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. 1996 Nov 2;135(4):1139–1150. doi: 10.1083/jcb.135.4.1139

Regulated clustering of variant CD44 proteins increases their hyaluronate binding capacity

PMCID: PMC2133377  PMID: 8922392

Abstract

Cell contact with the extracellular matrix component hyaluronic acid (HA) plays an important role in many developmental, physiological, and pathological processes, although the regulation of this contact is poorly understood. CD44 proteins carry an amino acid motif that mediates affinity to HA. Artificial clustering of the smallest 85-kD isoform of CD44 (CD44s) has previously been shown to promote binding of the protein to soluble HA (Lesley, J., R. Hyman, and P.W. Kincade. 1993. Adv. Immunol. 54:271-335; Persche, A., J. Lesley, N. English, I. Trowbridge, and R. Hyman. 1995. Eur. J. Immunol. 25:495-501). Here we show that in rat pancreatic carcinoma cells, splice variants of CD44 (CD44v), but not CD44s, form molecular aggregates in the plasma membrane. We demonstrate that reduction-sensitive dimerization of CD44v occurs, and also that larger aggregations of the protein can be stabilized by chemical cross-linking. Different CD44v proteins present on the same cell exclusively form homoaggregates. Molecular clustering does not require an intact cytoplasmic domain of the protein. The ability of cells to bind to soluble HA is upregulated more than one magnitude by the ectopic expression of CD44v4-v7, but only when the CD44v4-v7 protein forms intermolecular aggregates. Tunicamycin treatment inhibits HA binding by CD44v and at the same time destroys oligomerization. We propose that the regulation of clustering of CD44, mediated by factors including the presence of variant exons and glycosylation, allows cells in turn to regulate their HA binding properties.

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Selected References

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  1. Aruffo A., Stamenkovic I., Melnick M., Underhill C. B., Seed B. CD44 is the principal cell surface receptor for hyaluronate. Cell. 1990 Jun 29;61(7):1303–1313. doi: 10.1016/0092-8674(90)90694-a. [DOI] [PubMed] [Google Scholar]
  2. Bartolazzi A., Nocks A., Aruffo A., Spring F., Stamenkovic I. Glycosylation of CD44 is implicated in CD44-mediated cell adhesion to hyaluronan. J Cell Biol. 1996 Mar;132(6):1199–1208. doi: 10.1083/jcb.132.6.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bennett K. L., Modrell B., Greenfield B., Bartolazzi A., Stamenkovic I., Peach R., Jackson D. G., Spring F., Aruffo A. Regulation of CD44 binding to hyaluronan by glycosylation of variably spliced exons. J Cell Biol. 1995 Dec;131(6 Pt 1):1623–1633. doi: 10.1083/jcb.131.6.1623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Diamond M. S., Springer T. A. A subpopulation of Mac-1 (CD11b/CD18) molecules mediates neutrophil adhesion to ICAM-1 and fibrinogen. J Cell Biol. 1993 Jan;120(2):545–556. doi: 10.1083/jcb.120.2.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dougherty G. J., Cooper D. L., Memory J. F., Chiu R. K. Ligand binding specificity of alternatively spliced CD44 isoforms. Recognition and binding of hyaluronan by CD44R1. J Biol Chem. 1994 Mar 25;269(12):9074–9078. [PubMed] [Google Scholar]
  6. Dustin M. L., Olive D., Springer T. A. Correlation of CD2 binding and functional properties of multimeric and monomeric lymphocyte function-associated antigen 3. J Exp Med. 1989 Feb 1;169(2):503–517. doi: 10.1084/jem.169.2.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Goldstein L. A., Zhou D. F., Picker L. J., Minty C. N., Bargatze R. F., Ding J. F., Butcher E. C. A human lymphocyte homing receptor, the hermes antigen, is related to cartilage proteoglycan core and link proteins. Cell. 1989 Mar 24;56(6):1063–1072. doi: 10.1016/0092-8674(89)90639-9. [DOI] [PubMed] [Google Scholar]
  8. Günthert U., Hofmann M., Rudy W., Reber S., Zöller M., Haussmann I., Matzku S., Wenzel A., Ponta H., Herrlich P. A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell. 1991 Apr 5;65(1):13–24. doi: 10.1016/0092-8674(91)90403-l. [DOI] [PubMed] [Google Scholar]
  9. Hardwick C., Hoare K., Owens R., Hohn H. P., Hook M., Moore D., Cripps V., Austen L., Nance D. M., Turley E. A. Molecular cloning of a novel hyaluronan receptor that mediates tumor cell motility. J Cell Biol. 1992 Jun;117(6):1343–1350. doi: 10.1083/jcb.117.6.1343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. He Q., Lesley J., Hyman R., Ishihara K., Kincade P. W. Molecular isoforms of murine CD44 and evidence that the membrane proximal domain is not critical for hyaluronate recognition. J Cell Biol. 1992 Dec;119(6):1711–1719. doi: 10.1083/jcb.119.6.1711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Heider K. H., Hofmann M., Hors E., van den Berg F., Ponta H., Herrlich P., Pals S. T. A human homologue of the rat metastasis-associated variant of CD44 is expressed in colorectal carcinomas and adenomatous polyps. J Cell Biol. 1993 Jan;120(1):227–233. doi: 10.1083/jcb.120.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Herrlich P., Zöller M., Pals S. T., Ponta H. CD44 splice variants: metastases meet lymphocytes. Immunol Today. 1993 Aug;14(8):395–399. doi: 10.1016/0167-5699(93)90141-7. [DOI] [PubMed] [Google Scholar]
  13. Isacke C. M. The role of the cytoplasmic domain in regulating CD44 function. J Cell Sci. 1994 Sep;107(Pt 9):2353–2359. doi: 10.1242/jcs.107.9.2353. [DOI] [PubMed] [Google Scholar]
  14. Katoh S., Zheng Z., Oritani K., Shimozato T., Kincade P. W. Glycosylation of CD44 negatively regulates its recognition of hyaluronan. J Exp Med. 1995 Aug 1;182(2):419–429. doi: 10.1084/jem.182.2.419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Knudson C. B., Knudson W. Hyaluronan-binding proteins in development, tissue homeostasis, and disease. FASEB J. 1993 Oct;7(13):1233–1241. [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lesley J., English N., Perschl A., Gregoroff J., Hyman R. Variant cell lines selected for alterations in the function of the hyaluronan receptor CD44 show differences in glycosylation. J Exp Med. 1995 Aug 1;182(2):431–437. doi: 10.1084/jem.182.2.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lesley J., Hyman R. CD44 can be activated to function as an hyaluronic acid receptor in normal murine T cells. Eur J Immunol. 1992 Oct;22(10):2719–2723. doi: 10.1002/eji.1830221036. [DOI] [PubMed] [Google Scholar]
  19. Lesley J., Hyman R., Kincade P. W. CD44 and its interaction with extracellular matrix. Adv Immunol. 1993;54:271–335. doi: 10.1016/s0065-2776(08)60537-4. [DOI] [PubMed] [Google Scholar]
  20. Lesley J., Schulte R., Hyman R. Binding of hyaluronic acid to lymphoid cell lines is inhibited by monoclonal antibodies against Pgp-1. Exp Cell Res. 1990 Apr;187(2):224–233. doi: 10.1016/0014-4827(90)90085-o. [DOI] [PubMed] [Google Scholar]
  21. Liao H. X., Levesque M. C., Patton K., Bergamo B., Jones D., Moody M. A., Telen M. J., Haynes B. F. Regulation of human CD44H and CD44E isoform binding to hyaluronan by phorbol myristate acetate and anti-CD44 monoclonal and polyclonal antibodies. J Immunol. 1993 Dec 1;151(11):6490–6499. [PubMed] [Google Scholar]
  22. Lokeshwar V. B., Bourguignon L. Y. Post-translational protein modification and expression of ankyrin-binding site(s) in GP85 (Pgp-1/CD44) and its biosynthetic precursors during T-lymphoma membrane biosynthesis. J Biol Chem. 1991 Sep 25;266(27):17983–17989. [PubMed] [Google Scholar]
  23. Matzku S., Komitowski D., Mildenberger M., Zöller M. Characterization of BSp73, a spontaneous rat tumor and its in vivo selected variants showing different metastasizing capacities. Invasion Metastasis. 1983;3(2):109–123. [PubMed] [Google Scholar]
  24. Matzku S., Wenzel A., Liu S., Zöller M. Antigenic differences between metastatic and nonmetastatic BSp73 rat tumor variants characterized by monoclonal antibodies. Cancer Res. 1989 Mar 1;49(5):1294–1299. [PubMed] [Google Scholar]
  25. McCourt P. A., Ek B., Forsberg N., Gustafson S. Intercellular adhesion molecule-1 is a cell surface receptor for hyaluronan. J Biol Chem. 1994 Dec 2;269(48):30081–30084. [PubMed] [Google Scholar]
  26. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  27. Morgenstern J. P., Land H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 1990 Jun 25;18(12):3587–3596. doi: 10.1093/nar/18.12.3587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Murakami S., Shimabukuro Y., Miki Y., Saho T., Hino E., Kasai D., Nozaki T., Kusumoto Y., Okada H. Inducible binding of human lymphocytes to hyaluronate via CD44 does not require cytoskeleton association but does require new protein synthesis. J Immunol. 1994 Jan 15;152(2):467–477. [PubMed] [Google Scholar]
  29. Neame P. J., Christner J. E., Baker J. R. The primary structure of link protein from rat chondrosarcoma proteoglycan aggregate. J Biol Chem. 1986 Mar 15;261(8):3519–3535. [PubMed] [Google Scholar]
  30. Paterson D. J., Jefferies W. A., Green J. R., Brandon M. R., Corthesy P., Puklavec M., Williams A. F. Antigens of activated rat T lymphocytes including a molecule of 50,000 Mr detected only on CD4 positive T blasts. Mol Immunol. 1987 Dec;24(12):1281–1290. doi: 10.1016/0161-5890(87)90122-2. [DOI] [PubMed] [Google Scholar]
  31. Perschl A., Lesley J., English N., Trowbridge I., Hyman R. Role of CD44 cytoplasmic domain in hyaluronan binding. Eur J Immunol. 1995 Feb;25(2):495–501. doi: 10.1002/eji.1830250228. [DOI] [PubMed] [Google Scholar]
  32. Rudy W., Hofmann M., Schwartz-Albiez R., Zöller M., Heider K. H., Ponta H., Herrlich P. The two major CD44 proteins expressed on a metastatic rat tumor cell line are derived from different splice variants: each one individually suffices to confer metastatic behavior. Cancer Res. 1993 Mar 15;53(6):1262–1268. [PubMed] [Google Scholar]
  33. Sherman L., Sleeman J., Herrlich P., Ponta H. Hyaluronate receptors: key players in growth, differentiation, migration and tumor progression. Curr Opin Cell Biol. 1994 Oct;6(5):726–733. doi: 10.1016/0955-0674(94)90100-7. [DOI] [PubMed] [Google Scholar]
  34. Sleeman J. P. Xenopus A-myb is expressed during early spermatogenesis. Oncogene. 1993 Jul;8(7):1931–1941. [PubMed] [Google Scholar]
  35. Thomas L., Byers H. R., Vink J., Stamenkovic I. CD44H regulates tumor cell migration on hyaluronate-coated substrate. J Cell Biol. 1992 Aug;118(4):971–977. doi: 10.1083/jcb.118.4.971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Underhill C. B., Chi-Rosso G., Toole B. P. Effects of detergent solubilization on the hyaluronate-binding protein from membranes of simian virus 40-transformed 3T3 cells. J Biol Chem. 1983 Jul 10;258(13):8086–8091. [PubMed] [Google Scholar]
  38. Yang B., Yang B. L., Savani R. C., Turley E. A. Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein. EMBO J. 1994 Jan 15;13(2):286–296. doi: 10.1002/j.1460-2075.1994.tb06261.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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