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The PDGF receptor and signal transduction
Two PDGF receptor subunits have been identified. Both are transmembrane glycoproteins whose cytoplasmic domains display tyrosine kinase activity upon activation. The mature a-receptor contains 1066 amino acids and exhibits a molecular mass of 170 kDa. The b-receptor is slightly larger (1074 amino acid residues).
Binding of ligand promotes receptor dimerization and hence activation. Three isoforms of the receptor exist: aa, ab and bb. The particular dimeric form of ligand that binds dictates the
Table 7.8. The range of human PDGF receptor dimers which may form upon binding of different PDGF isoforms
PDGF ligand Receptor species formed
BB aa, ab, bb
AB aa, ab
GROWTH FACTORS 289
possible combination of dimers present in the corresponding receptor (Table 7.8). Ligand-induced dimerization triggers a variety of intracellular events, including receptor autophosphorylation, with subsequent activation of phospholipase C.
PDGF and wound healing
In vitro and in vivo studies support the thesis that PDGF is of value in wound management — particularly with regard to chronic wounds. All three isoforms of PDGF are available from a range of recombinant systems. In vitro studies, using various cell lines, suggest that PDGF AB or BB dimeric isoforms are most potent.
Normal skin appears to be devoid of PDGF receptors. Animal studies illustrate that rapid expression of both a- and b-receptor subunits is induced upon generation of an experimental wound (e.g. a surgical incision). Receptor expression is again switched off following re-epithelialization and complete healing of the wound.
Initial human trials have found that daily topical application of PDGF (BB isoform) stimulated higher healing rates of chronic pressure wounds, although the improvement recorded fell just short of being statistically significant. A second trial found that daily topical application of PDGF (BB) did promote statistically significant accelerated healing rates of chronic diabetic ulcers.
The product (tradename Regranex) was approved for general medical use in the late 1990s. Its active ingredient is manufactured by Chiron Corporation, in an engineered strain of Saccharomyces cerevisiae harbouring the PDGF B-chain gene. Regranex is notable in that it is formulated as a non-sterile (low bioburden) gel, destined for topical administration. The final formulation contains methylparaben, propylparaben and m-cresol as preservatives. In addition, as is the case with EGF, PDGF antagonists may also prove valuable in the treatment of some cancer types in which inappropriately high generation of PDGF-like mitogenic signals leads to the transformed state.
FIBROBLAST GROWTH FACTORS (FGFs)
Fibroblast growth factors (FGFs) constitute a family of about 20 proteins (numbered consecutively FGF-1 to FGF-20). Typically, they display a molecular mass in the region of 1828 kDa and induce a range of mitogenic, chemotactic and angiogenic responses. Classification as an FGF is based upon structural similarity. All display a 140 amino acid central core which is highly homologous between all family members. All FGFs also tightly bind heparin and heparin-like glycosaminoglycans found in the extracellular matrix. This property has been used to purify several such FGFs via heparin affinity chromatography. Subsequent to binding to the immobilized heparin, FGF can be eluted by inclusion of high NaCl concentrations in the eluting buffer. Although many of the original members of this family stimulate the growth/ development of fibroblasts (hence the name), several newer members have little or no effect upon fibroblasts.
The means by which FGFs are secreted from their producer cells remains to be fully elucidated. Several (e.g. FGF-1 and FGF-2, also known as acidic-FGF and basic-FGF, respectively; Figure 7.4) do not contain a classical signal sequence for secretion (i.e. a short N-terminus stretch of hydrophobic amino acid residues which direct newly synthesized polypeptides to the endoplasmic reticulum and hence ultimately facilitates their extracellular release). Interestingly, several such FGFs display a nucleas localization motif and have been
found in association with the nucleas. In this way, they may induce selected biological responses independent of the extracellular route.
FGFs induce the majority of their characteristic responses via binding to high-affinity cell surface receptors. Four such receptors, which show 55-72% homology at the amino acid level, have been identified to date. The receptors are multi-domain, consisting of three extracellular immunoglobulin-like domains (Ig domains), a highly hydrophobic transmembrane domain and two intracellular tyrosine kinase domains. Different receptors are found on different cell types and each receptor is either strongly, moderately, poorly or not activated by its own characteristic range of FGFs. This signalling complexity underscores the complexity of biological responses induced by FGFs.