Stimulation-induced Down-regulation of Tumor Necrosis Factor-α Converting Enzyme

John R Doedens,Roy A Black

doi:10.1074/jbc.275.19.14598

Abstract

The extracellular domains of many proteins, including growth factors, cytokines, receptors, and adhesion molecules, are proteolytically released from cells, a process termed "shedding." Tumor necrosis factor-alpha converting enzyme (TACE/ADAM-17) is a metalloprotease-disintegrin that sheds tumor necrosis factor-alpha and other proteins. To study the regulation of TACE-mediated shedding, we examined the effects of stimulation of cells on TACE localization and expression. Immunofluorescence microscopy revealed a punctate distribution of TACE on the surface of untreated cells, and stimulation of monocytic cells with lipopolysaccharide did not affect TACE staining. Phorbol 12-myristate 13-acetate (PMA), a potent inducer of shedding, decreased cell-surface staining for TACE. Surface biotinylation experiments confirmed and extended this observation; PMA decreased the half-life of surface-biotinylated TACE without increasing the turnover of total cell-surface proteins. Soluble fragments of TACE were not detected in the medium of cells that had down-regulated TACE, and TACE was not down-regulated when endocytosis was inhibited. Antibody uptake experiments suggested that cell-surface TACE was internalized in response to PMA. Surprisingly, a metalloprotease inhibitor prevented the PMA-induced turnover of TACE. Thus, PMA activates shedding and causes the down-regulation of a major "sheddase," suggesting that induced shedding may be regulated by a mechanism that decreases the amount of active TACE on the cell surface.

Highlights

A wide variety of proteins, including cytokines, growth factors, and their receptors, as well as cell adhesion molecules, are synthesized as transmembrane proteins that can be released from cells by proteolysis, a process termed “ectodomain shedding”
Phorbol 12-myristate 13-acetate (PMA) Treatment Results in the Turnover of Pre-existing TACE Molecules—To examine the fate of pre-existing TACE molecules in PMA-treated cells, we reversed the steps in the previous experiment such that Jurkat cells were surface-biotinylated before treatment with PMA
This approach is akin to a pulse-chase experiment; TACE molecules present on the surface of cells are labeled with biotin at the beginning of the experiment, and their fate is followed over time

Summary

Introduction

A wide variety of proteins, including cytokines, growth factors, and their receptors, as well as cell adhesion molecules, are synthesized as transmembrane proteins that can be released from cells by proteolysis, a process termed “ectodomain shedding” (reviewed in Ref. 1). Only the processed form of TACE was detected when immunoprecipitates of surface-biotinylated cells were probed with streptavidin-HRP conjugates, regardless of the precipitating antibody used (Fig. 1D).

Results

Conclusion