlabeled Opn in 250 μl before bound and free fractions were separated by centrifugation as described (6) in four separate experiments Similar results were obtained for binding of recombinant phosphorylated Opn to K8 cells
31. CD44 was expressed in the murine embryonic cell line A31 after amplification of cDNA from K8 cells with 5′-CAGAATTCCTCGATCTCCTGGTAAGGAG-3′ and 5′-TAGGATCCTTGCCTCAACTGTGCACTCA-3′ primers, resulting in a single species of cDNA containing exons 7v through 10v according to PCR and sequencing analysis This cDNA was cloned into the Bam HI-Eco RI sites of the eukaryotic expression vector pcDNA III/neo (Invitrogen, San Diego CA), which contains enhancer-promoter sequences from the early gene of human cytomegalovirus, the SV40 polyadenylation-transcription termination signal, and the neomycin resistance gene for selection. CD44 clones were transfected into the A31 mouse embryonic fibroblast cell line by electroporation and selection according to G418 resistance and adherence to HA
32. The AF3.G7 hybridoma, generated by fusing cow insulin-immune C57BL/6 lymph node cells with the BW5147 thymoma line, responds to cow insulin according to interleukin-2 production [D. G. Spinella et al. , J Immunol. 138, 3991 (1987)] and expresses CD44 on its cell surface as judged by flow cytomctry analysis with Pgp-1 antibody (clone IM7, Pharmingen). AF3.G7 cells were lysed in 0 1 % Triton X-100 buffer containing 25 mM potassium phosphate, pH 7.4, 4 mM EDTA, 10 mM sodium chloride, 5 mM magnesium chloride, 10 mM iodoacetamide, 0.025% (w/v) sodium azide, 0.2 U/ml of aprotinin. 20 mg/ml of pepstatin A, 1 mM phenylmethylsulfonyl fluoride. and 1 mM sodium orthovanadate, and centrifuged at 100,000g before the supernatant was filtered and loaded onto the indicated affinity resins overnight. After extensive washing, the bound protein was eluted with 3 M NaSCN and salt was removed in Centricon filter units and Excellulose GF-5 detergent removal columns (Pierce) followed by concentration in Microsep 10K filters (Fitron)
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39. 6) were incubated with biotinylated ligand in 150 μl of calcium- and magnesium-free phosphate-buffered saline (PBS) for 30 min at 37°C followed by fixation in 1% paraformaldehyde for 10 mim on ice. After resuspension in calcium- and magnesium-free PBS, phycoerythrin-streptavidin was added at 1.100 dilution for 20 min on ice, and cells were washed and analyzed with a Coulter Profile flow cytometer.
40. Plates (96 well) were coated with either 10 (μg/ml of Opn, 10 μg/ml of fibronectin, or 100 μg/ml of HA for 18 hours at 4°C followed by blocking with 1 mg/ml of BSA (bovine serum albumin) for 2 hours at room temperature. One thousand cells per well were incubated at 37°C in calcium-free and magnesium-free PBS containing 100 μg/ml of BSA. After 30 min the cells were fixed in 4% paraformaldehyde in PBS and stained with toluidine blue. Attachment was assessed by counting the total number of cells per well Soluble inhibitors were added at the following concentrations: GRGDS peptide, 1 mM; Opn, 500 μg/ ml; hyaluronic acid (HA), 1 mg/ml
41. 4 cells/well) in calcium- and magnesium-free PBS were incubated with either 100 μg of HA or 50 μg of Opn for 15 min at 4°C In experiments with blocking antibody cells were incubated with CD44 antibody KM 81 (TIB 241 supernatant; 1:20 dilution) for 15 min before addition of ligand. Cells were scored positive when more than 50% of the cells were in aggregates of four or more cells.
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43. 3 cells were added in 500 μl to the upper chamber and incubated at 37°C in the presence or absence of chemotactic agents in the lower chamber. After 4 hours, the membranes were fixed in methanol and stained with hematoxylin + toluidine blue Responding cells on the lower surface of the filter were counted microscopically and evaluated in triplicates.
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45. This study was supported in part by NIH research grants Al2184 and Al3600 to H C.; NIH grant PO1 AR34078 and a grant from the Peabody Foundation to M J.G.; and a Barr Program Small Grant to G.F.W J. Schmidt and L Gerstenfeld provided the K8 osteosarcoma cell line The authors are grateful to L Glimcher, C. Rudd, and G Dranoff for critical reading of the manuscript, to W. Fu for expert technical assistance, and to A Angel for assistance in the preparation of the manuscript.