Biophysical studies of cell membrane receptors for the growth factors PDGP and EGF : Aspects on lateral and aggregation

Abstract: When extracellular growth factors bind to their specific cell surface receptors, they elicit a cascade of biochemical events, including tyrosine kinase-mediated phosphorylation and changes in intracellular free ca2+, leading to cell differentiation and proliferation. However, direct effects of ligand-binding on the receptors are poorly understood. This thesis is aimed at investigating the dynamics of growth factor receptors in human fibroblasts after ligand binding, viz. the platelet-derived growth factor (PDGF), and after addition of serum. The effect of ultraviolet B (UV-B) irradiation on the lateral mobility of epidermal growth factor (EGF) receptors was assessed in fibroblasts and keratinocytes.Two techniques were mainly employed to study receptor dynamics. Lateral mobility of receptors was measured with fluorescence recovery after photobleaching (FRAP), and receptor aggregation was analysed with a novel technique, image correlation spectroscopy (ICS), based on confocal laser scanning microscopy.The results, obtained with FRAP for the lateral mobility of the WGA-labelled glycoconjugate and PDGF receptors showed, that: i) the diffusion rate (D) and mobile fraction (R) of the PDGF-receptors depend on the growth conditions, viz. absence or presence of serum in the growthmedium, and stimulation with PDGF or serum. In general, serum increased both D and R in serum-starved fibroblasts, whereas PDGF alone did not change the mobility to any appreciable extent; ii) inhibition of tyrosine kinase using an inhibitor (Erbstatin analog) gave only a slightalteration of the lateral mobility achieved with serum or PDGF addition; iii) UV-B irradiation, noxious to skin cells, caused higher diffusion rate and mobile fraction of EGF-receptors, but in combination with the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), the effects on D and R were counteracted; i v) modulation of ca2+ extraand/ or intracellularly, strongly affected the lateral diffusion of PDGFreceptors, as well as the response to PDGF and serum. In cells depleted intracellulary of ca2+ with a calcium chelating agent, D was reduced about 2-fold compared to control cells; after addition of serum or PDGF D increased again. When extracellular ca2+ was chelated with EGTA, R increased. In cells intra- and extracellularly depleted of ca2+, D wasslightly lower and R higher compared to the controls. In this case PDGF had no effect , whereas serum increased D and R dramatically.In the aggregation studies, using the novel ICS method for quantifying the aggregation state of the receptors, it was found that the surface density of the PDGF receptor clusters decreased when the temperature was raised from 4 oc to 37°C, viz. the density of the clusters was about 3-4 fold lower at 37°C than at 4°C. Tyrosine kinase inhibition with Erbstatin analog seemed to induce receptor dispersion, since the cluster density was several fold higher than without Erbstatin analog. PDGF did not induce further clustering at 37°C. PDGF partly abolished the dispersion effect of Erbstatin analog, in agreement with the involvement of tyrosine phosphorylation in PDGF stimulation of cells.