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Converting Enzyme (TACE) Is a Growth Hormone Binding Protein (GHBP) Sheddase: The Metalloprotease TACE/ADAM-17 Is Critical for (PMA-Induced) GH Receptor Proteolysis and GHBP Generation1
Department of Cell Biology (Y.Z., S.J.F.), University of Alabama at Birmingham, Birmingham, Alabama 35294; Department of Medicine (J.J., S.J.F.), Division of Endocrinology and Metabolism, University of Alabama at Birmingham, Birmingham, Alabama 35294; Immunex Corp. (R.A.B.), Seattle, Washington 98101; Center for Endocrinology (G.B.), Metabolism, and Molecular Medicine, Department of Medicine, Northwestern University Medical School, Chicago, Illinois 60611; and Veterans Affairs Medical Center (S.J.F.), Birmingham, Alabama 35294
Address all correspondence and requests for reprints to: Stuart J. Frank, University of Alabama at Birmingham, Room 756, DREB, 1808 7th Avenue South, Birmingham, Alabama 35294. E-mail: frank{at}endo.dom.uab.edu
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Abstract |
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(TNF-)-converting enzyme
(TACE), catalyzes the shedding of TNF- from its transmembrane
precursor, a process that is also inhibitable by IC3. TACE may hence be
a candidate for GHBP sheddase. In this study, we reconstitute
fibroblasts derived from a TACE knockout mouse (Null cells) with either
the rabbit (rb) GHR alone (Null/R) or rbGHR plus murine TACE
(Null/R+T). Although GHR in both cells was expressed at similar
abundance, dimerized normally and caused JAK2 activation in response to
GH independent of TACE expression, PMA was unable to generate GHBP from
Null/R cells. In contrast, PMA caused ample GHBP generation from TACE
reconstituted (Null/R+T) cells, and this GHBP shedding was
substantially inhibited by IC3 pretreatment. Corresponding to the
induced shedding of GHBP from Null/R+T cells, PMA treatment caused a
significant loss of immunoblottable GHR in Null/R+T, but not in Null/R
cells. We conclude that TACE is an enzyme required for PMA-induced GHBP
shedding and that PMA-induced down-regulation of GHR abundance may in
significant measure be attributable to TACE-mediated GHR proteolysis. |
Introduction |
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The GHBP is one particularly well-studied example of a large family of soluble, circulating ectodomains of cytokine receptors, many of which arise by similar mechanisms (9, 10). Thus, its generation and action can serve as a paradigm for a number of other binding protein/receptor pairs. Biological actions attributed to the GHBP are diverse. GHBP in plasma, through complex formation with GH, creates a circulating GH reservoir (11), protects GH from degradation and excretion, prolongs its half-life, and may enhance its bioactivity in vivo through these mechanisms (12, 13). GHBP may also act as a modulator/inhibitor of GH action at the tissue level by competing with GHRs for ligand (14) and by forming unproductive GHR-GHBP heterodimers that are unable to signal. In that capacity, the GHBP acts in a dominant negative manner. Additionally, the GHBP plasma level is thought to reflect GHR abundance, particularly in liver, as suggested by several indirect observations as well as by direct evidence (2, 3, 15). Finally, generation of GHBP from the GHR by proteolysis may be one mechanism for down-regulating GHR abundance.
Very little is known about the regulation of GHBP generation, the protease involved in shedding, the cellular site, or the tissues where proteolysis takes place. The fact that a naturally occurring, truncated GHR variant with a prolonged residence time at the cell surface is particularly efficient in generating GHBP suggests that proteolysis occurs at the cell surface (16, 17). Shedding can be promoted by sulfhydryl-inactivating agents, such as N-ethyl-maleimide (NEM) (18), but it is not clear whether this is fully representative of physiological shedding. Recent evidence from our laboratories has shown that phorbol 12-myristate,13-acetate (PMA) promotes GHBP shedding in human lymphoblasts (IM-9 cells) and other cells transfected with the GHR, implicating a protein kinase C pathway in this process (19).2 Furthermore, treatment with the metalloprotease inhibitor IC3 (Immunex Corp. compound 3) inhibits both PMAinduced and constitutive GHBP shedding in IM-9 cells (19). These findings implicated a metalloprotease of the metzincin family as a GHBP sheddase but fall short of positively identifying which protease is involved.
One of the metzincins known to be sensitive to IC3 is tumor necrosis
factor (TNF)--converting enzyme (TACE or ADAM-17), which has been
cloned and extensively characterized (20, 21). In addition
to its ability to liberate TNF- from its transmembrane precursor,
TACE is widely expressed (including in liver) and has also been shown
to catalyze the proteolytic shedding of several other cell surface
molecules in response to phorbol ester stimulation
(20, 21, 22). TACE was therefore considered to be a candidate
for the GHBP sheddase. To more directly address this possibility, we
used a genetic approach whereby fibroblasts derived from a TACE
knockout mouse
(TACE
Zn/Zn
EC-2 cells, referred to herein as Null cells) were transfected with the
rabbit (rb)GHR and examined for their ability to generate GHBP
spontaneously and after treatment with PMA. Null cells cotransfected
with rbGHR and murine TACE were similarly examined (TACE
reconstitution).
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Materials and Methods |
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Cells, cell culture, and transfections
TACE null cells (24) (referred to herein as Null
cells) were maintained in DMEM/F12 50/50 mixture (1 g/liter glucose)
(Cellgro, Inc.) supplemented with 5% FBS (Biofluids,
Rockville, MD) and 50 mg/ml gentamicin sulfate, 100 U/ml penicillin,
and 100 mg/ml streptomycin (all Biofluids). Stable
transfection of Null cells was achieved by introducing either pSX rbGHR
(25, 26) alone (20 µg) or pSX rbGHR and pXS mTACE (10
µg of each) (in 3 ml of DMEM medium in 60 x 15 mm dishes), each
along with 1 µg of pSP65-SR.2-HAtag-HygroHA-Hygro (empty vector
carrying the Hygromycin resistance marker, kindly provided by Dr. M.
Streuli, Dana-Farber Cancer Institute, Boston, MA), using Lipofectin
Plus (Life Technologies, Inc.) according to the
manufacturer’s suggestions. Transfected cells were grown in complete
DMEM/F12 50/50 mixture growth medium for 48 h. After dilution,
clones were negatively selected in medium supplemented with 300 µg/ml
Hygromycin B (Mediatech, Inc., Herndon, VA) and
screened for GHR or TACE expression by anti-GHR or anti-TACE
immunoblotting of detergent extracts, as detailed in text and figure
legends.
Antibodies
The 4G10 monoclonal antiphosphotyrosine (APT) antibody,
anti-MAPK affinity-purified rabbit antibody [directed at residues
333–367 of rat ERK1; recognizes both ERK1 and ERK2 (Upstate Biotechnology, Inc., Lake Placid, NY)] and anti-Phospho-MAPK
affinity-purified rabbit antibody (recognizing the dually
phosphorylated Thr183 and Tyr185 residues that correspond to the active
forms of ERK1 and ERK2) (Promega Corp., Madison, WI) were
all purchased commercially.
The rabbit polyclonal sera, anti-GHRcyt-AL37 (27) (referred to herein as anti-GHR) and anti-JAK2AL33 (27) (referred to herein as anti-JAK2), have been described. The rabbit polyclonal antibody, anti-TACEcyt-AL45 (referred to as anti-TACE), was raised against a bacterially expressed GST fusion protein incorporating the entire cytoplasmic domain (residues 695–827) of murine TACE (28). Construction of the plasmid encoding this GST fusion protein is described below.
Plasmid construction
The pSX plasmid (a gift of Dr. J. Bonifacino, NIH and Dr.
K. Arai, DNAX), which drives eukaryotic protein expression from
the SR promoter, has been described (29). Preparation
of the rbGHR complementary DNA (cDNA) (a gift of Dr. W. Wood,
Genentech, Inc.) and its ligation into pSX have been
described (25). The murine TACE cDNA (28) was
subcloned into the XhoI/BglII site of the pXS
vector (pSX vector with the polylinker in reverse orientation). cDNA
encoding GST fusion protein incorporating the entire cytoplasmic domain
was generated by ligating the PCR fragment corresponding to amino acid
695–827 of murine TACE into the BamHI/EcoRI site
of the pGEX-2TRS vector (30). This GST fusion protein was
expressed in Escherichia coli and purified as described
previously (25, 31, 32).
Inhibitor pretreatment, cell stimulation, protein extraction,
electrophoresis, and immunoblotting
Serum starvation of Null transfectants was accomplished by
substitution of 0.5% BSA for serum in the culture medium for 16–20 h
before experiments. IC3 (50 µM) or dimethyl sulfoxide
(DMSO) (as a vehicle control) was incubated with serum-starved
cells for 15 min before treatment with PMA (1 µg/ml, unless otherwise
noted), or DMSO. Both IC3 and PMA were diluted from DMSO-dissolved
stock solutions (IC3, 10 mM, PMA, 0.2 mg/ml). Pretreatment
and stimulation were carried out at 37 C in serum
free medium. hGH was used at a final concentration of 500 ng/ml.
Details of the treatment protocol have been described (19, 26). Briefly, stimulations were performed in serum starvation
medium and terminated by washing the cells once with ice-cold PBS and
then harvesting by scraping in ice-cold PBS in the presence of 0.4
mM sodium orthovanadate (PBS-vanadate). Pelleted cells were
collected by brief centrifugation and solubilized for 15 min at 4
C in fusion lysis buffer (19, 26). After centrifugation at 15,000x g for 15 min at 4
C, the detergent extracts were either subjected
to immunoprecipitation or were directly electrophoresed under
reducing (or, if indicated, nonreducing) conditions.
Anti-JAK2 immunoprecipitation was performed as previously described (26). Protein-A Sepharose (Amersham Pharmacia Biotech AB, Uppsala, Sweden) was used to adsorb immune complexes and, after extensive washing with lysis buffer, SDS sample buffer eluates were resolved by SDS-PAGE and immunoblotted. Resolution of proteins by SDS-PAGE, Western transfer of proteins, and blocking of Hybond-ECL (Amersham Pharmacia Biotech) with 2% BSA were performed as previously described (26, 33). Immunoblotting with antibodies, anti-GHR (1:2000), anti-JAK2 (1:2000), anti-MAPK (1:1000), anti-Phospho-MAPK (1:5000), anti-TACE (1:1000), and 4G10 (1:2500), with HRP-conjugated antirabbit or antimouse secondary antibodies (1:2000) and ECL detection reagents (all from Amersham Pharmacia Biotech) and stripping and reprobing of blots were accomplished according to manufacturer’s suggestions.
GHBP assay
GHBP activity was measured in conditioned media by a standard GH
binding assay, as previously reported (19, 34).
Conditioned medium (1 ml) from cells treated as indicated was incubated
with freshly labeled 125I-hGH (
0.5 ng) for 45
min at 37 C. Bound GH was then immediately
separated from free GH by gel chromatography on a Sephadex G-100 column
at 4 C. The fraction of GH bound was determined
by peak integration.
Densitometric analysis
Densitometric quantitation of ECL immunoblots was performed
using a video camera and the Image 1.49 program (developed by W.
S. Rasband, Research Services Branch, NIMH, Bethesda, MD). The fraction
of full-length GHRs remaining in extracts from PMA-treated cells (see
Fig. 4C
) was estimated by measuring the intensity of the GHR signal
relative to that present in unstimulated cell extracts from the same
experiment. As indicated when graphically shown, pooled data from
several experiments are displayed as the mean ±
SEM.
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Results |
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, either of the two TNF receptors, or
type II IL-1 receptor from their respective transmembrane precursors
(24). In our initial characterization of Null cells,
endogenous murine GHR was not detected by anti-GHR immunoblotting (Fig. 1A, lane 1). We therefore stably
expressed the full-length rabbit (rb) GHR alone (R) or coexpressed
rbGHR and wild-type murine TACE (R+T) by transfection of the respective
cDNA expression vectors into Null cells. Two resulting transfected
clones, Null/R and Null/R+T, were selected on the basis of similar
expression of the rbGHR and used for the majority of the studies that
followed. (We note, however, that all results obtained with these two
clones were verified using other similarly matched clone pairs.) As
seen in Fig. 1A, anti-GHR immunoblotting of SDS-PAGE-resolved detergent
extracts of Null/R and Null/R+T (lanes 2 and 3, respectively) showed
the rbGHR in each cell type to be present at similar levels as both a
diffuse band of roughly 120–140 kDa (bracket) and a more
discrete and somewhat faster migrating band of approximately 100 kDa
(arrow). These findings are consistent with previous studies
in which expression of the rbGHR yielded both a fully glycosylated
slower migrating form and a more rapidly migrating incompletely
glycosylated form (25, 35).
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, anti-TACE
immunoblotting of detergent extracts of Null/R and Null/R+T detected
forms of TACE in both cells. Null/R cells (Fig. 1B, lane 2) and Null
cells (lane 1) expressed only a TACE form migrating in SDS-PAGE at
roughly 110 kDa, representing exclusively the internally deleted TACE
in those cells. In contrast, Null/R+T cells (Fig. 1B, lane 3) expressed
in addition a more abundant anti-TACE-reactive species at 120 kDa,
which was also detected with a previously characterized
(20) anti-TACE extracellular domain antibody (data not
shown). This band, present in Null/R+T, but not Null/R, represents the
transfected full-length murine TACE molecule. The data in Fig. 1
indicated that Null/R and Null/R+T cells were reconstituted with
similar levels of rbGHR and that wild-type TACE was expressed only in
Null/R+T cells.
Functionality of transfected GHRs
We next confirmed that rbGHRs in Null/R and Null/R+T cells, in
addition to being expressed at comparable levels, were also similar
with regard to early GH-induced events. An important consequence of GH
binding is the dimerization of the GHR with formation of a tripartite
GH-GHR complex with a 1:2 stoichiometry (36). Using cells
expressing human, rabbit, and murine GHRs, we have previously described
that GH-induced receptor dimers undergo disulfide linkage and are
detectable as high Mr forms under nonreducing
conditions (26, 30); GH-induced disulfide-linked dimers of
heterologously expressed rbGHR exhibit a particularly indistinct
appearance (26), perhaps due to a more pronounced
variability in glycosylation in such transfected receptors. As shown in
Fig. 2A, GH treatment of both Null/R and
Null/R+T cells caused appearance of the high-Mr
GHR form when extracts were resolved under nonreducing (but not
reducing) conditions (Fig. 2A, lanes 2 vs. 6 and 4
vs. 8). The similar degree of GH-induced GHR disulfide
linkage in the two cell types (Fig. 2A, lanes 2 vs. 4)
strongly suggests that GH-induced receptor dimerization proceeded
independent of the expression of TACE. Similarly, GH-induced activation
of the GHR-associated cytoplasmic tyrosine kinase, JAK2, is an early
hormone-induced event that is required for GHR signaling (37, 38). As assessed by antiphosphotyrosine immunoblotting of
anti-JAK2 immunoprecipitates, GH also induced a similar degree of JAK2
activation in Null/R and Null/R+T cells (Fig. 2B, lanes 2 and 4). These
results indicate that the rbGHRs responded to GH with manifestations of
early activation without regard to TACE expression. By extension, the
receptors in each cell were apparently displayed at the cell surface
normally so as to be available to respond to GH.
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). The two cell
types were plated at equivalent density and treated with PMA under
serum-free conditions, after which the conditioned media were cleared
of cellular elements and assessed for specific binding of
125I-hGH. No GH binding activity (corresponding
to <10 pM GHBP) was detected in supernatants of
Null/R cells exposed to PMA for either 30 or 60 min. Dramatically
different results were obtained with Null/R+T cells. PMA promoted the
generation of ample GHBP in the conditioned media of the
TACE-containing cells after either 30 or 60 min. Notably, addition of
the metalloprotease inhibitor, IC3, during the 30-min period of PMA
exposure reduced by 80% the PMA-induced GHBP production by Null/R+T
cells, further corroborating that the metalloprotease activity of TACE
was indeed mediating GHBP shedding in these cells.
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). PMA promoted marked GHR loss
in Null/R+T cells after either 30 or 60 min (lanes 4–6). By contrast,
Null/R cells showed no loss of GHR abundance over the 60 min PMA
incubation (lanes 1–3). As a control for protein loading in each lane,
the same blot was stripped and reprobed with anti-TACE (Fig. 4B). PMA
caused no change in the abundance of either the transfected wild-type
TACE or the endogenous internally deleted TACE during the treatment
period studied. Figure 4C summarizes the differential effect of PMA on
GHR abundance in Null/R vs. Null/R+T cells, as measured
densitometrically in several experiments. Compared with the receptor
abundance in the absence of PMA, we observed that 68.4 ± 5.2%
and 66.3 ± 2.1% (mean ± SEM) of
immunoblottable rbGHR remained after 30 and 60 min of PMA treatment,
respectively, in Null/R+T cells. This time course is comparable to the
kinetics of PMA-induced GHR loss that we observed in IM-9 cells
(19) and in mouse fibroblasts and other rbGHR
transfectants.1 In contrast, Null/R cells exhibited
a modest increase in GHRs 30 min after PMA treatment, which declined
toward baseline at 60 min. Thus, the selective PMA-induced decrease in
GHR in Null/R+T indicates that, in addition to being responsible for
GHBP generation, TACE substantially contributes to PMA-induced GHR
down-regulation.
Maintained PMA responsiveness in TACE Null cells
In principle, the inability of PMA to induce GHBP shedding in
Null/R cells could be accounted for by a coincidental or TACE
deficiency-related defect in PMA responsiveness in those cells. As
shown in Fig. 4D, however, there was no difference between Null/R and
Null/R+T cells in the ability of PMA to cause activation of the MAP
kinases, ERK1 and ERK2, as assessed by immunoblotting with a
phosphorylation state specific anti-active MAPK antibody (lanes 1–3
vs. 4–6). Thus, the inability of the phorbol ester to cause
GHBP shedding in Null/R cells was not due to an inability of the cells
to respond to PMA. This is particularly notable in light of other
preliminary data1 indicating that the ability of PMA to
activate MAP kinases correlates to its induction of GHR
proteolysis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because of the specific nature of TACE deletion and restitution in this study design, it can be stated that TACE is necessary and probably sufficient for GHR proteolysis and GHBP shedding. Inhibition by IC3 provides corroborative evidence. It appears that the function of TACE with respect to GHR cleavage cannot be duplicated by other proteases, at least in these fibroblasts. What remains unanswered is whether TACE is the final enzyme in the pathway to GHBP shedding, or whether it acts upstream from the final step in a proteolytic cascade leading to GHR cleavage. To date, no other enzyme in this pathway has been identified. TACE is a transmembrane protease and thus in close proximity to the membrane-bound GHR, although the exact catalytic site/mechanism and substrate specificity remain to be determined. It is also not yet known how PMA (presumably via activation of PKC) activates the GHR proteolysis/GHBP shedding process. Interestingly, our preliminary data1 indicates that PMA-induced GHR proteolysis in murine fibroblasts and CHO cells stably expressing rbGHR is partially inhibited by MEK1 inhibitors. This suggests that the cleavage process may be influenced by MAP kinase activation. Further studies will be required to determine whether TACE is itself a substrate for PKC, MEK1, or MAP kinases.
Our finding that PMA-induced down-regulation of GHR abundance may in large measure be attributable to TACE activity is particularly interesting and potentially physiologically relevant. PKC-dependent loss of cell surface GHR has been observed in several systems and attributed to a variety of potential mechanisms including receptor internalization, phosphorylation-dependent degradation, or intracellular redistribution (43, 44, 45, 46). Our current results suggest that a component of GHR down-regulation is mechanistically linked to GHBP shedding, and that GHR inactivation in situ by TACE proteolysis is one mechanism for decreasing GH binding to cells (43, 44). These results are also in concert with the finding that TACE activity contributes to the PMA-induced down-regulation of other TACE substrates (24). It remains to be determined whether the contribution of TACE activity to down-regulation of GHR abundance is substantial in all cells which are targets for GH action.
TACE can mediate the regulated proteolysis of various cell surface molecules with shedding of the extracellular domains of these proteins (20, 21, 22, 24). Based on our current data, the GHR can also be considered a TACE substrate. We do not yet know the specific residues in the GHR at which TACE-mediated cleavage occurs; by analogy to other TACE substrates, it is likely that the extracellular catalytic domain of TACE is cleaving the GHR near its extracellular/transmembrane junction. This would also be consistent with the overall structure of the GHBP, although the exact carboxy terminus of the GHBP is not known (47). Interestingly, there is no clear consensus sequence for TACE cleavage even among the substrates for which the cleavage sites are known, suggesting that a three-dimensional conformation, rather than a linear sequence, is the target of TACE activity. In addition, the regions of TACE (other than the catalytic domain) required for cleavage of its substrates can vary among the different TACE targets (24). Future studies of GHR cleavage and GHBP shedding will address these structure-function issues as they pertain to the TACE-GHR interaction. Our TACE reconstitution system will also be useful in future studies aimed at comparing the relative susceptibilities of rabbit and rodent GHRs to PMA-induced proteolysis, although previous reports suggest markedly diminished GHBP shedding when rodent vs. rabbit receptors were expressed in COS and CHO cells (39, 48).
In summary, we established the identity of a metalloprotease of the ADAM family—TACE—that is crucial for the proteolytic shedding of GHBP from the rabbit GHR. TACE may account for a substantial degree of PMA-induced down-regulation of GHR abundance. The identification of TACE as the critical enzyme involved in GHBP generation will permit scrutiny of the mechanism of GHR cleavage as well as provide new insights into the regulation of GHR cleavage and GHBP production, and hence modulation of GH action by these processes.
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Acknowledgments |
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Footnotes |
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2 Guan, R., Y. Zhang, J. Jiang, C. A. Baumann, R.
A. Black, G. Baumann, and S. J. Frank, manuscript submitted.
Received July 20, 2000.
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from cells. Nature 385:729–733[CrossRef][Medline]
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Nature 385:733–736[CrossRef][Medline]
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X. Wang, K. He, M. Gerhart, Y. Huang, J. Jiang, R. J. Paxton, S. Yang, C. Lu, R. K. Menon, R. A. Black, et al. Metalloprotease-mediated GH Receptor Proteolysis and GHBP Shedding. DETERMINATION OF EXTRACELLULAR DOMAIN STEM REGION CLEAVAGE SITE J. Biol. Chem., December 20, 2002; 277(52): 50510 - 50519. [Abstract] [Full Text] [PDF]
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H. Ge, L. Huang, T. Pourbahrami, and C. Li Generation of Soluble Leptin Receptor by Ectodomain Shedding of Membrane-spanning Receptors in Vitro and in Vivo J. Biol. Chem., November 22, 2002; 277(48): 45898 - 45903. [Abstract] [Full Text] [PDF]
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P. van Kerkhof, M. Smeets, and G. J. Strous The Ubiquitin-Proteasome Pathway Regulates the Availability of the GH Receptor Endocrinology, April 1, 2002; 143(4): 1243 - 1252. [Abstract] [Full Text] [PDF]
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 L. Guo, J. R. Eisenman, R. M. Mahimkar, J. J. Peschon, R. J. Paxton, R. A. Black, and R. S. Johnson A Proteomic Approach for the Identification of Cell-surface Proteins Shed by Metalloproteases Mol. Cell. Proteomics, January 1, 2002; 1(1): 30 - 36. [Abstract] [Full Text] [PDF]
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M. Maamra, M. Bidlingmaier, M.-C. Postel-Vinay, Z. Wu, C. J. Strasburger, and R. J. M. Ross Generation of Human Soluble Leptin Receptor by Proteolytic Cleavage of Membrane-Anchored Receptors Endocrinology, October 1, 2001; 142(10): 4389 - 4393. [Abstract] [Full Text] [PDF]
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Y. Zhang, R. Guan, J. Jiang, J. J. Kopchick, R. A. Black, G. Baumann, and S. J. Frank Growth Hormone (GH)-induced Dimerization Inhibits Phorbol Ester-stimulated GH Receptor Proteolysis J. Biol. Chem., June 29, 2001; 276(27): 24565 - 24573. [Abstract] [Full Text] [PDF]
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B. Vincent, E. Paitel, P. Saftig, Y. Frobert, D. Hartmann, B. De Strooper, J. Grassi, E. Lopez-Perez, and F. Checler The Disintegrins ADAM10 and TACE Contribute to the Constitutive and Phorbol Ester-regulated Normal Cleavage of the Cellular Prion Protein J. Biol. Chem., October 5, 2001; 276(41): 37743 - 37746. [Abstract] [Full Text] [PDF]
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