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. 2003 Apr 15;371(Pt 2):289–299. doi: 10.1042/BJ20021500

Beta1 integrin and alpha-dystroglycan binding sites are localized to different laminin-G-domain-like (LG) modules within the laminin alpha5 chain G domain.

Hao Yu 1, Jan F Talts 1
PMCID: PMC1223287  PMID: 12519075

Abstract

Laminins are a group of extracellular-matrix proteins important in development and disease. They are heterotrimers, and specific domains in the different chains have specialized functions. The G domain of the alpha5 chain has now been produced in transfected mammalian cells as single modules and two tandem arrays, alpha5LG1-3 and alpha5LG4-5 (LG is laminin G domain-like). Using these fragments we produced specific polyclonal antibodies functional in immunoblotting and immunofluorescence studies and in solid-phase assays. Both alpha5LG tandem arrays had physiologically relevant affinities for sulphated ligands such as heparin and sulphatides. Cells adhered to these fragments and acquired a spread morphology when plated on alpha5LG1-3. Binding of integrins alpha3beta1 and alpha6beta1 was localized to the alpha5LG1-3 modules, and alpha-dystroglycan binding was localized to the alpha5LG4-5 modules, thus locating these activities to different LG modules within the laminin alpha5 G domain. However, both these activities were of relatively low affinity, indicating that integrin-mediated cell adhesion to the laminin 10/11 alpha5G domain depends on contributions from the other chains of the heterotrimer and that high-affinity alpha-dystroglycan binding could be dependent on specific Ca(2+)-ion-co-ordinating amino acids absent from alpha5LG4-5.

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Selected References

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  1. Andac Z., Sasaki T., Mann K., Brancaccio A., Deutzmann R., Timpl R. Analysis of heparin, alpha-dystroglycan and sulfatide binding to the G domain of the laminin alpha1 chain by site-directed mutagenesis. J Mol Biol. 1999 Mar 26;287(2):253–264. doi: 10.1006/jmbi.1999.2606. [DOI] [PubMed] [Google Scholar]
  2. Belkin A. M., Stepp M. A. Integrins as receptors for laminins. Microsc Res Tech. 2000 Nov 1;51(3):280–301. doi: 10.1002/1097-0029(20001101)51:3<280::AID-JEMT7>3.0.CO;2-O. [DOI] [PubMed] [Google Scholar]
  3. Colognato H., Yurchenco P. D. Form and function: the laminin family of heterotrimers. Dev Dyn. 2000 Jun;218(2):213–234. doi: 10.1002/(SICI)1097-0177(200006)218:2<213::AID-DVDY1>3.0.CO;2-R. [DOI] [PubMed] [Google Scholar]
  4. Deutzmann R., Aumailley M., Wiedemann H., Pysny W., Timpl R., Edgar D. Cell adhesion, spreading and neurite stimulation by laminin fragment E8 depends on maintenance of secondary and tertiary structure in its rod and globular domain. Eur J Biochem. 1990 Jul 31;191(2):513–522. doi: 10.1111/j.1432-1033.1990.tb19151.x. [DOI] [PubMed] [Google Scholar]
  5. Ferletta M., Ekblom P. Identification of laminin-10/11 as a strong cell adhesive complex for a normal and a malignant human epithelial cell line. J Cell Sci. 1999 Jan;112(Pt 1):1–10. doi: 10.1242/jcs.112.1.1. [DOI] [PubMed] [Google Scholar]
  6. Fox J. W., Mayer U., Nischt R., Aumailley M., Reinhardt D., Wiedemann H., Mann K., Timpl R., Krieg T., Engel J. Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV. EMBO J. 1991 Nov;10(11):3137–3146. doi: 10.1002/j.1460-2075.1991.tb04875.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Friedrich M. V., Göhring W., Mörgelin M., Brancaccio A., David G., Timpl R. Structural basis of glycosaminoglycan modification and of heterotypic interactions of perlecan domain V. J Mol Biol. 1999 Nov 19;294(1):259–270. doi: 10.1006/jmbi.1999.3259. [DOI] [PubMed] [Google Scholar]
  8. Geberhiwot T., Ingerpuu S., Pedraza C., Neira M., Lehto U., Virtanen I., Kortesmaa J., Tryggvason K., Engvall E., Patarroyo M. Blood platelets contain and secrete laminin-8 (alpha4beta1gamma1) and adhere to laminin-8 via alpha6beta1 integrin. Exp Cell Res. 1999 Dec 15;253(2):723–732. doi: 10.1006/excr.1999.4653. [DOI] [PubMed] [Google Scholar]
  9. Gu Y., Sorokin L., Durbeej M., Hjalt T., Jönsson J. I., Ekblom M. Characterization of bone marrow laminins and identification of alpha5-containing laminins as adhesive proteins for multipotent hematopoietic FDCP-Mix cells. Blood. 1999 Apr 15;93(8):2533–2542. [PubMed] [Google Scholar]
  10. Henry M. D., Campbell K. P. Dystroglycan inside and out. Curr Opin Cell Biol. 1999 Oct;11(5):602–607. doi: 10.1016/s0955-0674(99)00024-1. [DOI] [PubMed] [Google Scholar]
  11. Hohenester E., Tisi D., Talts J. F., Timpl R. The crystal structure of a laminin G-like module reveals the molecular basis of alpha-dystroglycan binding to laminins, perlecan, and agrin. Mol Cell. 1999 Nov;4(5):783–792. doi: 10.1016/s1097-2765(00)80388-3. [DOI] [PubMed] [Google Scholar]
  12. Kikkawa Y., Sanzen N., Fujiwara H., Sonnenberg A., Sekiguchi K. Integrin binding specificity of laminin-10/11: laminin-10/11 are recognized by alpha 3 beta 1, alpha 6 beta 1 and alpha 6 beta 4 integrins. J Cell Sci. 2000 Mar;113(Pt 5):869–876. doi: 10.1242/jcs.113.5.869. [DOI] [PubMed] [Google Scholar]
  13. Kikkawa Y., Sanzen N., Sekiguchi K. Isolation and characterization of laminin-10/11 secreted by human lung carcinoma cells. laminin-10/11 mediates cell adhesion through integrin alpha3 beta1. J Biol Chem. 1998 Jun 19;273(25):15854–15859. doi: 10.1074/jbc.273.25.15854. [DOI] [PubMed] [Google Scholar]
  14. Kohfeldt E., Maurer P., Vannahme C., Timpl R. Properties of the extracellular calcium binding module of the proteoglycan testican. FEBS Lett. 1997 Sep 15;414(3):557–561. doi: 10.1016/s0014-5793(97)01070-3. [DOI] [PubMed] [Google Scholar]
  15. Kortesmaa J., Yurchenco P., Tryggvason K. Recombinant laminin-8 (alpha(4)beta(1)gamma(1)). Production, purification,and interactions with integrins. J Biol Chem. 2000 May 19;275(20):14853–14859. doi: 10.1074/jbc.275.20.14853. [DOI] [PubMed] [Google Scholar]
  16. Lefebvre O., Sorokin L., Kedinger M., Simon-Assmann P. Developmental expression and cellular origin of the laminin alpha2, alpha4, and alpha5 chains in the intestine. Dev Biol. 1999 Jun 1;210(1):135–150. doi: 10.1006/dbio.1999.9270. [DOI] [PubMed] [Google Scholar]
  17. Libby R. T., Champliaud M. F., Claudepierre T., Xu Y., Gibbons E. P., Koch M., Burgeson R. E., Hunter D. D., Brunken W. J. Laminin expression in adult and developing retinae: evidence of two novel CNS laminins. J Neurosci. 2000 Sep 1;20(17):6517–6528. doi: 10.1523/JNEUROSCI.20-17-06517.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Matsumura K., Chiba A., Yamada H., Fukuta-Ohi H., Fujita S., Endo T., Kobata A., Anderson L. V., Kanazawa I., Campbell K. P. A role of dystroglycan in schwannoma cell adhesion to laminin. J Biol Chem. 1997 May 23;272(21):13904–13910. doi: 10.1074/jbc.272.21.13904. [DOI] [PubMed] [Google Scholar]
  19. Mayer U., Nischt R., Pöschl E., Mann K., Fukuda K., Gerl M., Yamada Y., Timpl R. A single EGF-like motif of laminin is responsible for high affinity nidogen binding. EMBO J. 1993 May;12(5):1879–1885. doi: 10.1002/j.1460-2075.1993.tb05836.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Miner J. H., Cunningham J., Sanes J. R. Roles for laminin in embryogenesis: exencephaly, syndactyly, and placentopathy in mice lacking the laminin alpha5 chain. J Cell Biol. 1998 Dec 14;143(6):1713–1723. doi: 10.1083/jcb.143.6.1713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Miner J. H., Patton B. L., Lentz S. I., Gilbert D. J., Snider W. D., Jenkins N. A., Copeland N. G., Sanes J. R. The laminin alpha chains: expression, developmental transitions, and chromosomal locations of alpha1-5, identification of heterotrimeric laminins 8-11, and cloning of a novel alpha3 isoform. J Cell Biol. 1997 May 5;137(3):685–701. doi: 10.1083/jcb.137.3.685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nielsen P. K., Gho Y. S., Hoffman M. P., Watanabe H., Makino M., Nomizu M., Yamada Y. Identification of a major heparin and cell binding site in the LG4 module of the laminin alpha 5 chain. J Biol Chem. 2000 May 12;275(19):14517–14523. doi: 10.1074/jbc.275.19.14517. [DOI] [PubMed] [Google Scholar]
  23. Nielsen P. K., Yamada Y. Identification of cell-binding sites on the Laminin alpha 5 N-terminal domain by site-directed mutagenesis. J Biol Chem. 2000 Nov 29;276(14):10906–10912. doi: 10.1074/jbc.M008743200. [DOI] [PubMed] [Google Scholar]
  24. Ott U., Odermatt E., Engel J., Furthmayr H., Timpl R. Protease resistance and conformation of laminin. Eur J Biochem. 1982 Mar;123(1):63–72. doi: 10.1111/j.1432-1033.1982.tb06499.x. [DOI] [PubMed] [Google Scholar]
  25. Parsons S. F., Lee G., Spring F. A., Willig T. N., Peters L. L., Gimm J. A., Tanner M. J., Mohandas N., Anstee D. J., Chasis J. A. Lutheran blood group glycoprotein and its newly characterized mouse homologue specifically bind alpha5 chain-containing human laminin with high affinity. Blood. 2001 Jan 1;97(1):312–320. doi: 10.1182/blood.v97.1.312. [DOI] [PubMed] [Google Scholar]
  26. Salmivirta Katriina, Talts Jan F., Olsson Magnus, Sasaki Takako, Timpl Rupert, Ekblom Peter. Binding of mouse nidogen-2 to basement membrane components and cells and its expression in embryonic and adult tissues suggest complementary functions of the two nidogens. Exp Cell Res. 2002 Oct 1;279(2):188–201. doi: 10.1006/excr.2002.5611. [DOI] [PubMed] [Google Scholar]
  27. Sasaki T., Costell M., Mann K., Timpl R. Inhibition of glycosaminoglycan modification of perlecan domain I by site-directed mutagenesis changes protease sensitivity and laminin-1 binding activity. FEBS Lett. 1998 Sep 18;435(2-3):169–172. doi: 10.1016/s0014-5793(98)01063-1. [DOI] [PubMed] [Google Scholar]
  28. Sasaki T., Timpl R. Domain IVa of laminin alpha5 chain is cell-adhesive and binds beta1 and alphaVbeta3 integrins through Arg-Gly-Asp. FEBS Lett. 2001 Dec 7;509(2):181–185. doi: 10.1016/s0014-5793(01)03167-2. [DOI] [PubMed] [Google Scholar]
  29. Shimizu H., Hosokawa H., Ninomiya H., Miner J. H., Masaki T. Adhesion of cultured bovine aortic endothelial cells to laminin-1 mediated by dystroglycan. J Biol Chem. 1999 Apr 23;274(17):11995–12000. doi: 10.1074/jbc.274.17.11995. [DOI] [PubMed] [Google Scholar]
  30. Sonnenberg A., Linders C. J., Modderman P. W., Damsky C. H., Aumailley M., Timpl R. Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8. J Cell Biol. 1990 Jun;110(6):2145–2155. doi: 10.1083/jcb.110.6.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sorokin L. M., Pausch F., Frieser M., Kröger S., Ohage E., Deutzmann R. Developmental regulation of the laminin alpha5 chain suggests a role in epithelial and endothelial cell maturation. Dev Biol. 1997 Sep 15;189(2):285–300. doi: 10.1006/dbio.1997.8668. [DOI] [PubMed] [Google Scholar]
  32. Spooncer E., Heyworth C. M., Dunn A., Dexter T. M. Self-renewal and differentiation of interleukin-3-dependent multipotent stem cells are modulated by stromal cells and serum factors. Differentiation. 1986;31(2):111–118. doi: 10.1111/j.1432-0436.1986.tb00391.x. [DOI] [PubMed] [Google Scholar]
  33. Talts J. F., Andac Z., Göhring W., Brancaccio A., Timpl R. Binding of the G domains of laminin alpha1 and alpha2 chains and perlecan to heparin, sulfatides, alpha-dystroglycan and several extracellular matrix proteins. EMBO J. 1999 Feb 15;18(4):863–870. doi: 10.1093/emboj/18.4.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Talts J. F., Aufderheide E., Sorokin L., Ocklind G., Mattsson R., Ekblom P. Induction of mouse tenascin expression by a human sarcomatoid Wilms' tumor cell line growing in nude mice. Int J Cancer. 1993 Jul 9;54(5):868–874. doi: 10.1002/ijc.2910540524. [DOI] [PubMed] [Google Scholar]
  35. Talts J. F., Mann K., Yamada Y., Timpl R. Structural analysis and proteolytic processing of recombinant G domain of mouse laminin alpha2 chain. FEBS Lett. 1998 Apr 10;426(1):71–76. doi: 10.1016/s0014-5793(98)00312-3. [DOI] [PubMed] [Google Scholar]
  36. Talts J. F., Sasaki T., Miosge N., Göhring W., Mann K., Mayne R., Timpl R. Structural and functional analysis of the recombinant G domain of the laminin alpha4 chain and its proteolytic processing in tissues. J Biol Chem. 2000 Nov 10;275(45):35192–35199. doi: 10.1074/jbc.M003261200. [DOI] [PubMed] [Google Scholar]
  37. Talts J. F., Timpl R. Mutation of a basic sequence in the laminin alpha2LG3 module leads to a lack of proteolytic processing and has different effects on beta1 integrin-mediated cell adhesion and alpha-dystroglycan binding. FEBS Lett. 1999 Sep 24;458(3):319–323. doi: 10.1016/s0014-5793(99)01180-1. [DOI] [PubMed] [Google Scholar]
  38. Tani T., Lehto V. P., Virtanen I. Expression of laminins 1 and 10 in carcinoma cells and comparison of their roles in cell adhesion. Exp Cell Res. 1999 Apr 10;248(1):115–121. doi: 10.1006/excr.1999.4399. [DOI] [PubMed] [Google Scholar]
  39. Taraboletti G., Rao C. N., Krutzsch H. C., Liotta L. A., Roberts D. D. Sulfatide-binding domain of the laminin A chain. J Biol Chem. 1990 Jul 25;265(21):12253–12258. [PubMed] [Google Scholar]
  40. Tiger C. F., Champliaud M. F., Pedrosa-Domellof F., Thornell L. E., Ekblom P., Gullberg D. Presence of laminin alpha5 chain and lack of laminin alpha1 chain during human muscle development and in muscular dystrophies. J Biol Chem. 1997 Nov 7;272(45):28590–28595. doi: 10.1074/jbc.272.45.28590. [DOI] [PubMed] [Google Scholar]
  41. Timpl R. Antibodies to collagens and procollagens. Methods Enzymol. 1982;82(Pt A):472–498. doi: 10.1016/0076-6879(82)82079-x. [DOI] [PubMed] [Google Scholar]
  42. Timpl R., Brown J. C. Supramolecular assembly of basement membranes. Bioessays. 1996 Feb;18(2):123–132. doi: 10.1002/bies.950180208. [DOI] [PubMed] [Google Scholar]
  43. Timpl R. Macromolecular organization of basement membranes. Curr Opin Cell Biol. 1996 Oct;8(5):618–624. doi: 10.1016/s0955-0674(96)80102-5. [DOI] [PubMed] [Google Scholar]
  44. Timpl R., Tisi D., Talts J. F., Andac Z., Sasaki T., Hohenester E. Structure and function of laminin LG modules. Matrix Biol. 2000 Aug;19(4):309–317. doi: 10.1016/s0945-053x(00)00072-x. [DOI] [PubMed] [Google Scholar]
  45. von der Mark Helga, Williams Inka, Wendler Olaf, Sorokin Lydia, von der Mark Klaus, Pöschl Ernst. Alternative splice variants of alpha 7 beta 1 integrin selectively recognize different laminin isoforms. J Biol Chem. 2001 Dec 14;277(8):6012–6016. doi: 10.1074/jbc.M102188200. [DOI] [PubMed] [Google Scholar]

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