Generation of Monoclonal Antibodies to Integrin-associated Proteins
EVIDENCE THAT α3β1 COMPLEXES WITH EMMPRIN/BASIGIN/OX47/M6*
Abstract
The α3β1integrin forms complexes with other cell-surface proteins, including transmembrane-4 superfamily (TM4SF) proteins (e.g. CD9, CD53, CD63, CD81, and CD82). To identify additional cell-surface proteins associated with α3β1 integrin, a monoclonal antibody selection protocol was developed. Mice were immunized with integrin α3β1-containing complexes isolated from HT1080 fibrosarcoma cells, and then 712 hybridoma clones were produced, and 95 secreted antibodies that recognized the HT1080 cell surface. Among these, 12 antibodies directly recognizing integrin α3 or β1 subunits were eliminated. Of the remaining 83, 16 co-immunoprecipitated proteins that resembled integrins under non-stringent detergent conditions. These 16 included 15 monoclonal antibodies recognizing EMMPRIN/basigin/OX-47/M6, a 45–55-kDa transmembrane protein with two immunoglobulin domains. The EMMPRIN protein associated with α3β1 and α6β1, but not α2β1 or α5β1, as shown by reciprocal immunoprecipitation experiments. Also, association with α3β1 was confirmed by cell-surface cross-linking and immunofluorescence co-localization experiments. Importantly, EMMPRIN-α3β1 complexes appear not to contain TM4SF proteins, suggesting that they are distinct from TM4SF protein-α3β1 complexes.
Footnotes
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↵* This work was supported by National Institutes of Health Grant CA42368, American Cancer Society Junior Faculty Research Award FRA-401 (to M. E. H.), and a fellowship from the Lady Tata Memorial Trust (to F. B.).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.
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↵‡ Present address: University of Birmingham, CRC Institute for Cancer Studies, Edgbaston, Birmingham, UK B15 2TA.
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↵§ The first two authors contributed equally to this work.
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↵¶ To whom correspondence should be addressed: Dana Farber Cancer Institute, Rm. M-613, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3410; Fax: 617-632-2662; E-mail:Martin_Hemler{at}DFCI.HARVARD.EDU.
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↵1 The abbreviations used are: TM4SF, transmembrane-4 superfamily; mAb, monoclonal antibody; FCS, fetal calf serum; CHO, Chinese hamster ovary cell; FITC, fluorescein isothiocyanate; MHC, major histocompatibility complex; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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- Received March 24, 1997.
- Revision received August 27, 1997.
