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Polypeptide growth factors- a growth area for biotechnology The spectacular recent upsurge in interest in polypeptide growth factors is due, in large part, to the techniques of molecular biology. These permit the production of large quantities of growth factors both as commercial products and as laboratory tools for investigation of cell regulation. The term 'growth factor' is usually used to describe a polypeptide synthesized and secreted by a cell, and having a regulatory effect on the proliferation or differentiation of nearby cells. Hormones are generally excluded on the basis of their endocrine, rather than paracrine or autocrine, action and, for convenience, cytokines (regulatory proteins secreted by cells of the immune system) are usually considered as a separate group. However, classification is somewhat arbitrary: cytokines are not restricted to the immune system, for example, and growth factors do not always have to be secreted to exert their effects, but can remain membrane-bound. Since growth factors influence every aspect of cell development, it follows that they will find applications in a wide variety of fields. Molecular biological techniques allow the economical production of large quantities of recombinant growth factors, in which the natural amino acid sequence and glycosytation can be modified to obtain a molecule tailored to meet specific requirements, The pharmaceutical industry is investigating therapeutic and diagnostic applications of recombinant growth factors (for a recent survey, see Ref. 1). In the field of cell culture, recombinant growth factors may provide a defined and economical alternative to the serum at present necessary in many systems. Of great theoretical and practical interest is the possibility of investigating the disruption of normal cell regulation causing malignancy 2 using engineered growth factors, growth factor receptors and transduction proteins. The interaction between the fields ~) 1990, Elsevier Science Publishers Ltd (UK)

of biotechnology and growth factor research was illustrated at the Biological Council's 5th Annual Symposium on Biotechnology*. This highly informative meeting was not limited to consideration of the role of polypeptide growth factors, but they, together with their signal transduction pathways, figured in many of the topics presented, a few examples of which are reported briefly here. Epidermal growth factor (EGF) has been the subject of much research, particularly in relation to its therapeutic action in wound healing and the possible involvement of its receptor in tumour development. An elegant functional analysis of the ligand-binding site of the EGF receptor using chimeric chicken/ human receptor molecules was described (for published details see Ref. 3). Murine EGF binds to the chicken EGF receptor with 100-fold lower affinity than to the human EGF receptor. Substitution of various domains of the chicken EGF receptor by domains from the human receptor reveal that extracellular domain III is the major ligand-binding domain, with domains I and II' probably exerting a modifying influence. [The exchange of domains did not disturb the overall folding of the receptor chimeras: the high affinity binding of transforming growth factor ~ (TGFe~) to the receptor was not impaired.] Monoctonal antibodies inhibiting EGF binding were shown to recognize specifically human domain III. The biological responses (tyrosine phosphorylation, receptor downregulation and DNA synthesis) of the transfected cells expressing the various receptors reflect the EGFbinding affinity of the constructed receptor. Similar construction of chimeras with substitutions in the *Biological Council's 5th Annual Symposium on Biotechnology (The Regulation of Growth and Development - The Elucidation of Signal Pathways), 14-15 December 1989, London, UK.

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cytoplasmic (kinase) domain, using, for example, kinase-minus mutants and truncated, constitutively active mutants has permitted study of the role of autophosphorylation and substrate phosphorylation in the signal transduction process. Platelet-derived growth factor (PDGF) is composed of two disulphide-bonded homologous polypeptide chains, A and B. It is a major mitogen of connective tissue cells, with w o u n d healing properties, and is secreted by several normal and malignant cell types. All three dimeric isoforms exist, and there are two structurally related receptor types, e~and h 4. The o~-receptors bind all three isoforms with high affinity; the h-receptors bind PDGF-BB with high affinity, PDGF-AB with lower affinity, and do not bind PDGF-AA. Both receptors, which are protein tyrosine kinases, mediate a mitogenic signal, but only the h-receptor mediates actin reorganization and chemotaxis. This provides an experimental method of distinguishing between activation of the two receptor types. Cross-competition exper: iments using the different PDGF isoforms have led to the proposal of a model in which receptor dimerization is linked to receptor activation. In the absence of available ~receptors, high concentrations of PDGF-AB inhibit the action of PDGFBB, suggesting that PDGF-AB activates the h-receptor via formation of a heterodimer complex. This model implies that it may be possible to manipulate the signal transduction by choice of appropriate isoforms of PDGF. Naturally occurring or specifically designed antagonists which prevent receptor dimerization might prove of clinical relevance in preventing autocrine or paracrine stimulation of tumours. Protein kinase C (PKC) is a protein involved in a multitude of physiological responses, including stimulation by growth factors such as EGF, PDGF and fibroblast growth factor (FGF) (for a review, see Ref. 5). Differences in activation and substrate specificity of the various PKCs must exist therefore, if different signals are to give rise to specific and distinct responses. Structural analysis has ted to the identification of a family of at least seven related gene products. In-vitro biochemical analysis, including characterization of

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TIBTECH - M A R C H 1990 [Vol. 8]

novel PKCs identified only by cDNA cloning, has been facilitated by the production of large amounts of PKC using the baculovirus expression system. PKC-e~, -[31, -82 and -y are Ca2+/phospholipid/diacylglyceroldependent protein kinases, while PKC-c shows no dependence on Ca2+. PKC-c (and possibly -6 and -~) might therefore be activated by diacylglycerol produced other than by the classical phosphatidylinositol pathway where inositol 1,4,5-trisphosphate (IP3) induces Ca2÷ release. Differences in substrate specificity for histone H1 and synthetic peptide substrates shown by PKC-e¢, -f~ and -? suggests that the different isotypes will phosphorylate different target proteins in vivo. The use of cDNAs to produce large quantities of pure PKCs, and the construction of chimeric and modified structures should facilitate the study of the role of PKC in signal transduction in different physiological and pathological-states. Several speakers discussed the changes in gene expression induced by growth factor stimulation. There is a set of at least 80 genes, such as c-fos

and c-jun, known as cellular 'immediate-early' genes, whose transcription is immediately and transiently activated on treatment of susceptible cells with growth factors or other mitogens. The product of the c-fos proto-oncogene, fos, is a transcription regulatory protein. Gene transfer experiments show that activation of c-fos transcription by many different growth factors is mediated by a short DNA sequence, the serum response element (SRE) that forms the binding site for a conserved nuclear protein, the serum response factor (SRF). Mutations that block SRF binding block the transcriptional activity of the SRE. Sequences related to SRE are found in the promoter regions of several other immediate-early genes. Some immediate-early genes may couple the short-term signals elicited by stimulation at the cell surface to long-term alterations in cellular phenotype by regulating expression of specific target genes. Rather than necessarily being limited to a specific role, they could be involved in general signal transduction processes within the nucleus, the specific target genes regulated being determined by the differentiated state of the cell.

These few examples, together with many other presentations, illustrated the ways in which molecular biology has made possible rapi'd,advances in the diverse fields of growth factor research, from ligand binding, receptor activation, signal transduction and changes in gene transcription, to regulation of cell proliferation and differentiation. Some of these discoveries will, in turn, be of direct benefit to biotechnology processes, permitting control and manipulation of cell growth and gene expression. C. JANE ROBINSON

National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hefts EN6 3QG, UK. References i Copsey, D. N. and Delnatte, S. Y. (1988) in Genetically Engineered Human Therapeutic Drugs, Stockton Press 2 Steel, C. M. (1989) Lancetii, 30-34

3 Lax, I., Bellot, F., Howk, R. etal. (1989) EMBO J. 8,421-427 4 Heldin, C-H. and Westermark, B. (1989) Trends Genet. 5, 108-111 5 Parker, P. J., Kour, G., Marais, R. M. et al. (1989) Mol. Cell. Endocrinol. 65, 1-11

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Polypeptide growth factors--a growth area for biotechnology.

TIBTECH - MARCH 1990 [Vol. 8] 59 Polypeptide growth factors- a growth area for biotechnology The spectacular recent upsurge in interest in polypep...
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