PROTEIN-KINASE C-DEPENDENT DOWN-REGULATION OF BASIC FIBROBLAST GROWTH-FACTOR (FGF-2) RECEPTOR BY PHORBOL ESTER AND EPIDERMAL GROWTH-FACTOR IN PORCINE GRANULOSA-CELLS

The regulation of the basic fibroblast growth factor (bFGF, or FGF-2) receptor on porcine granulosa cells was studied. Receptor levels befor e and after cell differentiation in vivo and in vitro did not show any significant changes. Dibutyryl cAMP and the protein kinase A (PKA) in hibitor H-8 had no effect on bFGF binding. These results suggest that PKA was not involved in the receptor expression. Treatment of the gran ulosa cells with phorbol 12-myristate 13-acetate (PMA), a protein kina se C (PKC) activator, progressively decreased the number of bFGF recep tors to about 20% of their initial levels after 8 h, and this effect o ccurred in a concentration- and time- dependent manner. Similarly, syn thetic diacylglycerol also inhibited bFGF binding. The highly specific PKC inhibitor GF109203X completely prevented the reduction of bFGF bi nding by PMA and diacylglycerol. Kinetic analyses of the turnover of c ell surface bFGF receptors in the presence of cycloheximide showed tha t PMA accelerated loss of receptors from the cell surface, suggesting the enhanced receptor internalization by PMA resulting in the receptor reduction. PMA did not influence steady-state FGF receptor messenger RNA levels. PMA induced an increased PKC activity in the membrane frac tion, and among PMA sensitive PKC alpha, beta II, delta and epsilon, o nly PKC alpha was readily detected by immunoblotting and translocated to the membrane fraction. PMA-pretreated cells showed negligible effec t on c-fos messenger RNA induction in response to bFGF stimulation, in dicating a functional reduction of receptors. When cells were incubate d with epidermal growth factor, receptor levels were reduced, but this effect was not observed in the presence of GF109203X. These results s uggest that the bFGF receptor in porcine granulosa cells is regulated by the PKC, not PKA, pathway in an isoenzyme-specific fashion and that its possible mechanism may involve regulation of receptor internaliza tion.