Possible regulation of epidermal growth factor-receptor tyrosine autophosphorylation by calcium and G proteins chemically permeabilized rat UMR106 cells

A model using chemically permeabilized cells was developed to examine mecha nisms that regulate protein tyrosine phosphorylation in osteoblastic cells. Using either permeabilized UMR106 osteoblastic or A431 (reference) cells, epidermal growth factor (EGF)-induced cellular tyrosine phosphorylation, an d whether there are previously unrecognized interactions between this trans duction pathway and Ca-24- or G protein-dependent signalling pathways, were investigated. Both permeabilized cell types, when maintained in non-supple mented cytoplasmic substitution solution (basic CSS), responded to EGF (1-1 00 ng/ml) with dose-dependent increases in tyrosine phosphorylation. A comp lex and time-dependent pattern of phosphotyrosine-containing proteins resul ted, but the profile of tyrosine phosphorylated proteins was appreciably le ss complex than in intact cells. Supplementation of basic CSS with MgATP re stored the normal complexity of the profiles for EGF-induced tyrosine phosp horylation proteins in both permeabilized cell lines and produced a more su stained accumulation of phosphoprotein products in A431 cells. Adding Ca2(less than or equal to 10(-6)M), with or without exogenous MgATP, dose-depe ndently attenuated EGF-induced tyrosine phosphorylation of EGF receptors (E GFR) and other substrates in UMR106 cells, but was less effective in A431 c ells. In both cell types, genistein, an inhibitor of tyrosine kinases, was more effective in attenuating EGF-induced receptor tyrosine phosphorylation in permeabilized cells. Similarly, orthovanadate, an inhibitor of protein tyrosine phosphatases, stimulated the accumulation of phosphoprotein produc ts more effectively in permeabilized cells. Thus, the permeabilization pres erves many features of intact cells while facilitating manipulation of intr acellular conditions. NaF reproducibly produced a significant vanadate-like action in permeabilized cells that was somewhat stronger than its effect o n intact cells. In contrast, the well-known inhibition of tyrosine phosphor ylation by phorbol 12-myristate 13-acetate (PMA) was less effective in perm eabilized cells than in intact cells; these actions of PMA were Ca2+-depend ent. In addition, guanylyl-imidodiphosphate (Gpp(NH)p) attenuated tyrosine phosphorylation in UMR106 cells, and this effect was specifically blocked b y guanosine 5'-O-(2-thiodiphosphate) (GDP beta s). These results strongly s uggest that there is crosstalk between EGFR-activated tyrosine phosphorylat ion/dephosphorylation pathways and both Ca2+- and G protein-mediated pathwa ys un UMR106 cells, revealing a previously unrecognized modulation of EGF s ignalling in osteoblast-like: cells that contrasts with the simpler regulat ory mechanisms found in A431 cells. (C) 1999 Elsevier Science Ltd. All righ ts reserved.