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Endocrinology Vol. 141, No. 12 4342-4348
Copyright © 2000 by The Endocrine Society

ARTICLES

Tumor Necrosis Factor-{alpha} 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

Yue Zhang, Jing Jiang, Roy A. Black, Gerhard Baumann and Stuart J. Frank

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The GH binding protein (GHBP), which exists in many vertebrates, is a circulating high affinity binding protein corresponding to the extracellular domain of the GH receptor (GHR). In humans, rabbits, and several other species, the GHBP is generated by proteolysis of the GHR and shedding of its extracellular domain. We previously showed that GHBP shedding is inducible by the phorbol ester phorbol 12-myristate,13-acetate (PMA) and inhibited by the metalloprotease inhibitor, Immunex Corp. Compound 3 (IC3). The metzincin metalloprotease, tumor necrosis factor-{alpha} (TNF-{alpha})-converting enzyme (TACE), catalyzes the shedding of TNF-{alpha} 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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GH binding protein (GHBP) corresponds to the circulating extracellular domain of the GH receptor (GHR) (1). It is found in the circulation of many vertebrates and has been evolutionarily conserved from at least the bony fish through humans (2, 3, 4). Depending on the species, different mechanisms are used to generate GHBP. In rodents, the GHBP is secreted as an alternative splicing product of the GHR gene that contains a short hydrophilic sequence in place of the transmembrane and intracellular domains (5, 6). This hydrophilic tail is encoded by a special exon (exon 8A) interposed between exons 7 (extracellular) and 8 (transmembrane domain) (7, 8). In many other species, including rabbits and humans, exon 8A is missing, and the GHBP is generated by proteolytic cleavage from the membrane-bound GHR, a process also known as shedding. Although the ultimate physiological role of the GHBP is not known, its evolutionary conservation and the employment of two different mechanisms for its production suggest that it subserves important function(s).

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)-{alpha}-converting enzyme (TACE or ADAM-17), which has been cloned and extensively characterized (20, 21). In addition to its ability to liberate TNF-{alpha} 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{Delta}Zn/{Delta}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).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant hGH was kindly provided by Eli Lilly & Co. (Indianapolis, IN). PMA, Hygromycin B, and other routine reagents were purchased from Sigma (St. Louis, MO) unless otherwise noted. Restriction endonucleases were obtained from New England Biolabs, Inc. (Beverly, MA). Immunex Corp. Compound 3 (IC3), supplied by Immunex Corp., is identical to Compound 2 (23), except that the napthylalanine side chain is replaced by a tert butyl group.

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{alpha}.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{alpha} 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. 4CGo) 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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  		Figure 4. PMA induces loss of immunologically detectable rbGHRs in Null/R+T cells. A and B, Null/R and Null/R+T cells (one-half 90% confluent 100 x 20 mm dish per sample) were treated with DMSO vehicle (0 min) or PMA (1 µg/ml) at 37 C for 30 or 60 min before solubilization in detergent-containing lysis buffer. Extracted proteins were resolved by SDS-PAGE and immunoblotted with anti-GHR (A) or anti-TACE (B). As in Fig. 1Go, the positions of the fully glycosylated and incompletely glycosylated forms of the rbGHR are indicated in A and the positions of the internally deleted endogenous Null cell TACE and transfected full-length murine TACE are indicated in B. C, Anti-GHR immunoblots from four experiments such as that in A were densitometrically analyzed. In each case, the rbGHR abundance present after the indicated duration of PMA treatment in each cell was compared with that present without PMA treatment (control, considered 100%). P < 0.02 for comparison of Null/R vs. Null/R+T at both 30 and 60 min of PMA treatment. D, PMA induces activation of MAP kinases in both Null/R and Null/R+T cells. Serum-starved cells (one-half 90% confluent 100 x 20 mm dish per sample) were treated with PMA for 0, 5, or 60 min, as indicated, before solubilization in 1%SDS sample buffer. Extracted proteins were resolved by SDS-PAGE and immunoblotted with anti-phosphoMAPK. Positions of activated ERK1 (upper arrow) and ERK2 (lower arrow) are indicated.

 
Statistical analysis
Statistical analysis was performed by unpaired t test or ANOVA, followed by Newman-Keuls test, as appropriate. A P value of <0.05 was accepted as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GHR expression in TACE Null cells
Null cells harbor a TACE molecule in which an exon encoding 51 residues of the conserved zinc binding domain was deleted by homologous recombination (22). They are thus unable to mediate significant shedding of TNF-{alpha}, 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. 1AGo, 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. 1AGo, 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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  		Figure 1. Expression of rabbit GHR (R) and murine TACE (T) in Null/R and Null/R+T cells. A and B, Null, Null/R, and Null/R+T cells (one-half 90% confluent 100 x 20 mm dish per sample) were solubilized in 1% Triton X-100-containing lysis buffer. Extracted proteins were resolved by SDS-PAGE and immunoblotted with anti-GHR (A) or anti-TACE (B). The positions of the fully glycosylated rbGHR (bracket) and an incompletely glycosylated rbGHR form (arrow) are indicated in A. The positions of the internally deleted catalytically inactive endogenous Null cell TACE (lower arrow) and the transfected full-length wild-type murine TACE (upper arrow) are indicated in B.

 
TACE reconstitution of Null cells
To characterize the expression of TACE in the two cells, we generated a polyclonal rabbit antiserum, anti-TACE, directed against a bacterially expressed GST fusion protein incorporating the entire cytoplasmic domain (residues 695–827) of murine TACE (as in Materials and Methods). This antibody detected human and rodent TACE in a variety of cell lines by both immunoprecipitation and immunoblotting (data not shown). As seen in Fig. 1BGo, anti-TACE immunoblotting of detergent extracts of Null/R and Null/R+T detected forms of TACE in both cells. Null/R cells (Fig. 1BGo, 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. 1BGo, 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. 1Go 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. 2AGo, 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. 2AGo, lanes 2 vs. 6 and 4 vs. 8). The similar degree of GH-induced GHR disulfide linkage in the two cell types (Fig. 2AGo, 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. 2BGo, 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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  		Figure 2. GH causes rbGHR disulfide linkage and JAK2 tyrosine phosphorylation in both Null/R and Null/R+T cells. Serum-starved cells [one-half 90% confluent 100 x 20 mm dish per sample for detergent extracts (A) and one 90% confluent 100 x 20 mm dish per sample for immunoprecipitations (B)] were treated without (-, lanes 1, 3, 5, 7) or with (+, lanes 2, 4, 6, 8) GH for 10 min. Detergent-extracted proteins were directly resolved by SDS-PAGE under nonreducing (A, lanes 1–4) or reducing (A, lanes 5–8) conditions or were immunoprecipitated with anti-JAK2 and resolved by reducing SDS-PAGE (B). Resolved proteins were immunoblotted with anti-GHR (A) or antiphosphotyrosine antibody (B). The positions in A of the nondisulfide-linked (lower bracket) and disulfide-linked (upper bracket) rbGHR are indicated, as is the position of tyrosine phosphorylated JAK2 (P-JAK2, arrow in B).

 
GHBP shedding in TACE Null and TACE reconstituted cells
We have previously shown that the phorbol ester, PMA, promoted the proteolytic shedding of the GHBP from the transmembrane GHR in IM-9 cells, murine 3T3-F442A cells and CHO cells stably expressing the rbGHR (19 and R. Guan, et al., manuscript in preparation). To test whether TACE mediates this PMA-induced GHBP shedding, we measured the GHBP content in the supernatants of PMA-treated Null/R and Null/R+T cells (Fig. 3Go). 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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  		Figure 3. PMA-induced shedding of GHBP in Null/R and Null/R+T cells and its inhibition by the metalloprotease inhibitor, IC3. Serum-starved cells (one 90% confluent 100 x 20 mm dish in 4 ml serum-free medium per sample) were treated with DMSO vehicle or PMA (1 µg/ml) at 37 C for 30 min and 60 min (left panel), or treated with PMA for 30 min in the presence of IC3 (50 µM), which was added 15 min before PMA treatment (right panel). GHBP activity was measured in conditioned medium as described in Materials and Methods. GHBP was not detectable in DMSO-treated samples of both cell types. Data are plotted as mean ± SEM for four determinations. P < 0.001 for comparison of PMA-treated Null/R with Null/R+T in the absence of IC3. P < 0.05 for comparison of Null/R+T treated with PMA vs. PMA in the presence of IC3.

 
Down-regulation of GHRs by PMA in TACE reconstituted cells
By virtue of release of the GHR extracellular domain, PMA-induced GHBP shedding is expected to decrease the number of full-length GHRs remaining after phorbol ester treatment. We tested for this effect by exposing Null/R and Null/R+T cells to PMA (1 µg/ml) for 30 or 60 min before cell harvesting, detergent solubilization of cellular proteins, and assessment of remaining GHR abundance by anti-GHR immunoblotting (Fig. 4AGo). 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. 4BGo). 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 4CGo 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. 4DGo, 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.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These findings clearly indicate that Null/R cells are unable to generate detectable amounts of GHBP from the rbGHR in response to PMA. The rbGHR is particularly prone to proteolysis and large amounts of GHBP are readily observed in a variety of systems (39, 40, 41, 42).1 Thus, the absence of detectable GHBP in the media from Null/R cells is particularly striking. This alone is substantial evidence that TACE is critical for the shedding process. When cells were reconstituted with TACE via transfection, their ability of generate GHBP was restored, further corroborating the importance of TACE in GHR proteolysis. Finally, treatment with IC3 inhibited GHBP production in TACE reconstituted cells, similar to its inhibitory action in IM-9 cells which endogenously express the human GHR.

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.


   Acknowledgments
 
The authors appreciate helpful conversations with Drs. J. Kudlow, A. Paterson, E. Chin, R. Guan, L. Liang, S.-O. Kim, and J. Messina. The authors thank Drs. P. Reddy and D. Cerretti for supplying the TACE Null cells and TACE cDNA.


   Footnotes
 
1 This work was supported by a VA Merit Review award (to S.J.F.), grants from the National Science Foundation and the Northwestern Memorial Foundation (G.B.), and in part by NIH Grant DK-46395 (to S.J.F.). Parts of this work were presented at the 82nd annual meeting of The Endocrine Society in Toronto, Ontario, Canada, 2000. Back

2 Guan, R., Y. Zhang, J. Jiang, C. A. Baumann, R. A. Black, G. Baumann, and S. J. Frank, manuscript submitted. Back

Received July 20, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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