PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
 
J Cell Biol. 1993 April 1; 121(1): 179–191.
PMCID: PMC2119779

Biochemical characterization and tissue distribution of the A and B variants of the integrin alpha 6 subunit

Abstract

Two cytoplasmic variants of the alpha 6 integrin, alpha 6A and alpha 6B, have been identified previously (Hogervorst, F., I. Kuikman, A. G. van Kessel, and A. Sonnenberg. 1991. Eur. J. Biochem. 199:425-433; Cooper, H. M., R. N. Tamura, and V. Quaranta. 1991. J. Cell Biol. 115:843-850). Using synthetic peptides, containing sequences of their cytoplasmic domains, we have produced mAbs specific for either of the variants. These antibodies reacted with a variety of different epithelial tissues. In some tissues (e.g., salivary gland) both variants could be detected while in others only one of the variants was found (e.g., alpha 6A in epidermis and alpha 6B in kidney). Among nonepithelial cells and tissues, perineural fibroblasts and Schwann cells in peripheral nerves and platelets reacted with anti-alpha 6A, while microvascular endothelia reacted with both anti-alpha 6A and anti- alpha 6B. From our immunohistochemical results there is not evidence that combination with beta 1 or beta 4 is restricted to one of the two variants of alpha 6. This was confirmed by immunoprecipitation studies which showed that both beta 1 and beta 4 were coprecipitated by both anti-alpha 6A or anti-alpha 6B antibodies from cells. Also, the distribution of alpha 6A and alpha 6B subunits associated with beta 1 on cells attached to laminin was similar: both were found in focal contacts colocalizing with vinculin. In contrast, the alpha 6A subunit, associated with beta 4 in cultures of a squamous cell carcinoma cell line, was found to codistribute with bullous pemphigoid antigen 230 in hemidesmosomal-like structures. The alpha 6A and alpha 6B variants, immunoprecipitated from various cell lines, exhibited slightly different electrophoretic mobilities. Analysis of the antigens under reducing conditions showed that the mobility of the light chains, but not of the heavy chains, is different. In addition, in some cells the light chains of alpha 6A and alpha 6B, each are of two different sizes. Treatment with N-glycanase showed that these two light chain variants of alpha 6A and alpha 6B are not due to differences in N-linked glycosylation, and may therefore represent alternative proteolytic products of the alpha 6 precursor. We further demonstrate that alpha 6A, but not alpha 6B, is a major target for PMA-induced phosphorylation. Phosphorylated alpha 6A contained phosphoserine and a small amount of phosphotyrosine. There are also two variants of the integrin alpha 3 subunit with different cytoplasmic domains, but in the cell lines examined only alpha 3A could be demonstrated by RT- PCR.(ABSTRACT TRUNCATED AT 400 WORDS)

Full Text

The Full Text of this article is available as a PDF (3.4M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Albelda SM, Buck CA. Integrins and other cell adhesion molecules. FASEB J. 1990 Aug;4(11):2868–2880. [PubMed]
  • Argraves WS, Pytela R, Suzuki S, Millán JL, Pierschbacher MD, Ruoslahti E. cDNA sequences from the alpha subunit of the fibronectin receptor predict a transmembrane domain and a short cytoplasmic peptide. J Biol Chem. 1986 Oct 5;261(28):12922–12924. [PubMed]
  • Aumailley M, Timpl R, Sonnenberg A. Antibody to integrin alpha 6 subunit specifically inhibits cell-binding to laminin fragment 8. Exp Cell Res. 1990 May;188(1):55–60. [PubMed]
  • Bogaert T, Brown N, Wilcox M. The Drosophila PS2 antigen is an invertebrate integrin that, like the fibronectin receptor, becomes localized to muscle attachments. Cell. 1987 Dec 24;51(6):929–940. [PubMed]
  • Boyle WJ, van der Geer P, Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. [PubMed]
  • Carter WG, Kaur P, Gil SG, Gahr PJ, Wayner EA. Distinct functions for integrins alpha 3 beta 1 in focal adhesions and alpha 6 beta 4/bullous pemphigoid antigen in a new stable anchoring contact (SAC) of keratinocytes: relation to hemidesmosomes. J Cell Biol. 1990 Dec;111(6 Pt 2):3141–3154. [PMC free article] [PubMed]
  • Cooper HM, Tamura RN, Quaranta V. The major laminin receptor of mouse embryonic stem cells is a novel isoform of the alpha 6 beta 1 integrin. J Cell Biol. 1991 Nov;115(3):843–850. [PMC free article] [PubMed]
  • de Curtis I, Quaranta V, Tamura RN, Reichardt LF. Laminin receptors in the retina: sequence analysis of the chick integrin alpha 6 subunit. Evidence for transcriptional and posttranslational regulation. J Cell Biol. 1991 Apr;113(2):405–416. [PMC free article] [PubMed]
  • De Luca M, Tamura RN, Kajiji S, Bondanza S, Rossino P, Cancedda R, Marchisio PC, Quaranta V. Polarized integrin mediates human keratinocyte adhesion to basal lamina. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6888–6892. [PubMed]
  • Hemler ME, Huang C, Schwarz L. The VLA protein family. Characterization of five distinct cell surface heterodimers each with a common 130,000 molecular weight beta subunit. J Biol Chem. 1987 Mar 5;262(7):3300–3309. [PubMed]
  • Hemler ME, Crouse C, Takada Y, Sonnenberg A. Multiple very late antigen (VLA) heterodimers on platelets. Evidence for distinct VLA-2, VLA-5 (fibronectin receptor), and VLA-6 structures. J Biol Chem. 1988 Jun 5;263(16):7660–7665. [PubMed]
  • Hemler ME, Crouse C, Sonnenberg A. Association of the VLA alpha 6 subunit with a novel protein. A possible alternative to the common VLA beta 1 subunit on certain cell lines. J Biol Chem. 1989 Apr 15;264(11):6529–6535. [PubMed]
  • Hieda Y, Nishizawa Y, Uematsu J, Owaribe K. Identification of a new hemidesmosomal protein, HD1: a major, high molecular mass component of isolated hemidesmosomes. J Cell Biol. 1992 Mar;116(6):1497–1506. [PMC free article] [PubMed]
  • Hogervorst F, Kuikman I, von dem Borne AE, Sonnenberg A. Cloning and sequence analysis of beta-4 cDNA: an integrin subunit that contains a unique 118 kd cytoplasmic domain. EMBO J. 1990 Mar;9(3):765–770. [PubMed]
  • Hogervorst F, Kuikman I, van Kessel AG, Sonnenberg A. Molecular cloning of the human alpha 6 integrin subunit. Alternative splicing of alpha 6 mRNA and chromosomal localization of the alpha 6 and beta 4 genes. Eur J Biochem. 1991 Jul 15;199(2):425–433. [PubMed]
  • Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. [PubMed]
  • Ill CR, Engvall E, Ruoslahti E. Adhesion of platelets to laminin in the absence of activation. J Cell Biol. 1984 Dec;99(6):2140–2145. [PMC free article] [PubMed]
  • Jones JC, Kurpakus MA, Cooper HM, Quaranta V. A function for the integrin alpha 6 beta 4 in the hemidesmosome. Cell Regul. 1991 Jun;2(6):427–438. [PMC free article] [PubMed]
  • Kajiji S, Tamura RN, Quaranta V. A novel integrin (alpha E beta 4) from human epithelial cells suggests a fourth family of integrin adhesion receptors. EMBO J. 1989 Mar;8(3):673–680. [PubMed]
  • Kantor RR, Mattes MJ, Lloyd KO, Old LJ, Albino AP. Biochemical analysis of two cell surface glycoprotein complexes, very common antigen 1 and very common antigen 2. Relationship to very late activation T cell antigens. J Biol Chem. 1987 Nov 5;262(31):15158–15165. [PubMed]
  • Kennel SJ, Foote LJ, Falcioni R, Sonnenberg A, Stringer CD, Crouse C, Hemler ME. Analysis of the tumor-associated antigen TSP-180. Identity with alpha 6-beta 4 in the integrin superfamily. J Biol Chem. 1989 Sep 15;264(26):15515–15521. [PubMed]
  • Kennel SJ, Godfrey V, Ch'ang LY, Lankford TK, Foote LJ, Makkinje A. The beta 4 subunit of the integrin family is displayed on a restricted subset of endothelium in mice. J Cell Sci. 1992 Jan;101(Pt 1):145–150. [PubMed]
  • Kolodziej MA, Vilaire G, Gonder D, Poncz M, Bennett JS. Study of the endoproteolytic cleavage of platelet glycoprotein IIb using oligonucleotide-mediated mutagenesis. J Biol Chem. 1991 Dec 5;266(34):23499–23504. [PubMed]
  • Kramer RH, McDonald KA, Crowley E, Ramos DM, Damsky CH. Melanoma cell adhesion to basement membrane mediated by integrin-related complexes. Cancer Res. 1989 Jan 15;49(2):393–402. [PubMed]
  • Lee EC, Lotz MM, Steele GD, Jr, Mercurio AM. The integrin alpha 6 beta 4 is a laminin receptor. J Cell Biol. 1992 May;117(3):671–678. [PMC free article] [PubMed]
  • Lotz MM, Korzelius CA, Mercurio AM. Human colon carcinoma cells use multiple receptors to adhere to laminin: involvement of alpha 6 beta 4 and alpha 2 beta 1 integrins. Cell Regul. 1990 Feb;1(3):249–257. [PMC free article] [PubMed]
  • Plummer TH, Jr, Elder JH, Alexander S, Phelan AW, Tarentino AL. Demonstration of peptide:N-glycosidase F activity in endo-beta-N-acetylglucosaminidase F preparations. J Biol Chem. 1984 Sep 10;259(17):10700–10704. [PubMed]
  • Poncz M, Eisman R, Heidenreich R, Silver SM, Vilaire G, Surrey S, Schwartz E, Bennett JS. Structure of the platelet membrane glycoprotein IIb. Homology to the alpha subunits of the vitronectin and fibronectin membrane receptors. J Biol Chem. 1987 Jun 25;262(18):8476–8482. [PubMed]
  • Ruoslahti E. Integrins. J Clin Invest. 1991 Jan;87(1):1–5. [PMC free article] [PubMed]
  • Shaw LM, Messier JM, Mercurio AM. The activation dependent adhesion of macrophages to laminin involves cytoskeletal anchoring and phosphorylation of the alpha 6 beta 1 integrin. J Cell Biol. 1990 Jun;110(6):2167–2174. [PMC free article] [PubMed]
  • Shimizu Y, Van Seventer GA, Horgan KJ, Shaw S. Regulated expression and binding of three VLA (beta 1) integrin receptors on T cells. Nature. 1990 May 17;345(6272):250–253. [PubMed]
  • Song WK, Wang W, Foster RF, Bielser DA, Kaufman SJ. H36-alpha 7 is a novel integrin alpha chain that is developmentally regulated during skeletal myogenesis. J Cell Biol. 1992 May;117(3):643–657. [PMC free article] [PubMed]
  • Sonnenberg A, Daams H, Van der Valk MA, Hilkens J, Hilgers J. Development of mouse mammary gland: identification of stages in differentiation of luminal and myoepithelial cells using monoclonal antibodies and polyvalent antiserum against keratin. J Histochem Cytochem. 1986 Aug;34(8):1037–1046. [PubMed]
  • Sonnenberg A, Janssen H, Hogervorst F, Calafat J, Hilgers J. A complex of platelet glycoproteins Ic and IIa identified by a rat monoclonal antibody. J Biol Chem. 1987 Jul 25;262(21):10376–10383. [PubMed]
  • Sonnenberg A, Modderman PW, Hogervorst F. Laminin receptor on platelets is the integrin VLA-6. Nature. 1988 Dec 1;336(6198):487–489. [PubMed]
  • Sonnenberg A, Hogervorst F, Osterop A, Veltman FE. Identification and characterization of a novel antigen complex on mouse mammary tumor cells using a monoclonal antibody against platelet glycoprotein Ic. J Biol Chem. 1988 Oct 5;263(28):14030–14038. [PubMed]
  • Sonnenberg A, Linders CJ, Daams JH, Kennel SJ. The alpha 6 beta 1 (VLA-6) and alpha 6 beta 4 protein complexes: tissue distribution and biochemical properties. J Cell Sci. 1990 Jun;96(Pt 2):207–217. [PubMed]
  • Sonnenberg A, Linders CJ, Modderman PW, Damsky CH, 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. [PMC free article] [PubMed]
  • Sonnenberg A, Calafat J, Janssen H, Daams H, van der Raaij-Helmer LM, Falcioni R, Kennel SJ, Aplin JD, Baker J, Loizidou M, et al. Integrin alpha 6/beta 4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion. J Cell Biol. 1991 May;113(4):907–917. [PMC free article] [PubMed]
  • Sonnenberg A, Gehlsen KR, Aumailley M, Timpl R. Isolation of alpha 6 beta 1 integrins from platelets and adherent cells by affinity chromatography on mouse laminin fragment E8 and human laminin pepsin fragment. Exp Cell Res. 1991 Dec;197(2):234–244. [PubMed]
  • Stanley JR, Hawley-Nelson P, Yuspa SH, Shevach EM, Katz SI. Characterization of bullous pemphigoid antigen: a unique basement membrane protein of stratified squamous epithelia. Cell. 1981 Jun;24(3):897–903. [PubMed]
  • Stepp MA, Spurr-Michaud S, Tisdale A, Elwell J, Gipson IK. Alpha 6 beta 4 integrin heterodimer is a component of hemidesmosomes. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8970–8974. [PubMed]
  • Suzuki S, Naitoh Y. Amino acid sequence of a novel integrin beta 4 subunit and primary expression of the mRNA in epithelial cells. EMBO J. 1990 Mar;9(3):757–763. [PubMed]
  • Suzuki S, Argraves WS, Pytela R, Arai H, Krusius T, Pierschbacher MD, Ruoslahti E. cDNA and amino acid sequences of the cell adhesion protein receptor recognizing vitronectin reveal a transmembrane domain and homologies with other adhesion protein receptors. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8614–8618. [PubMed]
  • Suzuki S, Argraves WS, Arai H, Languino LR, Pierschbacher MD, Ruoslahti E. Amino acid sequence of the vitronectin receptor alpha subunit and comparative expression of adhesion receptor mRNAs. J Biol Chem. 1987 Oct 15;262(29):14080–14085. [PubMed]
  • Takada Y, Elices MJ, Crouse C, Hemler ME. The primary structure of the alpha 4 subunit of VLA-4: homology to other integrins and a possible cell-cell adhesion function. EMBO J. 1989 May;8(5):1361–1368. [PubMed]
  • Takada Y, Murphy E, Pil P, Chen C, Ginsberg MH, Hemler ME. Molecular cloning and expression of the cDNA for alpha 3 subunit of human alpha 3 beta 1 (VLA-3), an integrin receptor for fibronectin, laminin, and collagen. J Cell Biol. 1991 Oct;115(1):257–266. [PMC free article] [PubMed]
  • Tamura RN, Rozzo C, Starr L, Chambers J, Reichardt LF, Cooper HM, Quaranta V. Epithelial integrin alpha 6 beta 4: complete primary structure of alpha 6 and variant forms of beta 4. J Cell Biol. 1990 Oct;111(4):1593–1604. [PMC free article] [PubMed]
  • Tamura RN, Cooper HM, Collo G, Quaranta V. Cell type-specific integrin variants with alternative alpha chain cytoplasmic domains. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10183–10187. [PubMed]
  • Tanaka T, Korman NJ, Shimizu H, Eady RA, Klaus-Kovtun V, Cehrs K, Stanley JR. Production of rabbit antibodies against carboxy-terminal epitopes encoded by bullous pemphigoid cDNA. J Invest Dermatol. 1990 May;94(5):617–623. [PubMed]
  • Teixidó J, Parker CM, Kassner PD, Hemler ME. Functional and structural analysis of VLA-4 integrin alpha 4 subunit cleavage. J Biol Chem. 1992 Jan 25;267(3):1786–1791. [PubMed]
  • Tetteroo PA, Lansdorp PM, Leeksma OC, von dem Borne AE. Monoclonal antibodies against human platelet glycoprotein IIIa. Br J Haematol. 1983 Nov;55(3):509–522. [PubMed]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press