Characterization and regulation of receptors for epidermal growth factor in mouse calvaria

Epidermal growth factor (EGF) has been shown to stimulate bone resorption in mouse calvaria in organ culture by a prostaglandin-mediated mechanism. In this report we demonstrate and characterize specific receptors for EGF in mouse bone. Binding of tracer quantities of [125I]iodo-EGF reached a maximum after 2 h of incubation at 37 C and began to decrease only after 8 h of continuous exposure to EGF. Scatchard analysis of equilibrium binding data showed a single class of binding sites with a Kd = 2 X 10(-9) M present at a concentration of 3.8 X 10(10) sites/calvarium. The association (ki = 4.4 X 10(6) M-1 min-1) and dissociation (k2 = 0.015 min-1) rate constants for the approach to equilibrium were calculated, and there was no evidence of cooperativity between binding sites. Pretreatment of bones with EGF for 12 h produced a decrease in EGF binding of about 20%, and a maximum decrease to 50-60% of control binding after 48 h of treatment. Decreased binding was due solely to a decrease in the number of receptors rather than to a change in receptor affinity. This down-modulation of receptors occurred even when the bone resorption-stimulating action of EGF was blocked completely by indomethacin, and thus seemed to be triggered only by occupancy of receptors by EGF. Treatment of calvaria with parathyroid hormone, 1,25-dihydroxyvitamin D3, calcitonin, and prostaglandin E2 produced no net change in the number or affinity of EGF receptors in bone. EGF also produced no change in the total DNA content of mouse calvaria. We conclude that mouse bone contains specific high affinity receptors for EGF, and that this experimental system is useful for investigations on EGF receptor modulation and the biological actions of EGF on bone.