(Received for publication, November 20, 1995; and in revised form, January 22, 1996)
From the
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 precursor (proTGF-
) 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-
, cell
adhesion receptor L-selectin, interleukin 6 receptor
subunit,
-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.
The extracellular domain of a large number of transmembrane
proteins can be proteolytically released into the medium. This shedding
process regulates the fate and physical location of membrane-anchored
growth factors(1) , growth factor receptors(2) , cell
adhesion molecules, ectoenzymes(3) , and proteins of unknown
function such as the -amyloid precursor protein (
APP) (
)(4) . Many of these proteins are of practical
importance. For example,
APP is implicated in the pathogenesis of
Alzheimer's disease(5) , angiotensin converting enzyme
plays an important role in the regulation of blood
pressure(6) , and tumor necrosis factor
(TNF-
) and
the homing receptor L-selectin are implicated in inflammatory
responses(7, 8) . Ectodomain shedding can convert
membrane-anchored growth factors into diffusible factors, membrane
receptors into soluble competitors of their own ligand (9) or
accessories to ligand binding(2) , and cell adhesion receptors
into products no longer capable of mediating physical interactions with
other cells or the extracellular matrix(8) . Membrane protein
ectodomain shedding is now recognized as an important aspect of cell
regulation and cell-cell interaction.
Despite its broad interest, this shedding mechanism involves molecular components of unknown identity. Shedding appears to occur at or near the cell surface and does not require cytosolic factors that are essential for many forms of membrane traffic(10) . Shedding is often stimulated by protein kinase C activators and other agents(11, 12, 13, 14, 15, 16, 17, 18, 19, 20) . However, the proteins that are shed are not the targets of phosphorylation in this process. The nature of the proteases involved is of great interest because they might constitute ideal targets for therapy in various disease conditions. Given the diversity of amino acid sequences that are cleaved, many different proteases could be involved in this process, each endowed with a specific substrate recognition capacity. This notion has been reinforced by recent reports that the shedding of different ectodomains appears to be inhibited by different proteinase inhibitors(19, 20, 21, 22, 23, 24, 25, 26, 27, 28) .
Despite expectations that many different proteolytic activities may
be involved in membrane protein ectodomain shedding, recent genetic
evidence suggests that these processes may share certain components. We
recently isolated a mutant cell line that is defective in the shedding
of at least two unrelated molecules, APP and proTGF-
, thus
providing evidence that the shedding mechanisms of these two molecules
share a common component(29) . In the present report, we show
that independent selection of cell mutants defective in proTGF-
shedding yields cell lines that have identical phenotypes and belong to
the same genetic complementation group, indicating a repeated isolation
of mutations in the same gene. The defect in these cells prevents
shedding of all membrane proteins tested. Furthermore, recently
described compounds that inhibit certain metalloproteases and prevent
shedding of TNF-
(25) and the 80-kDa TNF-
receptor (26) prevent also the shedding of TGF-
,
APP,
L-selectin, IL-6 R
, and a large group of endogenous membrane
proteins in parental CHO cells. Previously observed differences in
protease inhibitor sensitivity of these various molecules are shown
here to result from effects of the inhibitors on membrane protein
maturation and transport. These results suggest the existence of a
common shedding mechanism involving one or several components sensitive
to metalloprotease inhibitors.
For cell fusions, 2 10
cells of a hygromicin-resistant clone and 2
10
cells of a histidinol-resistant clone were plated in 60-mm
dishes. 16 h later, the cultures were briefly covered with 3 ml of 45%
polyethylene glycol (M
1300-1600, American
Type Culture Collection) in MEM, 10 mM Hepes with a final pH
of 7.3. The polyethylene glycol/MEM solution was immediately aspirated,
leaving only the minimum amount needed to cover the cells, and the
cultures were incubated for 10 min at 37 °C. Cells were washed
three times with MEM and twice with MEM containing nonessential amino
acids and 10% fetal bovine serum using warm medium. After 10 h of
incubation in the latter medium, the cultures were trypsinized and
plated into 150-mm dishes. Hybrid cell clones were selected in
histidine-free MEM containing nonessential amino acids, 10% dialyzed
fetal bovine serum, 0.5 mM histidinol, and 800 µg/ml of
hygromicin for 2 weeks.
For biotinylation of cell surface proteins,
cells were labeled with 250 µCi/ml of
[S]methionine and 250 µCi/ml of
[
S]cysteine for 2 h in methionine- and
cysteine-free medium, chased for 30 min in complete medium, shifted to
4 °C, and incubated with 1 mg/ml sulfo-NHS-LC-biotin (Pierce) for 1
h at 4 °C. Unreacted biotinylating agent was quenched by washing
the cells with PBS containing 50 mM Tris. Cells were then
incubated for 30 min at 37 °C in complete medium, 5 min with or
without 200 µM TAPI-2, and an additional 20 min with or
without 1 µM PMA and/or 200 µM TAPI-2. Cells
were washed three times with cold PBS and lysed in lysis buffer. Medium
samples and cell lysates were incubated with streptavidin-agarose
beads, and the beads were washed with 0.1% Triton X-100 and 0.1% SDS
and analyzed on 12-18% gradient polyacrylamide gels.
Although M1 and other cell lines established from the original
mutant pool have the same phenotype, the entire mutant pool could be
derived from a single mutant clone enriched during the consecutive
rounds of sorting. In order to determine the frequency of isolation of
a shedding defective phenotype by mutation of the same gene, we
isolated an independent cell line, M2, by repeating the mutagenesis and
sorting protocol with a fresh batch of CHO cells. Like M1 cells, M2
cells were unable to shed cell surface proTGF- (Fig. 1A) or
APP (data not shown) in response to
PMA. Furthermore, hybrids generated by fusion of M1 or M2 with parental
CHO cells had wild type shedding activity (Fig. 1A). M1
M2 cell hybrids lacked shedding activity, showing that they
belong to the same genetic complementation group (Fig. 1A). Thus, the M1 and M2 cell lines have the same
recessive phenotype and belong to the same complementation group. These
results argue that the defect in membrane protein ectodomain cleavage
in these independent cell lines is caused by recessive mutations in the
same gene.
Figure 1:
Shedding of L-selectin, IL-6 R,
and TGF-
in wild type and mutant CHO cells and cell hybrids. A, wild type CHO cells (WT), their mutant derivatives
M1 and M2, and hybrids between the indicated cell lines, all expressing
HA-tagged proTGF-
, were treated with or without PMA for 20 min.
The levels of cell surface immunostaining with anti-HA antibody were
analyzed by flow cytometry. The results are expressed as percentages
relative to the mean fluorescence of cells not treated with PMA and are
the averages ± S.D. of triplicate determinations. B,
wild type or mutant (M1) CHO cells transfected with L-selectin, IL-6
R
, or proTGF-
were metabolically labeled with
[
S]cysteine and
[
S]methionine and then chased in complete medium
for 45 min with or without PMA. Cell lysates were immunoprecipitated
with antibodies against the against the L-selectin cytoplasmic domain
or against the IL-6 R
or proTGF-
ectodomains, respectively.
Medium samples were immunoprecipitated with antibodies against the
ectodomains of L-selectin, IL-6 R
, or proTGF-
, respectively.
Immunoprecipitates were analyzed by SDS-PAGE and
autoradiography.
Immunoprecipitation of metabolically labeled
L-selectin transfectants with antibodies against the L-selectin
intracellular domain yielded products of 50 and 74 kDa (Fig. 1B). Based on previous characterization, these
products correspond to the biosynthetic precursor of L-selectin and the
fully glycosilated cell surface form, respectively (19) (see
also Fig. 3B). Upon cell treatment with PMA, the cell
surface form is converted into a soluble form (Fig. 1B), leaving a cell-associated 6-kDa
transmembrane/cytoplasmic fragment(19) . In contrast to
parental CHO cells, M1 and M2 cells showed a lack of basal or activated
L-selectin shedding activity (Fig. 1B and data not
shown). Likewise, IL-6 R, which is expressed in transfected CHO
cells as an 80-kDa cell surface protein and shed as a 57-kDa soluble
ectodomain in response to PMA(28) , was not shed in the mutants (Fig. 1B, middle panel). The results obtained
with M1 and M2 transfectants were identical, and only one of the
mutants (M2) is shown here for simplicity. These results indicate that
the gene affected in these mutants is essential not only for
proTGF-
and
APP shedding but also for the shedding of
L-selectin and IL-6 R
.
Figure 3:
3,4-DCI does not prevent shedding of
L-selectin or proTGF-. A, flow cytometry analysis of CHO
cells expressing L-selectin and HA/proTGF-
treated with or without
PMA and/or 100 µM 3,4-DCI for 20 min. Cells were
immunostained with antibodies against the L-selectin ectodomain or the
HA epitope and analyzed by flow cytometry. B, metabolically
labeled CHO cells expressing HA/proTGF-
and L-selectin were chased
for 25 min in complete medium and then incubated with anti HA
monoclonal antibodies or polyclonal antibodies against the ectodomain
of L-selectin at 4 °C. Cells were then shifted to 37 °C,
briefly washed with medium with or without 3,4-DCI, and incubated in
the presence or the absence of PMA and/or 100 µM 3,4-DCI
for 15 min as indicated. Immune complexes present in cell lysates and
medium were precipitated with protein A-agarose and analyzed by
SDS-PAGE.
Figure 2:
Effect of 3,4-DCI on the release of
L-selectin, IL-6 R, and proTGF-
ectodomains. CHO cells
transfected with L-selectin, IL-6 R
, or HA/proTGF-
were
metabolically labeled and chased for 45 min in the presence or the
absence of 100 µM 3,4-DCI. Where indicated, PMA was added
during the last 30 min of the chase period. Aliquots from medium
samples were immunoprecipitated with antiserum against the
corresponding protein ectodomains.
The current availability of a HA-tagged proTGF-
construct that can be recognized on the cell surface by anti-HA
antibody allowed us to assess the effect of these protease inhibitors
on the shedding of cell surface proTGF-
using FACS analysis. In
marked contrast with the results obtained by immunoprecipitation of
metabolically labeled products, FACS analysis of CHO transfectants
surface-stained with anti-HA antibody showed that 3,4-DCI did not
inhibit the PMA-induced shedding of cell surface proTGF-
(Fig. 3A). As described previously(21) ,
3,4-DCI did not prevent the PMA-induced loss of cell surface L-selectin
as determined by FACS analysis. The same results were obtained when
TPCK or DFP were analyzed in this type of assay (data not shown).
To
confirm the inability of 3,4-DCI to prevent ectodomain cleavage at the
cell surface, a protocol was designed to specifically follow the fate
of proteins that are present on the cell surface at the time of PMA
addition. Cells were metabolically labeled and then chased long enough
to allow labeled membrane proteins to reach the cell surface. Cells
were then incubated with antibodies against proteins of interest and
treated with PMA and/or 3,4-DCI. The immune complexes formed on the
cell surface were recovered by precipitation from cell lysates and
medium samples and analyzed by SDS-PAGE. When tested in this manner,
3,4-DCI did not inhibit PMA-induced loss of either proTGF- or
L-selectin from the cell surface (Fig. 3B, left
panels) and did not inhibit the release of soluble L-selectin and
TGF-
into the medium (Fig. 3B, right
panels).
These results suggested that 3,4-DCI and related
protease inhibitors prevented the release of newly synthesized membrane
proteins but not the shedding of these proteins once they have reached
the cell surface. To determine whether these protease inhibitors
interfered with transport of membrane proteins to the cell surface, a
proTGF- pulse-chase metabolic labeling experiment was done in the
presence or the absence of the inhibitors. ProTGF-
is synthesized
as an 18-kDa precursor that matures into forms of 20-22 kDa that
reach the cell surface and are converted to a 17-kDa product by removal
of the N-terminal pro-region (30) (Fig. 4, 60 min
lanes). Under basal conditions, this 17-kDa proTGF-
form
slowly turns over without significant release of TGF-
into the
medium(30) . The addition of 3,4-DCI at the start of the chase
period markedly delayed the maturation of the 18-kDa precursor into the
20-22-kDa form, as seen by immunoprecipitation from cell lysates (Fig. 4, right panels). Furthermore, 3,4-DCI delayed
the appearance of these forms on the cell surface, as determined by
precipitation of cell surface anti-HA immune complexes (Fig. 4, left panels). Similar effects were observed when TPCK and DFP
were tested under these conditions. Moreover, IL-6 R
,
APP,
and L-selectin also showed deficient maturation in the presence of
these serine protease inhibitors (data not shown). These results argue
that 3,4-DCI, TPCK, and DFP prevent ectodomain release by inhibiting
membrane protein maturation and transport rather than by specifically
blocking cell surface shedding activity.
Figure 4:
Effect of protease inhibitors on the
biosynthesis and transport of TGF-. CHO cells expressing
HA/proTGF-
were pulsed with [
S]cysteine for
20 min and chased for the indicated times in complete medium with or
without 100 µM 3,4-DCI or 200 µM TAPI-2 as
indicated. To immunoprecipitate cell surface HA/proTGF-
, cells
were incubated with anti-HA antibody at 4 °C and lysed, and immune
complexes were precipitated with protein A-agarose. For
immunoprecipitation of total cell HA/proTGF-
, cell lysates were
immunoprecipitated with antiserum against the cytoplasmic domain of
proTGF-
. See the text for a description of the various
proTGF-
forms indicated by the arrows.
CHO cells expressing transfected
proTGF-, L-selectin, or IL-6 R
were metabolically labeled,
chased, and then treated with or without PMA or TAPI-2. It should be
noted that TAPI-2 was added to the cells 30 min after the radioactive
pulse; this period of time is enough to allow transport of proTGF-
(Fig. 4), L-selectin (Fig. 3), and
-APP (31) to the cell surface. Therefore the effects of TAPI cannot
be due to interference with maturation or transport of these molecules.
In order to analyze the membrane forms of these proteins or endogenous
APP, cell lysates were immunoprecipitated with antibodies against
the extracellular domain of IL-6 R
or against the cytoplasmic
domains of L-selectin,
APP, or proTGF-
. In order to analyze
the soluble forms, medium samples were immunoprecipitated with the same
antibodies against IL-6 R
or antibodies against soluble L-selectin
or TGF-
. Cleavage of TGF-
and
APP was also followed by
the appearance of their residual 15-kDa transmembrane/cytoplasmic
fragments in the immunoprecipitates with anti-cytoplasmic domain
antibodies. The results of these experiments show that TAPI-2 inhibited
the PMA-induced shedding of all these proteins (Fig. 5).
Half-maximal inhibition of shedding activity was observed with 10
µM TAPI-2 (Fig. 6), a concentration that is
comparable with that required for half-maximal inhibition of TNF-
cleavage (25) or TNF-
receptor cleavage(26) .
Among other known metalloprotease inhibitors tested,
1,10-phenanthroline inhibited the shedding of TGF-
,
APP,
L-selectin, and IL-6 R
, whereas EDTA, EGTA, or phosphoramidon were
without effect at the concentrations tested (Table 1). The effect
of 1,10-phenanthroline on the shedding of TGF-
was prevented by
the addition of 5 mM ZnCl
(data not shown),
suggesting that the inhibitory effect of 1,10-phenanthroline is due to
its metal-chelating activity.
Figure 5:
Effect of TAPI-2 on the shedding of
L-selectin, IL-6 R,
APP, and TGF-
. CHO cells expressing
these proteins were metabolically labeled and chased in complete medium
with or without TAPI-2 for 25 min with PMA addition for the final 20
min, as indicated. Cell lysates (top panel in each set) were
immunoprecipitated with antibodies against the IL-6 R
extracellular domain or against the cytoplasmic domains of L-selectin,
proTGF-
or
APP, respectively. The cytoplasmic tail of
APP (
APP, bottom panel) is visualized in a
longer exposure of the corresponding portion of the gel. Medium samples (bottom panels in other sets) were immunoprecipitated with
antibodies against the ectodomain of L-selectin, IL-6 R
, or
TGF-
, respectively.
Figure 6:
Effect of different concentrations of
TAPI-2 on the shedding of APP, L-selectin, TGF-
, and IL-6
R
. CHO cells expressing expressing the indicated proteins were
metabolically labeled and treated with the indicated concentrations of
TAPI-2 as described in the legend to Fig. 5. Shedding of the
corresponding proteins was analyzed by determining the relative level
of immunoprecipitable L-selectin or IL-6 R
released into the
medium or proTGF-
or
APP cytoplasmic tails, respectively. The
results are expressed as percentages relative to controls that received
no TAPI-2 and are the averages of duplicate
determinations.
Additional experiments were designed to determine the effect of TAPI-2 on endogenous CHO proteins that are shed in response to PMA. For these experiments, metabolically labeled proteins were chased to allow their transport to the cell surface. The cell surface was then biotinylated by treatment with sulfo-NHS-LC-biotin and washed. Cells were incubated for a short period with or without PMA and/or TAPI-2. Biotinylated proteins were retrieved from the medium using immobilized streptavidin. The results show that numerous labeled proteins with molecular masses ranging from 30 to >200 kDa were released into the medium of PMA-treated cells but not that of control cells (Fig. 7). On the basis of cpm bound to streptavidin-agarose beads, the amount of biotinylated cell surface proteins released to the medium corresponds to 2-4% of biotinylated proteins present in the corresponding cell lysates. The SDS-PAGE profiles of metabolically labeled, biotinylated proteins recovered from the medium and cell lysates did not match, arguing that this technique specifically detects the minority of cell surface proteins that undergo PMA-induced ectodomain cleavage (data not shown). The addition of TAPI-2-inhibited PMA induced release of all these proteins (Fig. 7), arguing that all membrane protein ectodomain shedding in these cells is catalyzed by metalloprotease activity. Collectively, these results favor the hypothesis that one or several proteases with characteristics of a metalloprotease are responsible for the shedding of and entire set of transmembrane proteins, and their action can be disrupted by mutations in a single gene.
Figure 7: Effect of TAPI-2 on the PMA-induced release of endogenous CHO cell surface proteins. Metabolically labeled cells were incubated with sulfo-NHS-LC-biotin to label cell surface proteins, briefly washed with medium with or without TAPI-2, and then incubated with or without TAPI-2 and PMA for 20 min. Biotinylated proteins were recovered from the medium with streptavidin-agarose beads and analyzed on 12-18% gradient polyacrylamide gels.
Shedding of cell surface protein ectodomains is a general
mechanism that affects the activity of many membrane molecules with
different functions(1, 3) . The four membrane proteins
(proTGF-,
APP, L-selectin, and IL-6 receptor
) chosen
for the present studies are well known examples of this phenomenon. A
sense of how many different proteins are affected by this process is
provided by present (Fig. 7) and previous (29) experiments showing that treatment with the protein kinase
C activator PMA leads to the rapid shedding of approximately 2% of the
surface protein in CHO cells. The shed proteins are of widely different
molecular weights, and their electrophoretic profiles are distinct from
that of the general cell surface protein population. These observations
indicate that a significant subset of cell surface proteins including
proTGF-
,
APP, L-selectin, IL-6 R
, and many others are
shed into the medium, and often, if not always, this process can be
activated through protein kinase C-dependent and -independent
mechanisms.
Despite their common fate, no clear similarity can be
found between the various membrane proteins that undergo ectodomain
shedding. In particular, no sequence similarity can be found in the
cleavage site of these molecules to suggest that a common protease
mediates the shedding of all these molecules. Based on these
considerations and due to previously noted differences in sensitivity
to protease inhibitors(21, 27) , it had been proposed
that different proteases are involved in the shedding of different
membrane
proteins(20, 23, 24, 25, 32) .
However, the phenotype of our two independent mutant cell lines
indicate that one component of the shedding machinery that lies
downstream of protein kinase C is required for shedding of all proteins
tested (this report and (29) ). Moreover, the present results
show that in our mutant selection experiments we repeatedly isolated
independent mutant cell lines that have the same recessive shedding
phenotype and belong to the same complementation group. These
observations strongly suggest that shedding of membrane protein
ectodomains is mediated by a common system with an essential component
encoded by a nonredundant gene in CHO cells. The substrates of this
shedding system include TGF-,
APP, IL-6 R
, L-selectin,
and essentially all other cell surface proteins that are shed in
response to PMA.
Among the original lines of evidence suggesting
that shedding of different membrane protein ectodomains may be mediated
by different proteases is the observation that certain serine protease
inhibitors (3,4-DCI, TPCK, and DFP) can inhibit the release of newly
synthesized proTGF- (27) but are ineffective against
shedding of L-selectin (21) . The inhibitory effect on
TGF-
release was observed by adding these compounds to
metabolically labeled cells and measuring the conversion of newly
synthesized proTGF-
into TGF-
and the 15-kDa cell-associated
fragment. However, shedding of membrane protein ectodomains takes place
at or near the cell surface(10, 33) . In the present
studies, these protease inhibitors were ineffective against PMA-induced
cleavage of cell surface proTGF-
, L-selectin, IL-6 R
, and
APP. This result was obtained irrespective of whether shedding was
assayed by immunoprecipitating metabolically labeled cell surface
proteins and soluble ectodomains or by performing flow cytometry of
cell surface proTGF-
or L-selectin immunostaining. When added to
cells immediately after a metabolic pulse, 3,4-DCI, TPCK, and DFP did
prevent the release of various ectodomains. However, it is now clear
that this effect is due to inhibition of membrane protein transport to
the cell surface by these agents. Whether this is mediated by their
antiprotease activity remains to be determined. We conclude that
3,4-DCI, TPCK, and DFP are ineffective against the cell surface
shedding of proTGF-
, L-selectin, IL-6 R
, and
APP
ectodomains. Therefore, the previously noted differences in sensitivity
to these protease inhibitors no longer constitute evidence that
different proteases catalyze the shedding of these proteins.
Recently, several compounds with the characteristics of
metalloprotease inhibitors have been shown to block the shedding of
TNF-(23, 24, 25) and the 80-kDa
TNF-
receptor ectodomain(26) . These compounds are
derivatives of hydroxamic acid that are known to inhibit
metalloproteases by binding to their active site(34) . In the
present studies, we tested the effects of one such compound,
TAPI-2(26) , on the diverse group of membrane proteins whose
shedding is activated by PMA in CHO cells. TAPI-2 does not interfere
with the biosynthesis of proTGF-
(present work) or TNF-
receptor(26) . The results show that TAPI-2 inhibits basal as
well as PMA-activated shedding of TGF-
,
APP, L-selectin, and
IL-6 R
and is equally potent in each case. Furthermore, TAPI-2
also inhibits the shedding of all endogenous CHO membrane proteins that
undergo this process in response to PMA. The general metalloprotease
inhibitor 1,10-phenanthroline, which acts by chelating heavy metals, is
also an effective inhibitor of PMA-activated ectodomain cleavage,
whereas other ion chelators including EDTA and EGTA are ineffective. It
has been previously shown that the shedding of CD43, CD44, and CD16 is
inhibited by 1,10-phenanthroline but not by EDTA or EGTA(21) .
This result might be explained by a higher affinity of
1,10-phenanthroline for the zinc ion found in the active site of most
metalloproteases. In our experiments, the effect of 1,10-phenanthroline
could be prevented by the addition of Zn
, indicating
that the inhibitory effect of 1,10-phenanthroline is due to its
metal-chelating activity. Phosphoramidon, which inhibits certain
metalloproteases by binding to their active site, is without effect on
the shedding of all the membrane proteins tested here. The evidence
argues that TAPI-2 and 1,10-phenanthroline specifically inhibit a
regulated shedding activity likely to be catalyzed by one or several
metalloproteases that release the ectodomains of a diverse group of
unrelated cell surface proteins.
The present results provide strong evidence for the existence of a single general mechanism for membrane protein ectodomain shedding. The evidence at hand is consistent with two alternative models. In one model, a family of metalloproteases with different substrate specificity would catalyze the shedding of a diverse group of membrane proteins in the same cell; all these proteases would depend on a shared regulatory component that can be disrupted by mutations in a single gene. Because extracellular proteases are often activated by proteolysis, one possibility is that this gene encodes a protease that activates all the others. In the second model, the target of the mutations would be the shedding protease itself. This model would imply the existence of a protease with unusually broad substrate specificity, perhaps one that would recognize a certain secondary structure (or a lack thereof) as the cleavage site. The mutant cell lines described here may help identify these components of the shedding machinery.