Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1995 Apr 2;129(2):357–366. doi: 10.1083/jcb.129.2.357

Specific receptor detection by a functional keratinocyte growth factor- immunoglobulin chimera

PMCID: PMC2199909  PMID: 7721940

Abstract

Fibroblast growth factor receptors (FGFRs) are encoded by at least four distinct highly conserved genes, and alternative splicing generates multiple gene products. The close relationship among different FGFRs has greatly increased the difficulty in generating specific immunochemical probes. As an alternative strategy, we constructed a fusion protein comprising keratinocyte growth factor (KGF) and an IgG1 Fc domain (HFc). The chimeric molecule was efficiently secreted from transfectants as a disulfide-linked dimer that bound KGFRs with high affinity. Moreover, the KGF-HFc, like native KGF, induced DNA synthesis by epithelial cells implying normal functional receptor activation. Because it retained the convenient detection properties of an immunoglobulin, it was possible to use the KGF-HFc in ligand-mediated histochemical analysis of KGFRs. Flow cytometry revealed KGF-HFc chimera detection of the KGFR, an alternative FGFR2 product, but not FGFR1 (flg) or FGFR2 (bek). Histochemical analysis of normal skin demonstrated the specific localization of KGFRs within the spinous layer, a zone of epithelial cell differentiation. KGFRs were also localized to epithelial cells within a specific region of the hair follicle, and they were not detectable in cells of the sweat gland. Tissue sections of soft palate and tonsil, two examples of nonkeratinizing epithelium, revealed staining of stratum spinosum and some staining of the basal cell layer as well. Neither salivary gland epithelium nor lymphoid cells were positive. The ciliated epithelium of the trachea exhibited KGFR expression in intermediate and basal cell layers. In striking contrast to the normal pattern of staining in the adjacent epithelium, a squamous cell carcinoma of skin lacked detectable KGFRs. Our present findings suggest that growth factor-Ig fusion proteins may be generally applicable in ligand-mediated histochemical detection and localization of growth factor receptors.

Full Text

The Full Text of this article is available as a PDF (6.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aaronson S. A. Growth factors and cancer. Science. 1991 Nov 22;254(5035):1146–1153. doi: 10.1126/science.1659742. [DOI] [PubMed] [Google Scholar]
  2. Alarid E. T., Rubin J. S., Young P., Chedid M., Ron D., Aaronson S. A., Cunha G. R. Keratinocyte growth factor functions in epithelial induction during seminal vesicle development. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):1074–1078. doi: 10.1073/pnas.91.3.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ashmun R. A., Look A. T., Roberts W. M., Roussel M. F., Seremetis S., Ohtsuka M., Sherr C. J. Monoclonal antibodies to the human CSF-1 receptor (c-fms proto-oncogene product) detect epitopes on normal mononuclear phagocytes and on human myeloid leukemic blast cells. Blood. 1989 Feb 15;73(3):827–837. [PubMed] [Google Scholar]
  4. Bennett B. D., Bennett G. L., Vitangcol R. V., Jewett J. R., Burnier J., Henzel W., Lowe D. G. Extracellular domain-IgG fusion proteins for three human natriuretic peptide receptors. Hormone pharmacology and application to solid phase screening of synthetic peptide antisera. J Biol Chem. 1991 Dec 5;266(34):23060–23067. [PubMed] [Google Scholar]
  5. Bottaro D. P., Rubin J. S., Ron D., Finch P. W., Florio C., Aaronson S. A. Characterization of the receptor for keratinocyte growth factor. Evidence for multiple fibroblast growth factor receptors. J Biol Chem. 1990 Aug 5;265(22):12767–12770. [PubMed] [Google Scholar]
  6. Burton D. R. Immunoglobulin G: functional sites. Mol Immunol. 1985 Mar;22(3):161–206. doi: 10.1016/0161-5890(85)90151-8. [DOI] [PubMed] [Google Scholar]
  7. Capon D. J., Chamow S. M., Mordenti J., Marsters S. A., Gregory T., Mitsuya H., Byrn R. A., Lucas C., Wurm F. M., Groopman J. E. Designing CD4 immunoadhesins for AIDS therapy. Nature. 1989 Feb 9;337(6207):525–531. doi: 10.1038/337525a0. [DOI] [PubMed] [Google Scholar]
  8. Cheon H. G., LaRochelle W. J., Bottaro D. P., Burgess W. H., Aaronson S. A. High-affinity binding sites for related fibroblast growth factor ligands reside within different receptor immunoglobulin-like domains. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):989–993. doi: 10.1073/pnas.91.3.989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cogné M., Silvain C., Khamlichi A. A., Preud'homme J. L. Structurally abnormal immunoglobulins in human immunoproliferative disorders. Blood. 1992 May 1;79(9):2181–2195. [PubMed] [Google Scholar]
  10. Eilat D., Kikuchi G. E., Coligan J. E., Shevach E. M. Secretion of a soluble, chimeric gamma delta T-cell receptor-immunoglobulin heterodimer. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6871–6875. doi: 10.1073/pnas.89.15.6871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fantl W. J., Johnson D. E., Williams L. T. Signalling by receptor tyrosine kinases. Annu Rev Biochem. 1993;62:453–481. doi: 10.1146/annurev.bi.62.070193.002321. [DOI] [PubMed] [Google Scholar]
  12. Finch P. W., Rubin J. S., Miki T., Ron D., Aaronson S. A. Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth. Science. 1989 Aug 18;245(4919):752–755. doi: 10.1126/science.2475908. [DOI] [PubMed] [Google Scholar]
  13. Fuchs E. Epidermal differentiation: the bare essentials. J Cell Biol. 1990 Dec;111(6 Pt 2):2807–2814. doi: 10.1083/jcb.111.6.2807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Givol D., Yayon A. Complexity of FGF receptors: genetic basis for structural diversity and functional specificity. FASEB J. 1992 Dec;6(15):3362–3369. [PubMed] [Google Scholar]
  15. Hamers-Casterman C., Atarhouch T., Muyldermans S., Robinson G., Hamers C., Songa E. B., Bendahman N., Hamers R. Naturally occurring antibodies devoid of light chains. Nature. 1993 Jun 3;363(6428):446–448. doi: 10.1038/363446a0. [DOI] [PubMed] [Google Scholar]
  16. Heidaran M. A., Mahadevan D., Larochelle W. J. Beta PDGFR-IgG chimera demonstrates that human beta PDGFR Ig-like domains 1 to 3 are sufficient for high affinity PDGF BB binding. FASEB J. 1995 Jan;9(1):140–145. doi: 10.1096/fasebj.9.1.7821753. [DOI] [PubMed] [Google Scholar]
  17. LaRochelle W. J., Giese N., May-Siroff M., Robbins K. C., Aaronson S. A. Molecular localization of the transforming and secretory properties of PDGF A and PDGF B. Science. 1990 Jun 22;248(4962):1541–1544. doi: 10.1126/science.2163109. [DOI] [PubMed] [Google Scholar]
  18. LaRochelle W. J., Jensen R. A., Heidaran M. A., May-Siroff M., Wang L. M., Aaronson S. A., Pierce J. H. Inhibition of platelet-derived growth factor autocrine growth stimulation by a monoclonal antibody to the human alpha platelet-derived growth factor receptor. Cell Growth Differ. 1993 Jul;4(7):547–553. [PubMed] [Google Scholar]
  19. LaRochelle W. J., Robbins K. C., Aaronson S. A. Immunochemical localization of the epitope for a monoclonal antibody that neutralizes human platelet-derived growth factor mitogenic activity. Mol Cell Biol. 1989 Aug;9(8):3538–3542. doi: 10.1128/mcb.9.8.3538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Landolfi N. F. A chimeric IL-2/Ig molecule possesses the functional activity of both proteins. J Immunol. 1991 Feb 1;146(3):915–919. [PubMed] [Google Scholar]
  21. Liu Y. C., Kawagishi M., Kameda R., Ohashi H. Characterization of a fusion protein composed of the extracellular domain of c-kit and the Fc region of human IgG expressed in a baculovirus system. Biochem Biophys Res Commun. 1993 Dec 30;197(3):1094–1102. doi: 10.1006/bbrc.1993.2590. [DOI] [PubMed] [Google Scholar]
  22. Lyman S. D., James L., Vanden Bos T., de Vries P., Brasel K., Gliniak B., Hollingsworth L. T., Picha K. S., McKenna H. J., Splett R. R. Molecular cloning of a ligand for the flt3/flk-2 tyrosine kinase receptor: a proliferative factor for primitive hematopoietic cells. Cell. 1993 Dec 17;75(6):1157–1167. doi: 10.1016/0092-8674(93)90325-k. [DOI] [PubMed] [Google Scholar]
  23. Marchese C., Rubin J., Ron D., Faggioni A., Torrisi M. R., Messina A., Frati L., Aaronson S. A. Human keratinocyte growth factor activity on proliferation and differentiation of human keratinocytes: differentiation response distinguishes KGF from EGF family. J Cell Physiol. 1990 Aug;144(2):326–332. doi: 10.1002/jcp.1041440219. [DOI] [PubMed] [Google Scholar]
  24. Mark M. R., Lokker N. A., Zioncheck T. F., Luis E. A., Godowski P. J. Expression and characterization of hepatocyte growth factor receptor-IgG fusion proteins. Effects of mutations in the potential proteolytic cleavage site on processing and ligand binding. J Biol Chem. 1992 Dec 25;267(36):26166–26171. [PubMed] [Google Scholar]
  25. Miki T., Fleming T. P., Bottaro D. P., Rubin J. S., Ron D., Aaronson S. A. Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop. Science. 1991 Jan 4;251(4989):72–75. doi: 10.1126/science.1846048. [DOI] [PubMed] [Google Scholar]
  26. Ron D., Bottaro D. P., Finch P. W., Morris D., Rubin J. S., Aaronson S. A. Expression of biologically active recombinant keratinocyte growth factor. Structure/function analysis of amino-terminal truncation mutants. J Biol Chem. 1993 Feb 5;268(4):2984–2988. [PubMed] [Google Scholar]
  27. Rubin J. S., Osada H., Finch P. W., Taylor W. G., Rudikoff S., Aaronson S. A. Purification and characterization of a newly identified growth factor specific for epithelial cells. Proc Natl Acad Sci U S A. 1989 Feb;86(3):802–806. doi: 10.1073/pnas.86.3.802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shin S. U., Morrison S. L. Expression and characterization of an antibody binding specificity joined to insulin-like growth factor 1: potential applications for cellular targeting. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5322–5326. doi: 10.1073/pnas.87.14.5322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Werner S., Peters K. G., Longaker M. T., Fuller-Pace F., Banda M. J., Williams L. T. Large induction of keratinocyte growth factor expression in the dermis during wound healing. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6896–6900. doi: 10.1073/pnas.89.15.6896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Werner S., Weinberg W., Liao X., Peters K. G., Blessing M., Yuspa S. H., Weiner R. L., Williams L. T. Targeted expression of a dominant-negative FGF receptor mutant in the epidermis of transgenic mice reveals a role of FGF in keratinocyte organization and differentiation. EMBO J. 1993 Jul;12(7):2635–2643. doi: 10.1002/j.1460-2075.1993.tb05924.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wigler M., Silverstein S., Lee L. S., Pellicer A., Cheng Y. c., Axel R. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell. 1977 May;11(1):223–232. doi: 10.1016/0092-8674(77)90333-6. [DOI] [PubMed] [Google Scholar]
  32. Wilkinson D. G., Bailes J. A., Champion J. E., McMahon A. P. A molecular analysis of mouse development from 8 to 10 days post coitum detects changes only in embryonic globin expression. Development. 1987 Apr;99(4):493–500. doi: 10.1242/dev.99.4.493. [DOI] [PubMed] [Google Scholar]
  33. Williams A. F., Barclay A. N. The immunoglobulin superfamily--domains for cell surface recognition. Annu Rev Immunol. 1988;6:381–405. doi: 10.1146/annurev.iy.06.040188.002121. [DOI] [PubMed] [Google Scholar]
  34. Yayon A., Zimmer Y., Shen G. H., Avivi A., Yarden Y., Givol D. A confined variable region confers ligand specificity on fibroblast growth factor receptors: implications for the origin of the immunoglobulin fold. EMBO J. 1992 May;11(5):1885–1890. doi: 10.1002/j.1460-2075.1992.tb05240.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES