Ligand-induced activation of A431 cell epidermal growth factor receptors occurs primarily by an autocrine pathway that acts upon receptors on the surface rather than intracellularly.

M J Van De Vijver,R Kumar,J Mendelsohn

doi:10.1016/s0021-9258(20)89475-7

Abstract

A431 cells express high numbers of epidermal growth factor (EGF) receptors and produce a ligand for these receptors, transforming growth factor-alpha (TGF-alpha). We have obtained evidence that the EGF receptors on these cells may be activated through an "autocrine" pathway by ligand and have investigated whether activation of phosphorylation of the receptor by the endogenously produced TGF-alpha occurs intracellularly or at the cell surface. When A431 cells were cultured under serum-free conditions, in the absence of exogenous ligand, EGF receptors were found to have a basal level of phosphorylation. When cells were labeled by culturing with 32Pi in the continuous presence of monoclonal antibodies that block binding of TGF-alpha to the EGF receptor, phosphorylation decreased to 30 +/- 10% of the basal level. This reduction could not be accounted for by the decrease in receptor content attributable to down-regulation and catabolism of EGF receptors that resulted from the binding of anti-receptor monoclonal antibodies. The reduction in receptor phosphorylation mediated by antibody was accompanied by the accumulation of increased levels of secreted TGF-alpha species in the culture medium. We also pulse-labeled A431 cells for 15 min with [35S]cysteine and immunoprecipitated the cell lysate with anti-phosphotyrosine antibody after various chase periods. Tyrosine-phosphorylated EGF receptor became detectable after 40 min of chase and reached a maximum after 4-6 h; these times are in agreement with the intervals required for EGF receptors to reach the cell surface after synthesis and then to achieve maximal expression. In addition, only the 170-kDa, mature EGF receptor species, and not the 160-kDa intracellular precursor, was immunoprecipitated with the anti-phosphotyrosine antibody. The results of these pulse-chase experiments and the finding that anti-receptor monoclonal antibody can block receptor phosphorylation suggest that activation of EGF receptors can result from the binding of an endogenous ligand (presumably TGF-alpha), which occurs at the cell surface and not during receptor biosynthesis and intracellular processing.

Highlights

A431 cells expresshighnumbers of epidermal Autocrine stimulation by growth factors has been postugrowth factor (EGF) receptors and produce a ligand lated as a mechanism contributing to the growth of tumor forthesereceptorst,ransforminggrowthfactor-a cells [1]
Effect of monoclonal anrosine-phosphorylated EGF receptor became detecta- tibodies (mAbs) 225 and 528 onEGF Receptor Phosphoryla- ously exposed to anti-receptormAb, parallel cultures of A431 tion-Having established that the EGreFceptor in A431 cells cells were labeled for 16 h with "'Pi or [:"S]cysteine, in the is phosphorylated on tyrosine in the absence of exogenous absence or continuouspresence of varying concentrations of ligand, we wanted to investigate whether this phosphorylatiomnAb 528
AutoAcrcitnievation of EGF Receptors in A431 Cells ducedreceptorcatabolism is of critical importance to our accumulation of TGF-a-related secreted proteins in the meobservations, the question was addressed with an alternative dium of A431 cells cultured in the presence of mAbs which assay procedure

Summary

Introduction

A431 cells expresshighnumbers of epidermal Autocrine stimulation by growth factors has been postugrowth factor (EGF) receptors and produce a ligand lated as a mechanism contributing to the growth of tumor forthesereceptorst,ransforminggrowthfactor-a cells [1]. We transformed growth but is dependent on exogenous addition pulse-labeled A431 cells for 15 min with [36S]cysteine of ligand [8,9,10] or co-expression of EGF [11]or TGF-a [3].

Results

Conclusion