Diverse Cell Surface Protein Ectodomains Are Shed by a System Sensitive to Metalloprotease Inhibitors

doi:10.1074/jbc.271.19.11376

Diverse Cell Surface Protein Ectodomains Are Shed by a System Sensitive to Metalloprotease Inhibitors (*)

From the (1)Cell Biology and Genetics Program and Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, the
(2)I. Medizinische Klinik, Johannes Gutenberg-Universität-Medizinische Fakultät, D-55101 Mainz, Germany, and the
(3)Department of Immunology, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877

^¶Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed:
Box 116, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021.
Tel.: 212-639-8975; Fax: 212-717-3298.

Abstract

The extracellular domains of a diverse group of membrane proteins are shed in response to protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA). The lack of sequence similarity in the cleavage sites suggests the involvement of many proteases of diverse specificity in this process. However, a mutant Chinese hamster ovary cell line recently isolated for being defective in PMA-activated shedding of the membrane-anchored growth factor transforming growth factor α precursor (proTGF-α) is concomitantly defective in the shedding of many other unrelated membrane proteins. Here we show that independent mutagenesis and selection experiments yield shedding mutants having the same recessive phenotype and belonging to the same genetic complementation group. Furthermore, two structurally distinct agents, TAPI-2 and 1,10-phenanthroline, which are known to inhibit metalloproteases, block PMA-activated shedding of proTGF-α, cell adhesion receptor L-selectin, interleukin 6 receptor α subunit, β-amyloid precursor protein, and an entire set of anonymous Chinese hamster ovary cell surface proteins. Certain serine protease inhibitors prevent release of these proteins by interfering with their maturation and transport to the cell surface but do not inhibit ectodomain shedding from the cell surface. The results suggest the existence of a common system for membrane protein ectodomain shedding involving one or several proteolytic activities sensitive to metalloprotease inhibitors, whose ability to act can be disrupted by recessive mutations in a single gene.

Footnotes

↵^§ Recipient of a postdoctoral fellowship from the Spanish Ministry of Education and Science.
↵* This work was supported by Grant CA53559 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

βAPP

β-amyloid precursor protein

TNF-α

tumor necrosis factor α

IL-6

interleukin 6

IL-6 R

IL-6 receptor

CHO

Chinese hamster ovary

PMA

phorbol 12-myristate 13-acetate

DFP

diisopropylfluorophosphate

TPCK

tosylphenylalanyl chloromethyl ketone

MEM

minimum essential medium

PBS

phosphate-buffered saline

PAGE

polyacrylamide gel electrophoresis

FACS

fluorescence-activated cell sorter

DCI

dichloroisocoumarine

TAPI

N-{D,L-[2-9hydroxyamino-carbonyl)methyl]-4-methypentanoyl}L-3(2′naphthyl)-alanyl-L-alanine, 2-aminoethyl amide

TAPI-2

analog of TAPI with the naphthyl-alanine side chain replaced by a terbutyl group

proTGF-α

transforming growth factor α precursor.
- Received November 20, 1995.
- Revision received January 22, 1996.