The presence of epidermal growth factor binding sites in the intracellular organelles of term human placenta.

Abstract

Highly purified lysosomes, rough and smooth endoplasmic reticulum, and Golgi apparatus, as well as microvillus plasma membranes, bound 125I-labelled epidermal growth factor ([125I]EGF) with similar affinity. Scatchard plots for all the organelles were curvilinear. The apparent number of available binding sites per mg protein of intracellular organelles was 27-71% of that found in microvillus plasma membranes. The bound and free [125I]EGF were not degraded by any of the organelles. Binding and dissociation of [125I]EGF in all organelles were dependent on the time and temperature of incubation. The specificity of [125I]EGF binding was similar in all organelles. The optimal pH for binding to lysosomes was 6.0, in contrast to 7.0 for all the other organelles. Exposure of different organelles to enzymes and protein-modifying reagents resulted in numerous binding differences between the intracellular organelles and microvillus plasma membranes. Covalent affinity labelling with [125I]EGF revealed two major proteins of 155 and 140(X10(3)) Mr in all the organelles. The 155 X 10(3) Mr protein was labelled predominantly in all organelles except rough endoplasmic reticulum, where both proteins were equally labelled. Addition of proteolytic inhibitors during isolation of organelles did not alter the pattern of [125I]EGF-labelled binding proteins found in the organelles. EGF also stimulated phosphorylation of the 155 and 140(X10(3)) Mr proteins in all the organelles. The 155 X 10(3) Mr protein was phosphorylated more than the 140 X 10(3) Mr protein in microvillus plasma membranes and smooth endoplasmic reticulum, whereas the 140 X 10(3) Mr protein was phosphorylated more than the 155 X 10(3) Mr protein in lysosomes and both proteins were equally phosphorylated in rough endoplasmic reticulum. Several organelles also contained minor [125I]EGF-binding proteins that did not show phosphorylation response and proteins that showed phosphorylation response but did not bind [125I]EGF. Thus, the present study demonstrates by a number of different criteria, that several intracellular organelles of term human placenta also contain EGF-binding and kinase activities.