Superagonistic activation of ErbB-1 by EGF-related growth factors with enhanced association and dissociation rate constants.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) are mitogenic hormones that exert their activity primarily by binding to the EGF receptor, also known as ErbB-1. We have recently characterized a set of EGF/TGFalpha chimeric molecules with similar high affinity for ErbB-1 as EGF and TGFalpha and shown that three of these chimeras induce mitogenic cell stimulation at already a 10-fold lower concentration than their wild-type counterparts (Lenferink, A. E., Kramer, R. H., van Vugt, M. J., Königswieser, M., DiFiore, P. P., van Zoelen, E. J., and van de Poll, M. L. (1997) Biochem. J. 327, 859-865). In the present study we show that these so-called superagonistic chimeras do not differ from EGF and TGFalpha in their ability to induce ErbB-1 tyrosine phosphorylation but are considerably more potent in activation of mitogen-activated protein kinase phosphorylation. Direct cell binding studies and analysis of ligand-receptor interaction by surface plasmon resonance measurements revealed that both the association rate constant (k(on)) and the dissociation rate constant (k(off)) of these superagonists is 3-5-fold higher in comparison with the wild-type ligands and nonsuperagonistic chimeras. These data indicate that the dynamic on and off rate constants for receptor binding may be more specific parameters for determining the mitogenic activity of peptide hormones than their constants for equilibrium receptor binding.

Bradykinin-induced growth inhibition of normal rat kidney (NRK) cells is paralleled by a decrease in epidermal-growth-factor receptor expression.

Normal rat kidney fibroblasts, grown to density arrest in the presence of epidermal growth factor (EGF), can be induced to undergo phenotypic transformation by treatment with transforming growth factor beta or retinoic acid. Here we show that bradykinin blocks this growth-stimulus-induced loss of density-dependent growth arrest by a specific receptor-mediated mechanism. The effects of bradykinin are specific, and are not mimicked by other phosphoinositide-mobilizing agents such as prostaglandin F2 alpha. Northern-blot analysis and receptor-binding studies demonstrate that bradykinin also inhibits the retinoic acid-induced increase in EGF receptor levels in these cells. These studies provide additional evidence that EGF receptor levels modulate EGF-induced expression of the transformed phenotype in these cells.

Differential effects of PDGF isoforms on proliferation of normal rat kidney cells.

The effects of the PDGF isoforms AA, AB and BB have been studied on the proliferation of normal rat kidney cells, a non-tumorigenic fibroblast cell line which contains both type alpha and type beta PDGF receptors. On monolayer cells made quiescent by serum deprivation, PDGF-AA is a relatively poor mitogen compared to PDGF-AB and PDGF-BB. When these cells are made density-arrested following continuous incubation with epidermal growth factor, however, they can be restimulated to proliferate by all three PDGF isoforms with similar activity when added at sufficiently high concentration, resulting in phenotypic cellular transformation. Binding of radiolabelled isoforms to confluent NRK monolayers obeys the predictions of an induced receptor dimerisation model, and increases in the order AA < AB < BB. Upon preincubation of the cells with PDGF-AA, the dose-response curve for mitogenic activity of PDGF-AB is shifted to higher concentrations, indicating that PDGF-AA can partly antagonize the growth stimulating activity of PDGF-AB, as has also been observed in ligand binding studies. This observation has subsequently been confirmed using fluorescence cytometric analysis. PDGF-AB is highly active in inducing anchorage-independent proliferation of NRK cells, but in all such assays PDGF-AA is at least as potent as PDGF-BB. Intriguingly, PDGF-BB is almost devoid of activity in inducing soft agar growth of these cells, in contrast to PDGF-AA. When compared to substrate-attached cells, enhanced expression of the type alpha PDGF receptor was observed under anchorage-independent conditions. These results show that the relative potency of the three PDGF isoforms to stimulate proliferation of NRK cells is different for quiescent cells in monolayer, density-arrested cells and anchorage-independent cells. Moreover it is shown that the biological activity of PDGFs can be impaired by the additional presence of other isoforms.

Phenotypic transformation of normal rat kidney cells by transforming growth factor beta is not paralleled by enhanced production of a platelet-derived growth factor.

Phenotypic transformation of normal rat kidney (NRK) cells requires the concerted action of multiple polypeptide growth factors. Serum-deprived NRK cells cultured in the presence of epidermal growth factor (EGF) become density-inhibited at confluence, but they can be restimulated by a number of defined polypeptide growth factors, resulting in phenotypic cellular transformation. Kinetic data show that restimulation by transforming growth factor beta (TGF-beta) and retinoic acid is delayed when compared to induction by platelet-derived growth factor (PDGF), indicating that both TGF beta and retinoic acid may exert their growth-stimulating action by an indirect mechanism. Northern blot analysis shows that NRK cells express the genes for various polypeptide growth factors, including TGF beta 1, PDGF A-chain and basic fibroblast growth factor, but that the levels of expression are not affected by TGF beta or retinoic acid treatment. NRK cells also secrete low amounts of a PDGF-like growth factor into their extracellular medium, but the levels of secretion are insufficient to induce mitogenic stimulation and are unaffected by agents inducing phenotypic transformation. In combination with studies on the effects of anti-PDGF antibodies, it is concluded that phenotypic transformation of NRK cells by TGF beta and retinoic acid is not the result of enhanced production of a PDGF-like growth factor.

The role of hydrophobic interactions in the phospholipid-dependent activation of protein kinase C.

The effects of hydrophobic interaction on the activation of Ca2+-stimulated phospholipid-dependent protein kinase (protein kinase C), isolated from mouse brain, by phosphatidylserine (PS) and diacylglycerol (DAG) or phorbol 12-myristate 13-acetate were studied. To maintain bilayer structure during assay conditions, phosphatidylcholine was added to the PS vesicles. The vesicular structure of all types of PS was confirmed by freeze-fracture electron microscopy. The PS-dependent activation of purified protein kinase C from mouse brain is affected by the fatty acid composition of PS: an inverse relationship between the unsaturation index of PS (isolated from bovine heart, bovine spinal cord or bovine brain) and the ability to activate protein kinase C was demonstrated. In highly saturated PS lipid dispersions, only slight additional activation of protein kinase C by DAG was found, in contrast with highly unsaturated PS lipid dispersion, where DAG increased protein kinase C activity by 2-3-fold at optimal PS concentrations. We quantified the formation of the protein kinase C-Ca2+-PS-phorbol ester complex by using [3H]phorbol 12,13-dibutyrate [( 3H]PDBu). The efficiency of complex-formation, determined as the amount of [3H]PDBu bound, is not affected by variations in the hydrophobic part of PS. These results indicate a role of the hydrophobic part of the activating phospholipid in the activation mechanism of protein kinase C and in the action of cofactors.