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. 1991 Jul;2(7):503–512. doi: 10.1091/mbc.2.7.503

Identification of a cell retention signal in the B-chain of platelet-derived growth factor and in the long splice version of the A-chain.

A Ostman 1, M Andersson 1, C Betsholtz 1, B Westermark 1, C H Heldin 1
PMCID: PMC361840  PMID: 1782212

Abstract

The B-chain homodimer of platelet-derived growth factor (PDGF) is only very inefficiently secreted and remains largely associated with the producer cell; in contrast, the dimer of the short, and most common, splice variant of the A-chain is secreted. To identify the structural background to the differences in the secretory pattern between the different isoforms of PDGF, a set of chimeric PDGF A/B cDNAs was generated and expressed in COS cells. Analyses of the biosynthesis and processing of the corresponding products led to the identification of a determinant for cell association in the carboxy-terminal third of the PDGF B-chain precursor. Introduction of stop codons at various positions in the carboxy-terminal prosequence of the PDGF B-chain localized this determinant to an 11-amino-acid-long region (amino acids 219-229). This region contains an 8-amino-acid-long basic sequence that is homologous to a sequence present in an alternatively spliced longer version of the PDGF A-chain. In contrast to the short splice variant, the long splice A-chain version, like the B-chain, was found to remain predominantly cell associated. Thus, we have identified a conserved sequence that inhibits the secretion of some of the PDGF isoforms. Our data also suggest that switching of splicing patterns can be a mechanism to regulate the formation of secreted or cell-associated forms of PDGF-AA and possibly other growth factors.

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Selected References

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

  1. Beckmann M. P., Betsholtz C., Heldin C. H., Westermark B., Di Marco E., Di Fiore P. P., Robbins K. C., Aaronson S. A. Comparison of biological properties and transforming potential of human PDGF-A and PDGF-B chains. Science. 1988 Sep 9;241(4871):1346–1349. doi: 10.1126/science.2842868. [DOI] [PubMed] [Google Scholar]
  2. Betsholtz C., Johnsson A., Heldin C. H., Westermark B. Efficient reversion of simian sarcoma virus-transformation and inhibition of growth factor-induced mitogenesis by suramin. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6440–6444. doi: 10.1073/pnas.83.17.6440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Betsholtz C., Rorsman F., Westermark B., Ostman A., Heldin C. H. Analogous alternative splicing. Nature. 1990 Mar 22;344(6264):299–299. doi: 10.1038/344299a0. [DOI] [PubMed] [Google Scholar]
  4. Bonthron D. T., Morton C. C., Orkin S. H., Collins T. Platelet-derived growth factor A chain: gene structure, chromosomal location, and basis for alternative mRNA splicing. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1492–1496. doi: 10.1073/pnas.85.5.1492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bywater M., Rorsman F., Bongcam-Rudloff E., Mark G., Hammacher A., Heldin C. H., Westermark B., Betsholtz C. Expression of recombinant platelet-derived growth factor A- and B-chain homodimers in rat-1 cells and human fibroblasts reveals differences in protein processing and autocrine effects. Mol Cell Biol. 1988 Jul;8(7):2753–2762. doi: 10.1128/mcb.8.7.2753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Claesson-Welsh L., Eriksson A., Morén A., Severinsson L., Ek B., Ostman A., Betsholtz C., Heldin C. H. cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules. Mol Cell Biol. 1988 Aug;8(8):3476–3486. doi: 10.1128/mcb.8.8.3476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Claesson-Welsh L., Eriksson A., Westermark B., Heldin C. H. cDNA cloning and expression of the human A-type platelet-derived growth factor (PDGF) receptor establishes structural similarity to the B-type PDGF receptor. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4917–4921. doi: 10.1073/pnas.86.13.4917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Coussens L., Van Beveren C., Smith D., Chen E., Mitchell R. L., Isacke C. M., Verma I. M., Ullrich A. Structural alteration of viral homologue of receptor proto-oncogene fms at carboxyl terminus. Nature. 1986 Mar 20;320(6059):277–280. doi: 10.1038/320277a0. [DOI] [PubMed] [Google Scholar]
  9. Fleming T. P., Matsui T., Molloy C. J., Robbins K. C., Aaronson S. A. Autocrine mechanism for v-sis transformation requires cell surface localization of internally activated growth factor receptors. Proc Natl Acad Sci U S A. 1989 Oct;86(20):8063–8067. doi: 10.1073/pnas.86.20.8063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hammacher A., Hellman U., Johnsson A., Ostman A., Gunnarsson K., Westermark B., Wasteson A., Heldin C. H. A major part of platelet-derived growth factor purified from human platelets is a heterodimer of one A and one B chain. J Biol Chem. 1988 Nov 5;263(31):16493–16498. [PubMed] [Google Scholar]
  11. Hart C. E., Bailey M., Curtis D. A., Osborn S., Raines E., Ross R., Forstrom J. W. Purification of PDGF-AB and PDGF-BB from human platelet extracts and identification of all three PDGF dimers in human platelets. Biochemistry. 1990 Jan 9;29(1):166–172. doi: 10.1021/bi00453a022. [DOI] [PubMed] [Google Scholar]
  12. Hart C. E., Forstrom J. W., Kelly J. D., Seifert R. A., Smith R. A., Ross R., Murray M. J., Bowen-Pope D. F. Two classes of PDGF receptor recognize different isoforms of PDGF. Science. 1988 Jun 10;240(4858):1529–1531. doi: 10.1126/science.2836952. [DOI] [PubMed] [Google Scholar]
  13. Heldin C. H., Bäckström G., Ostman A., Hammacher A., Rönnstrand L., Rubin K., Nistér M., Westermark B. Binding of different dimeric forms of PDGF to human fibroblasts: evidence for two separate receptor types. EMBO J. 1988 May;7(5):1387–1393. doi: 10.1002/j.1460-2075.1988.tb02955.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Heldin C. H., Johnsson A., Wennergren S., Wernstedt C., Betsholtz C., Westermark B. A human osteosarcoma cell line secretes a growth factor structurally related to a homodimer of PDGF A-chains. Nature. 1986 Feb 6;319(6053):511–514. doi: 10.1038/319511a0. [DOI] [PubMed] [Google Scholar]
  15. Heldin C. H., Westermark B. Platelet-derived growth factor: mechanism of action and possible in vivo function. Cell Regul. 1990 Jul;1(8):555–566. doi: 10.1091/mbc.1.8.555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Heldin C. H., Westermark B., Wasteson A. Demonstration of an antibody against platelet-derived growth factor. Exp Cell Res. 1981 Dec;136(2):255–261. doi: 10.1016/0014-4827(81)90003-3. [DOI] [PubMed] [Google Scholar]
  17. Johnsson A., Betsholtz C., Heldin C. H., Westermark B. Antibodies against platelet-derived growth factor inhibit acute transformation by simian sarcoma virus. Nature. 1985 Oct 3;317(6036):438–440. doi: 10.1038/317438a0. [DOI] [PubMed] [Google Scholar]
  18. Josephs S. F., Guo C., Ratner L., Wong-Staal F. Human-proto-oncogene nucleotide sequences corresponding to the transforming region of simian sarcoma virus. Science. 1984 Feb 3;223(4635):487–491. doi: 10.1126/science.6318322. [DOI] [PubMed] [Google Scholar]
  19. Keating M. T., Williams L. T. Autocrine stimulation of intracellular PDGF receptors in v-sis-transformed cells. Science. 1988 Feb 19;239(4842):914–916. doi: 10.1126/science.2829358. [DOI] [PubMed] [Google Scholar]
  20. Keck P. J., Hauser S. D., Krivi G., Sanzo K., Warren T., Feder J., Connolly D. T. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science. 1989 Dec 8;246(4935):1309–1312. doi: 10.1126/science.2479987. [DOI] [PubMed] [Google Scholar]
  21. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Lee B. A., Maher D. W., Hannink M., Donoghue D. J. Identification of a signal for nuclear targeting in platelet-derived-growth-factor-related molecules. Mol Cell Biol. 1987 Oct;7(10):3527–3537. doi: 10.1128/mcb.7.10.3527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Leung D. W., Cachianes G., Kuang W. J., Goeddel D. V., Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science. 1989 Dec 8;246(4935):1306–1309. doi: 10.1126/science.2479986. [DOI] [PubMed] [Google Scholar]
  25. Lokeshwar V. B., Huang S. S., Huang J. S. Intracellular turnover, novel secretion, and mitogenically active intracellular forms of v-sis gene product in simian sarcoma virus-transformed cells. Implications for intracellular loop autocrine transformation. J Biol Chem. 1990 Jan 25;265(3):1665–1675. [PubMed] [Google Scholar]
  26. Maher D. W., Lee B. A., Donoghue D. J. The alternatively spliced exon of the platelet-derived growth factor A chain encodes a nuclear targeting signal. Mol Cell Biol. 1989 May;9(5):2251–2253. doi: 10.1128/mcb.9.5.2251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Matoskova B., Rorsman F., Svensson V., Betsholtz C. Alternative splicing of the platelet-derived growth factor A-chain transcript occurs in normal as well as tumor cells and is conserved among mammalian species. Mol Cell Biol. 1989 Jul;9(7):3148–3150. doi: 10.1128/mcb.9.7.3148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Matsui T., Heidaran M., Miki T., Popescu N., La Rochelle W., Kraus M., Pierce J., Aaronson S. Isolation of a novel receptor cDNA establishes the existence of two PDGF receptor genes. Science. 1989 Feb 10;243(4892):800–804. doi: 10.1126/science.2536956. [DOI] [PubMed] [Google Scholar]
  29. Mercola M., Melton D. A., Stiles C. D. Platelet-derived growth factor A chain is maternally encoded in Xenopus embryos. Science. 1988 Sep 2;241(4870):1223–1225. doi: 10.1126/science.3413486. [DOI] [PubMed] [Google Scholar]
  30. Mercola M., Wang C. Y., Kelly J., Brownlee C., Jackson-Grusby L., Stiles C., Bowen-Pope D. Selective expression of PDGF A and its receptor during early mouse embryogenesis. Dev Biol. 1990 Mar;138(1):114–122. doi: 10.1016/0012-1606(90)90181-h. [DOI] [PubMed] [Google Scholar]
  31. Ostman A., Rall L., Hammacher A., Wormstead M. A., Coit D., Valenzuela P., Betsholtz C., Westermark B., Heldin C. H. Synthesis and assembly of a functionally active recombinant platelet-derived growth factor AB heterodimer. J Biol Chem. 1988 Nov 5;263(31):16202–16208. [PubMed] [Google Scholar]
  32. Robbins K. C., Antoniades H. N., Devare S. G., Hunkapiller M. W., Aaronson S. A. Structural and immunological similarities between simian sarcoma virus gene product(s) and human platelet-derived growth factor. Nature. 1983 Oct 13;305(5935):605–608. doi: 10.1038/305605a0. [DOI] [PubMed] [Google Scholar]
  33. Robbins K. C., Leal F., Pierce J. H., Aaronson S. A. The v-sis/PDGF-2 transforming gene product localizes to cell membranes but is not a secretory protein. EMBO J. 1985 Jul;4(7):1783–1792. doi: 10.1002/j.1460-2075.1985.tb03851.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rorsman F., Bywater M., Knott T. J., Scott J., Betsholtz C. Structural characterization of the human platelet-derived growth factor A-chain cDNA and gene: alternative exon usage predicts two different precursor proteins. Mol Cell Biol. 1988 Feb;8(2):571–577. doi: 10.1128/mcb.8.2.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Thyberg J., Ostman A., Bäckström G., Westermark B., Heldin C. H. Localization of platelet-derived growth factor (PDGF) in CHO cells transfected with PDGF A- or B-chain cDNA: retention of PDGF-BB in the endoplasmic reticulum and Golgi complex. J Cell Sci. 1990 Oct;97(Pt 2):219–229. doi: 10.1242/jcs.97.2.219. [DOI] [PubMed] [Google Scholar]
  36. Truett M. A., Blacher R., Burke R. L., Caput D., Chu C., Dina D., Hartog K., Kuo C. H., Masiarz F. R., Merryweather J. P. Characterization of the polypeptide composition of human factor VIII:C and the nucleotide sequence and expression of the human kidney cDNA. DNA. 1985 Oct;4(5):333–349. doi: 10.1089/dna.1985.4.333. [DOI] [PubMed] [Google Scholar]
  37. Yarden Y., Escobedo J. A., Kuang W. J., Yang-Feng T. L., Daniel T. O., Tremble P. M., Chen E. Y., Ando M. E., Harkins R. N., Francke U. Structure of the receptor for platelet-derived growth factor helps define a family of closely related growth factor receptors. Nature. 1986 Sep 18;323(6085):226–232. doi: 10.1038/323226a0. [DOI] [PubMed] [Google Scholar]
  38. Yarden Y., Kuang W. J., Yang-Feng T., Coussens L., Munemitsu S., Dull T. J., Chen E., Schlessinger J., Francke U., Ullrich A. Human proto-oncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO J. 1987 Nov;6(11):3341–3351. doi: 10.1002/j.1460-2075.1987.tb02655.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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