Both Intra- and Extracellular Ca2+ Participate in the Regulation of the Lateral Diffusion of the PDGF-β2 Receptor

Abstract

When the receptors for platelet-derived growth factor (PDGF) are activated they aggregate, become tyrosine-phosphorylated and elicit a cascade of down-stream signals, including mobilization of Ca2+ from intra- and extracellular stores. Receptor mobility in the plane of the membrane is a prerequisite for receptor aggregation and further signalling. Using human foreskin fibroblasts (AG 1523) and fluorescence recovery after photobleaching (FRAP), we therefore assessed the lateral mobility characteristics of PDGF-beta2 receptors by their diffusion coefficient (D), and fraction of mobile receptors (R). This was done on cells stimulated with either normal human serum (NHS) or PDGF under different Ca2+-conditions. The results suggest that both intra- and extracellular free Ca2+ influence the mobility characteristics of the PDGF-beta2 receptor. Interestingly, the extracellular Ca2+ seems to impose general restrictions on the mobility of receptors, since R increased when extracellular Ca2+ was quenched with EGTA, whereas intracellular clamping of Ca2+ transients with MABTAM (BAPT/AM) primarily affected D. When both intra- and extracellular Ca2+ were quenced, D remained low and R high, further supporting the proposition that they achieve distinct effects. Inhibition of tyrosine phosphorylation with Erbstatin, partly inhibited the NHS effects and released PDGF-induced receptor immobilization. Ratio imaging with Fura-2 displayed that both NHS and PDGF induced changes in intracellular free [Ca2+]. In view of the present data it might have important effects on the state of the receptor in the membrane, for instance by regulating its lateral mobility, communication with other receptors and signalling functions in the membrane.