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Endocrinology Vol. 142, No. 3 1137-1147
Copyright © 2001 by The Endocrine Society

ARTICLES

Phorbol Ester- and Growth Factor-Induced Growth Hormone (GH) Receptor Proteolysis and GH-Binding Protein Shedding: Relationship to GH Receptor Down-Regulation1

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

Department of Medicine, Division of Endocrinology and Metabolism (J.J., S.J.F.), and Department of Cell Biology (R.G., Y.Z., S.J.F.), University of Alabama, and Veterans Affairs Medical Center (S.J.F.), Birmingham, Alabama 35294; Center for Endocrinology, Metabolism, and Molecular Medicine, Department of Medicine, Northwestern University Medical School (C.A.B., G.B.), Chicago, Illinois 60611; and Immunex Corp. (R.A.B.), Seattle, Washington 98101

Address all correspondence and requests for reprints to: Dr. Stuart J. Frank, University of Alabama, 1530 3rd Avenue South, BDB 731, Birmingham, Alabama 35294-0012. E-mail: frank{at}endo.dom.uab.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GH signals by interacting with GH receptor (GHR). A substantial fraction of circulating GH complexes with GH-binding protein (GHBP), which corresponds to the GHR extracellular domain. GHBP is generated by 1) alternative splicing of a common GHR precursor messenger RNA to encode secreted GHBP (the source of the vast majority of GHBP in rodents); and 2) proteolysis of the cell-associated GHR with shedding of GHBP (a mechanism operative in rabbits and humans). We previously observed that phorbol ester (PMA)-induced activation of protein kinase C (PKC) causes metalloprotease-mediated GHR proteolysis and GHBP shedding in human IM-9 lymphocytes. We now demonstrate that PMA-induced hydroxamate (IC3)-inhibitable GHR proteolysis and GHBP shedding were also detected in murine 3T3-F442A and 3T3-L1 preadipocytes and in Chinese hamster ovary (CHO) cells stably expressing rabbit GHR (rbGHR), although the degree of GHBP shedding was much smaller for murine GHR than for rabbit or human GHRs. PMA-induced GHR proteolysis in 3T3-F442A, 3T3-L1, and CHO-rbGHR cells was significantly reduced by pretreatment with mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 inhibitors, suggesting involvement of the mitogen-activated protein kinase pathway in regulating this PKC-dependent effect. In contrast, GHR proteolysis promoted by N-ethylmaleimide, although inhibited by IC3, was unaffected by inhibition of either PKC or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1. Thus, different pathways leading to metalloprotease-mediated receptor proteolysis are accessed by PMA vs. N-ethylmaleimide. To determine whether other, possibly more physiologically relevant, stimuli induce GHR proteolysis, we tested effects of platelet-derived growth factor (PDGF) and serum. Treatment of serum-deprived cells with PDGF (in 3T3-F442A cells) or serum (in 3T3-F442A and CHO-rbGHR cells) promoted GHR proteolysis, which was inhibited by IC3. Interestingly, PMA-, PDGF-, and serum-induced GHR proteolysis was associated with substantial decreases in GH-induced activation of Janus kinase-2, which were also prevented by IC3. These findings suggest that inducible metalloprotease-mediated GHR proteolysis constitutes an important mechanism of receptor down-regulation and modulation of GH signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH IS REQUIRED for normal postnatal growth and development and exerts significant metabolic effects in vertebrates (1). Like many important hormones, the actions of GH are highly regulated in several ways. Secretion of GH from the anterior pituitary gland (its major source) is tightly controlled by several mechanisms so as to provide appropriate levels of the circulating hormone. Although less well understood, regulation of the bioavailability of GH in the circulation is affected by the presence of the high affinity GH-binding protein (GHBP). GHBP is a circulating version of the extracellular domain of the cell surface GH receptor (GHR) and can be derived by two distinct mechanisms, alternative splicing of a common GHR precursor messenger RNA (mRNA) to yield a message encoding a secreted GHBP (believed to be the source of the vast majority, if not all, of serum GHBP in mice and rats) (2, 3, 4) and proteolysis of the full-length GHR (or truncated GHR forms) (5, 6) with shedding of the GHBP (the operative mechanism in rabbits and humans) (reviewed in Ref. 7). Roughly half of the circulating GH in the human is bound to GHBP (8), and GHBP levels vary in different physiological and pathophysiological states (7).

A further level of regulation of GH action is modulation of GHR availability at GH target tissues. Down-regulation of GHRs by GH is well described, although the mechanisms underlying it are incompletely understood (9). GHR levels and/or cell surface availability can also be regulated by hormones other than GH (10, 11). The proteolytic process leading to GHBP by shedding might also be expected to regulate GHR levels, even in cell types and species that do not generate the majority of their circulating GHBP by a proteolytic mechanism.

We recently described in human IM-9 cells a phorbol ester-induced protein kinase C (PKC)-dependent shedding of GHBP that is inhibited by compound 3 (IC3; Immunex Corp., Seattle, WA), a hydroxamate-based inhibitor of metzincin metalloproteases (12). This GHBP generation is accompanied by loss of cell-associated immunoreactive GHR and accumulation of a cell-associated GHR remnant protein detected with antibodies directed at the receptor’s cytoplasmic, but not extracellular, domain. Although it is PKC dependent, the signaling pathways that mediate such phorbol 12,13-myristate acetate (PMA)-induced GHR processing are as yet unknown. It is also not known whether other stimuli promote GHR proteolysis and if GHR proteolysis can lead to receptor down-regulation.

We now explore these processes further and demonstrate that PMA-induced IC3-inhibitable GHR proteolysis and GHBP shedding can also be detected in murine 3T3-F442A and 3T3-L1 preadipocytes that endogenously express GHRs and in Chinese hamster ovary (CHO) cells stably expressing the rabbit GHR (rbGHR), although the degree of GHBP shedding is much smaller for the murine GHR than for the rabbit or human GHRs. The PMA-induced GHR proteolysis in 3T3-F442A, 3T3-L1, and CHO-rbGHR cells is significantly reduced by pretreatment with inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK1), the upstream activator of mitogen-activated protein (MAP) kinases (MAPKs), suggesting involvement of the MAPK pathway in regulation of this PKC-dependent effect.

To determine whether other, possibly more physiologically relevant, stimuli induce GHR proteolysis, we also tested the effects of platelet-derived growth factor (PDGF) and serum on this process. Treatment of serum-deprived cells with PDGF (in 3T3-F442A cells) or serum (in 3T3-F442A and CHO-rbGHR cells) promotes GHR proteolysis, which is inhibited by IC3. PMA-, PDGF-, and serum-induced GHR processing is associated with substantial decreases in GH- induced activation of Janus kinase-2 (JAK2), which are also nearly completely prevented by IC3. These findings strongly suggest that inducible metalloprotease-mediated GHR proteolysis constitutes an important mechanism of receptor down-regulation and modulation of GH signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
PMA, N-ethylmaleimide (NEM), and hygromycin B were purchased from Sigma (St. Louis, MO), as were routine reagents unless otherwise noted. The PKC inhibitor, GF109203X (Calbiochem, San Diego, CA), and the MEK1 inhibitors, PD98059 (New England Biolabs, Inc., Beverly, MA) and U0126 (Promega Corp., Madison, WI), were purchased commercially. Recombinant human PDGF-BB was purchased from Intergen (Purchase, NY). Recombinant human GH (hGH) was provided by Eli Lilly & Co. (Indianapolis, IN). IC3, supplied by Immunex Corp., is identical to compound 2 (13), except that the napthylalanine side-chain is replaced by a tert-butyl group.

Cells and cell culture
3T3-F442A cells, kindly provided by Drs. H. Green (Harvard University, Boston, MA) and C. Carter-Su (University of Michigan, Ann Arbor, MI), and 3T3-L1 cells (a gift from Dr. R. Hardy, University of Alabama at Birmingham, Birmingham, AL) were cultured in DMEM (4.5 g/liter glucose; Cellgro, Inc.) supplemented with 10% calf serum (Biofluids, Rockville, MD) and 50 µg/ml gentamicin sulfate, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Biofluids). CHO cells (a gift from Dr. J. Kudlow, University of Alabama at Birmingham, Birmingham, AL) were maintained in DMEM (1 g/liter glucose; Cellgro, Mediatech, Herndon, VA) supplemented with 7% FBS (Biofluids) and 50 µg/ml gentamicin sulfate, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Biofluids). Stable transfection of CHO cells was achieved by introducing pSX rbGHR (14) (20 µg in 3 ml DMEM in 60 x 15-mm dishes) along with 1 µg pSP65-SR{alpha}.2-HAtag-Hygro (empty vector carrying the hygromycin resistance marker, provided by Dr. M. Streuli, Dana-Farber Cancer Institute, Boston, MA), using lipofectin (Life Technologies, Inc., Grand Island, NY), selected in 500 µg/ml hygromycin B, and characterized as reported previously (15).

Antibodies
The rabbit polyclonal sera, anti-GHRcyt-AL37 (directed at residues 271–620 of the hGHR (the entire cytoplasmic domain)), anti-GHRcyt (directed at residues 317–620 of hGHR), and anti-JAK2AL33 (directed at residues 746-1129 of murine JAK2), have been described, as has the procedure for affinity purification of anti-GHRcyt-AL37 (16, 17). Anti-MAPK affinity-purified rabbit antibody [directed at residues 333–367 of rat extracellular signal-regulated kinase 1 (ERK1); recognizes both ERK1 and ERK2], antiphosphotyrosine (APT) monoclonal antibody 4G10 (both from Upstate Biotechnology, Inc., Lake Placid, NY) and antiphospho-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.) were all purchased commercially.

Plasmid construction
The pSX plasmid (a gift from Dr. J. Bonifacino, NIH, and Dr. K. Arai, DNAX, Palo Alto, CA), which drives eukaryotic protein expression from the SR{alpha} promoter (composed of the simian virus 40 early promoter and the R-U5 segment of the human T cell lymphotrophic virus-1 long terminal repeat), has been described previously (18). Preparation of the rbGHR complementary DNA (a gift from W. Wood, Genentech, Inc., South San Francisco, CA) and its ligation into the pSX vector have been described previously (14, 19).

Inhibitor pretreatment, cell stimulation, protein extraction, electrophoresis, and immunoblotting
Serum starvation of 3T3-F442A cells and CHO transfectants was accomplished by substitution of 0.5% (wt/vol) BSA (fraction V, Roche Molecular Biochemicals, Indianapolis, IN) for serum in their respective culture media for 16–20 h before experiments. Pretreatment and stimulation were carried out at 37 C in serum-free medium. GF109203X (0.5 µM), PD98059 (100 µM), U0126 (10 µM), IC3 (50 µM), or dimethylsulfoxide (DMSO; as a vehicle control) were incubated with serum-starved cells for 15, 30, 30, or 15 min, respectively, before treatment with PMA (1 µg/ml, unless otherwise noted), NEM (5 mM), GH, PDGF-BB, calf serum (at the indicated concentrations), or vehicle controls. The inhibitors and stimulators were diluted from DMSO-dissolved stock solutions (GF109203X, 1 mM; PD98059, 50 mM; U0126, 10 mM; IC3, 10 mM; PMA, 1 mg/ml; NEM, 1 M), 10 mM acetic acid stock solution (PDGF-BB, 1 mg/ml), or undiluted calf serum. Stimulations were performed at 37 C. Details of the treatment protocol have been described previously (14, 15). Briefly, cells were stimulated in confluent 60 x 15-mm dishes (or, for immunoprecipitation experiments, 100 x 20-mm dishes; Falcon, Becton Dickinson & Co., Franklin Lakes, NJ) in serum starvation medium. Stimulations were terminated by washing the cells once with 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. For each cell type, pelleted cells were solubilized for 15 min at 4 C in fusion lysis buffer [1% (vol/vol) Triton X-100, 150 mM NaCl, 10% (vol/vol) glycerol, 50 mM Tris-HCl (pH 8.0), 100 mM NaF, 2 mM EDTA, 1 mM PMSF, 1 mM sodium orthovanadate, 10 mM benzamidine, and 10 µg/ml aprotinin] as indicated. After centrifugation at 15,000 x g for 15 min at 4 C, the detergent extracts were either subjected to immunoprecipitation or directly electrophoresed under reducing conditions, as indicated below. In some experiments harvested cells were solubilized directly in reducing SDS-PAGE sample buffer, as indicated, before electrophoresis.

Anti-GHRcyt-AL37 and anti-JAK2AL33 immunoprecipitations were performed as previously described (15, 16). Resolution of proteins under reducing conditions by SDS-PAGE, Western transfer of proteins, and blocking of Hybond-ECL (Amersham Pharmacia Biotech, Arlington Heights, IL) with 2% BSA were performed as previously described (14, 15, 20). Immunoblotting with affinity-purified anti-GHRcyt-AL37 and anti-GHRcyt, anti-JAK2AL33 (1:1000), 4G10 (1:1000), anti-MAPK (1:1000), and antiphospho-MAPK (1:5000) with horseradish peroxidase-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 the manufacturer’s suggestions.

GHBP assay
GHBP activity was measured in conditioned medium by a standardized GH binding assay, as previously reported (12, 21). Medium from CHO transfectant cells was directly assayed, whereas medium from 3T3-F442A cells required concentration to yield detectable GHBP activity. In the latter case, 7 ml medium were concentrated by ultrafiltration on Amicon Centricon 10 devices to a final volume of approximately 100 µl. GHBP recovery under those conditions is more than 95%. Conditioned medium from cells treated as indicated was incubated either undiluted (CHO transfectant cells; 1 ml) or after concentration (3T3-F442A cells; final incubation volume, 150 µl) with freshly labeled [125I]hGH (~0.5 ng, 15 µl) 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. In experiments in which treatment with PMA or PMA plus inhibitors was tested, the GHBP present in negative control (DMSO-treated) samples, which was minimal, was subtracted. Statistical analysis was performed with unpaired t test or ANOVA, followed by Newman-Keuls test, as appropriate. P < 0.05 was accepted as significant.

Densitometric analysis
Densitometry of ECL immunoblots was performed using a solid state video camera (Sony-77, Sony Corp.) and a 28-mm MicroNikkor lens over a light box of variable intensity (Northern Light Precision 890, Imaging Research, Inc., Toronto, Canada). Quantification was performed using a Macintosh II-based image analysis program (Image 1.49, developed by W. S. Rasband, Research Services Branch, NIMH, Bethesda, MD). The fraction of full-length GHRs remaining in extracts from PMA-, PDGF-, or calf serum-treated cells was estimated for each condition by measuring by densitometry the intensity of the specifically detected GHR signal relative to that signal present within the same experiment in extracts from unstimulated cells. Relative specific JAK2 tyrosine phosphorylation was estimated by normalizing the densitometric signal for tyrosine phosphorylation (APT immunoblot) of immunoprecipitated JAK2 by the abundance of JAK2 (anti-JAK2AL33 immunoblot) in that precipitate; this ratio for GH-stimulated samples without prior pretreatment was considered equal to 100%. As indicated when graphically shown, pooled data from several experiments are displayed as the mean ± SEM. The significance of differences of pooled results was estimated using unpaired t tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PMA-induced proteolysis of GHRs and shedding of GHBP in murine preadipocytes and in rabbit GHR-transfected CHO cells is inhibited by IC3
We have previously shown that the phorbol ester PMA induces rapid proteolysis of GHRs in IM-9 cells (12). This processing results in the coincident shedding of the receptor extracellular domain as a GHBP and the transient accumulation of cell-associated receptor fragments that are consistent with being GHR remnants (transmembrane GHRs devoid of the extracellular domain) (12). In that study constitutive and PMA-induced receptor proteolysis were blocked by both a PKC inhibitor and IC3, a hydroxamate-based metalloprotease inhibitor. These results were consistent with the likelihood that the GHBP-generating enzyme may be a member of the adamolysin family of proteases (22) and that its activity toward the GHR is influenced by PKC activity.

Despite evidence that GHR proteolysis does not account for a significant portion of the circulating GHBP in rodents (4), we wondered whether PMA might also promote GHR proteolysis in murine cell lines. The 3T3-F442A fibroblast expresses immunologically detectable GHRs and displays robust biochemical (e.g. cellular tyrosine phosphorylation) and functional (e.g. initiation of adipocyte differentiation) responses upon GH treatment (14, 23, 24), and hence was selected as a study model. We treated 3T3-F442A cells with PMA for 30 min, after which detergent-soluble proteins were extracted and resolved by SDS-PAGE (Fig. 1AGo). Immunoblotting with anti-GHRcyt-AL37, our previously described antibody to the GHR cytoplasmic domain (15, 16), revealed that PMA acutely promoted substantial loss of the full-length GHR (bracketed protein in lane 1 vs. lane 2). This loss of GHR was accompanied by increased abundance of an anti-GHRcyt-AL37-reactive protein of roughly 65 kDa (arrowhead), which, by analogy to our previous findings in IM-9 cells, we refer to as a GHR remnant protein. This same pattern of GHR loss and remnant accumulation in response to PMA was also specifically detectable when detergent-extracted proteins from the 3T3-F442A cells were first immunoprecipitated with anti-GHRcyt-AL37 before SDS-PAGE and anti-GHRcyt-AL37 immunoblotting (not shown).



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  		Figure 1. PMA-induced proteolysis of GHRs in 3T3-F442A cells is sensitive to metalloprotease and PKC inhibitors. A–C, PMA-induced loss of GHRs and appearance of GHR remnant protein. Serum-starved 3T3-F442A cells (one 90% confluent 60 x 15-mm dish/sample) were exposed to PMA (+) or the DMSO vehicle (-) for 30 min (A) or the indicated durations (B and C) after pretreatment for 15 min in the presence (+) of the metalloprotease (MP) inhibitor, IC3 (A); the PKC inhibitor, GF109203X (B); or their DMSO vehicle (-). In A and B, detergent extracts were resolved by SDS-PAGE and immunoblotted with affinity-purified anti-GHRcyt-AL37. The full-length GHR and GHR remnant protein appearing in response to PMA are indicated by a bracket and an arrowhead, respectively. In A and B, positions of prestained molecular mass markers (in kilodaltons) are indicated. In C, densitometric analysis of the full-length detergent-soluble GHRs remaining after 15 or 60 min of PMA treatment (1 µg/ml), as assessed by immunoblotting with anti-GHRcyt-AL37, in multiple experiments is presented. In each experiment, the amount of receptor present in DMSO-treated samples was considered 100%. The mean ± SEM is shown for nine independent determinations. P < 0.01 for comparison of the 15 and 60 min treatment samples each with the DMSO-treated samples. D, PMA-induced shedding of GHBP and its inhibition by the metalloprotease inhibitor, IC3. Serum-starved 3T3-F442A cells (one 90% confluent 150 x 25-mm dish in 7 ml serum-free medium/sample) were treated with DMSO vehicle (0 min PMA) or PMA (1 µg/ml) at 37 C for 15 and 60 min (left panel) or treated with or without IC3 (50 µM) for 15 min before treatment with DMSO or PMA for 90 min at 37 C (right panel). Conditioned medium (7 ml) from each sample was concentrated 50-fold and incubated with [125I]hGH as described in Materials and Methods. The fraction of GH bound to high affinity GHBP was derived by peak integration. Data are plotted as the mean ± SEM for several determinations (left panel: 15 min, n = 2; 60 min, n = 4; right panel: n = 2 each with and without IC3).

 
The time course of this PMA-induced GHR processing is shown in Fig. 1BGo, lanes 1–3, and is graphically displayed in Fig. 1CGo, in which the abundance of remaining GHR detectable by anti-GHRcyt-AL37 immunoblotting in several experiments was densitometrically determined. The profile of this rapid and substantial PMA-induced loss of immunologically detectable GHR (>60% loss in 15 min) is quite similar to the inhibitory effect of PMA on [125I]GH binding and GH-induced JAK2 tyrosine phosphorylation observed by King et al. previously in these same cells (25). PMA-induced GHR proteolysis was also observed in 3T3-L1 preadipocytes with a time course similar to that seen for 3T3-F442A cells (Fig. 3AGo and data not shown) and was seen in both cell types regardless of whether detergent-soluble extracts (as in Fig. 1Go) or total cell extracts (directly lysed in 1% SDS-containing PAGE buffer; not shown) were examined.



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  		Figure 3. Effects of MEK1 inhibition on PMA- and NEM-induced GHR proteolysis. A, Effect of PD98059 on PMA-induced GHR proteolysis in 3T3-L1 and 3T3-F442A cells. Serum-starved 3T3-L1 (lanes 1–4) or 3T3-F442A (lanes 5–8) cells (one 90% confluent 60 x 15-mm dish/sample) were exposed to PMA (+) or the DMSO vehicle (-) for 30 min after pretreatment in the presence (+) of PD98059 (30 min; MEK inhibitor), GF109203X (15 min; PKC inhibitor), or their DMSO vehicle (-). Detergent extracts were resolved by SDS-PAGE and sequentially immunoblotted with affinity-purified anti-GHRcyt-AL37 (upper panel), antiphospho-MAPK (middle panel), and anti-MAPK (lower panel). The full-length GHR and GHR remnant protein are indicated by a bracket and an arrowhead, respectively. The positions of the p44 (ERK1) and p42 (ERK2) MAPK species are also indicated. Note partial inhibition of proteolysis by PD98059 vs. near-complete inhibition by GF109203X. B, Effect of PD98059 on PMA-induced GHBP shedding in CHO-rbGHR cells. Serum-starved cells (one 90% confluent 100 x 20-mm dish in 4 ml serum-free medium/sample) were treated with DMSO vehicle (-) or PMA (1 µg/ml) at 37 C for 60 min in the presence or absence of the MEK inhibitor PD98059 (100 µM added 30 min previously). Conditioned medium (1 ml) from each sample was incubated with [125I]hGH, and the fraction of GH bound to high affinity GHBP was determined as described in Fig. 2BGo. Data are plotted as the mean ± SEM for three independent determinations. C, Inhibition of NEM-induced 3T3-F442A cell GHR proteolysis by IC3, but not by MEK1 or PKC inhibitors. Serum-starved 3T3-F442A cells (one 90% confluent 60 x 15-mm dish/sample) were exposed to NEM (+) or the DMSO vehicle (-) for 5 min after pretreatment with PD98059 (30 min; MEK inhibitor), GF109203X (15 min; PKC inhibitor), IC3 (15 min; MP inhibitor), or their DMSO vehicle (-). Detergent extracts were resolved by SDS-PAGE and immunoblotted with affinity-purified anti-GHRcyt-AL37. The full-length GHR and GHR remnant protein appearing in response to PMA are indicated by a bracket and an arrowhead, respectively. D, NEM does not activate MAPK(s) in 3T3-F442A cells. Serum-starved 3T3-F442A cells were treated as described in A with PMA (30 min), NEM (5 min), or their DMSO vehicle, and detergent extracts were resolved by SDS-PAGE and immunoblotted with antiphospho-MAPK. Arrows indicate the positions of p44 and p42 MAPK species. The experiments shown in C and D are each representative of three such experiments.

 
The PMA-induced GHR proteolysis in 3T3-F442A cells was inhibited by GF109203X, a potent inhibitor of most PKC isozymes (Fig. 1BGo, lanes 4–6 vs. 1–3). Murine GHR proteolysis induced by PMA was also prevented when the hydroxamate-based inhibitor of metalloproteases, IC3, was present. IC3 strongly inhibited both the PMA-induced loss of full-length GHR and the corresponding appearance of the cytoplasmic domain-containing GHR remnant (Fig. 1AGo, lanes 3 and 4 vs. lanes 1 and 2).

To confirm that the PMA-induced IC3-inhibitable GHR proteolysis in 3T3-F442A cells occurred during the period of incubation rather than adventitiously after cell harvesting and lysis, we measured the content of GHBP present in the cell supernatants collected after PMA treatment periods and before cell harvesting. It would be expected that GHR proteolysis in these cells, by analogy to that described by us and others in human cells (12, 26, 27, 28, 29), might be accompanied by release from the cells into the supernatant of shed GHR extracellular domain that maintains the capacity to bind GH. As graphically displayed in Fig. 1DGo, PMA treatment for as little as 15 min promoted release of GHBP from 3T3-F442A cells. Although the abundance of the GHBP shed from 3T3-F442A cells was low and only detectable after 50-fold concentration of medium (expressed in this case as percentage of GH bound per 150 µl of a 50-fold concentrate of an original 7 ml cell supernatant2), its specifically detectable appearance mirrored the pattern of PMA-induced loss of immunologically detectable GHRs (Fig. 1Go, D vs. C). Moreover, this release, like GHR proteolysis, was nearly completely (94%) inhibited by IC3 (Fig. 1Go, D and A). Although we do not interpret our data to indicate that GHR proteolysis contributes quantitatively to the pool of GHBP found in the rodent circulation (4), these results clearly indicate that PMA-induced metalloprotease-mediated GHR cleavage and GHBP shedding can occur in murine cells.

To study GHR proteolysis and GHBP shedding further, we generated another model system by stably expressing the rabbit (rb) GHR in Chinese hamster ovary (CHO) cells. As shown by others, CHO cells express no endogenous GHR and have proven to be a useful vehicle for GHR expression and studies of GH signaling and GHR trafficking (15, 30, 31). Immunoprecipitation and immunoblotting of rbGHRs from serum-starved CHO-rbGHR cells revealed the fully glycosylated (bracket) and incompletely glycosylated (arrow) forms of the transfected receptor (Fig. 2AGo, lane 1), as we have previously shown in COS-7 cells transfected with the rbGHR (32). As found in 3T3-F442A cells, PMA treatment acutely promoted substantial loss of the GHR and corresponding accumulation of an anti-GHRcyt-AL37-reactive lower Mr protein (arrowhead) of roughly 68 kDa (Fig. 2AGo, lane 2). Although we do not yet know the precise composition of this lower form, it migrates similarly to a recombinant version of the rbGHR that lacks the receptor extracellular domain (rbGHRdel ext), which we have previously characterized and referred to as the GHR remnant (12). Also, as we observed in 3T3-F442A cells, both the PMA-induced loss of GHR and the accumulation of cell-associated remnant were inhibited by pretreatment of the cells with either GF109203X (not shown) or IC3 (Fig. 2AGo, lane 3). In concert with these results, analysis of the cell supernatants revealed easily detectable PMA-induced shedding of GHBP that was inhibited by over 80% by IC3 (Fig. 2BGo; see Footnote 1). Thus, our previous observations and those presented herein indicate a susceptibility to PKC- and metalloprotease-mediated GHR proteolysis and GHBP shedding among several species of GHRs and cell types.



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  		Figure 2. PMA-induced proteolysis of rabbit GHRs and shedding of GHBP in CHO-rbGHR cells. A, PMA-induced GHR proteolysis is inhibited by IC3. Serum-starved CHO-rbGHR cells (one 90% confluent 60 x 15-mm dish/sample) were exposed to PMA (+; 1 µg/ml) or DMSO vehicle (-) for 10 min at 37 C with or without IC3 (50 µM) for 15 min before treatment. Detergent extracts were immunoprecipitated with anti-GHRcyt-AL37, and eluates were resolved by SDS-PAGE and immunoblotted with affinity-purified anti-GHRcyt. The full-length GHR and its incompletely glycosylated forms are indicated by the upper bracket and arrows, respectively. The GHR remnant protein that appears in response to PMA in an IC3-sensitive fashion is indicated by the arrowhead. The experiment shown is representative of four such experiments. B, PMA-induced GHPB shedding is inhibited by IC3. Serum-starved cells (one 90% confluent 100 x 20-mm dish in 4 ml serum-free medium/sample) were treated with DMSO vehicle (D) or PMA (1 µg/ml) at 37 C for 60 min in the presence or absence of IC3 (50 µM added 15 min previously). Conditioned medium (1 ml) from each sample was incubated with [125I]hGH, and the fraction of GH bound to high affinity GHBP was determined as described in Fig. 1DGo. Data are plotted as the mean ± SEM for three independent determinations.

 
Effects of MEK1 inhibition on PMA- and NEM-induced proteolysis of GHRs
Although some enzymes involved in the shedding of a variety of cell surface proteins have been identified (33, 34, 35, 36, 37), much less is known about the signaling events leading to activation of the shedding process. In many instances of shedding, including GHBP shedding, phorbol ester treatment activates this process, and PKC or particular PKC isozymes have been implicated (Refs. 12, 29, 36, 38 , and 39 and data herein). As PKC can activate MAPKs, we tested whether inhibitors of MAPK activation might influence PMA-induced GHR proteolysis and GHBP shedding. PD98059 is a well characterized inhibitor of MEK1, the upstream activator of the MAPKs, ERK1 and ERK2 (40). As shown in Fig. 3AGo, pretreatment of both 3T3-L1 (upper left panel) and 3T3-F442A (upper right panel) fibroblasts with PD98059 (100 µM) partially inhibited the PMA (1 µg/ml)-induced GHR loss and remnant accumulation observed with anti-GHRcyt-AL37 immunoblotting of cell extracts (lanes 1–3 and 5–7). The partial nature of inhibition by PD98059 is highlighted by comparison to the more complete inhibition rendered by GF109203X (lanes 4 and 8). In these same samples, PD98059 and GF109203X conferred a very similar and nearly complete degree of inhibition of PMA-induced activation of the MAPKs, ERK1 and ERK2 (as assessed in the left and right middle panels by immunoblotting with a state-specific antibody (antiphospho-MAPK) that specifically recognizes the phosphorylated threonine 183 and tyrosine 185 residues in the MAPK molecule that correlate to its enzymatic activation). Yet in this and other such experiments, densitometric analysis of anti-GHRcyt-AL37 immunoblots revealed that PMA-induced loss of GHR was inhibited, on the average, by 56% by PD98059 compared with 94% by GF109203X; similarly, the PMA-induced increase in remnant abundance was diminished by 39% by PD98059 vs. 85% by GF109203X.

The differences in the inhibition of PMA-induced receptor proteolysis relative to MAPK activation state could not be accounted for by a change in the abundance of ERK1 and ERK2 (bottom panel, lanes 1–4 and 5–8) in any of the samples. In other experiments with 3T3-F442A cells, acute incubation with PD98059 or GF109203X had no effect on cell viability or on the ability of the cells to respond to GH stimulation with JAK2 activation (41); thus, we think it unlikely that the effects of each inhibitor on PMA-induced GHR proteolysis can be attributed to drug toxicity. We also tested the effect of a distinct MEK1 inhibitor, U0126 (42), and found that, similarly to PD98059, U0126 partially inhibited the PMA-induced loss of GHR and accumulation of remnant in 3T3-F442A cells (data not shown).

We also examined the effect of MEK1 inhibition on PMA-induced GHBP shedding. We measured GHBP in the conditioned medium of CHO-rbGHR cells treated with PMA after pretreatment in the presence or absence of PD98059 (Fig. 3BGo). Again, pretreatment with PD98059 resulted in a significant, but partial (54.8 ± 6.3%) reduction of GHBP shed into the medium in response to PMA. Thus, inhibitors of MEK1 partially inhibited both PMA-induced GHR proteolysis and GHBP shedding, further substantiating the linkage between these two phenomena and indicating that at least part of the PMA-induced GHR processing in 3T3-F442A and CHO-rbGHR cells is probably attributable to MEK1, ERK(s), or other MEK1-dependent kinase(s).

As we and others have previously demonstrated, the sulfhydryl alkylating reagent, NEM, also promotes GHR proteolysis and GHBP shedding (12, 26, 27, 43). Notably, we have previously shown that NEM- and PMA-induced GHR processing was inhibited by IC3, but that, unlike PMA, NEM-induced receptor proteolysis was not affected by inhibition of PKC activity. The two stimuli were thus proposed to achieve activation of a common protease activity by potentially distinct pathways (12). We extended this analysis by testing NEM’s effects on GHR proteolysis in 3T3-F442A cells (Fig. 3Go, C and D). Acute treatment with NEM (5 mM, 5 min) promoted substantial loss of anti-GHRcyt-AL37 immunoblottable GHR and an increase in detectable remnant protein (Fig. 3CGo, lane 2 vs. lane 1). Interestingly, unlike the findings for PMA-induced GHR proteolysis (Fig. 3AGo), NEM-induced GHR proteolysis in 3T3-F442A cells was unaffected by pretreatment with either the MEK inhibitor (PD98059) or the PKC inhibitor (GF109203X; Fig. 3CGo, lanes 3 and 4 vs. lanes 1 and 2). However, consistent with our previous findings in IM-9 cells, NEM-induced GHR proteolysis was inhibited by IC3 (lane 5 vs. lane 2). Further, the NEM-induced proteolysis was not associated with activation of MAPK, as shown in the antiphospho-MAPK blot in Fig. 3DGo. These findings strongly support the proposition that NEM and PMA operate via distinct mechanisms to promote GHR cleavage.

PDGF and serum induce IC3-inhibitable GHR proteolysis
Our results to date indicate that GHR proteolysis and shedding of the receptor’s extracellular domain can be detected in several cell types in response to pharmacological activation of PKC with phorbol ester or in response to sulfhydryl alkylators. To further understand other potential regulators of this process, we tested whether growth factor stimulation might also activate GHR proteolysis; such stimuli have been recently implicated in the shedding of several other cell surface proteins (44, 45, 46). PDGF, in particular, was recently shown to diminish surface GHR expression in 3T3-F442A cells, although the mechanism(s) of this receptor loss remains uncertain (47). Our treatment of 3T3-F442A cells with PDGF-BB (40 ng/ml) for 30 min led to substantial loss of GHR abundance, as assessed by anti-GHRcyt-AL37 immunoblotting (Fig. 4AGo, upper panel, lane 2 vs. lane 1). Densitometric evaluation indicated that PDGF induced, on the average, a 56% decrease in GHR abundance (Fig. 4AGo, lower panel), consistent with the degree of loss of surface GHR observed by Rui et al. (46) in similar experiments. This GHR loss was associated with substantial generation of the GHR remnant protein (Fig. 4AGo, upper panel, lane 2 vs. 1). Notably, both the loss of GHR and the accumulation of remnant induced by PDGF were blocked by IC3 (upper panel, lane 3 vs. lanes 1 and 2, and lower panel), strongly suggesting that the PDGF-induced GHR loss was caused by receptor proteolysis. Similarly, treatment of serum-starved cells with 10% calf serum also caused GHR loss (36%) and remnant accumulation, both of which were prevented by pretreatment with IC3 (Fig. 4BGo).



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  		Figure 4. PDGF- and calf serum-induced metalloprotease-mediated GHR proteolysis and GHBP shedding. A and B, PDGF- and calf serum-induced GHR proteolysis in 3T3-F442A cells. Upper panels, Serum-starved 3T3-F442A cells (one 90% confluent 60 x 15-mm dish/sample) were exposed to PDGF (40 ng/ml; A, lanes 2 and 3), calf serum (10%, vol/vol; B, lanes 2 and 3), or vehicle (lane 1) for 30 min after pretreatment for 15 min in the presence (lane 3) or absence (lanes 1 and 2) of the metalloprotease (MP) inhibitor, IC3. Detergent extracts were resolved by SDS-PAGE and immunoblotted with affinity-purified anti-GHRcyt-AL37. Lower panels, Densitometric analysis of the full-length detergent-soluble GHRs remaining after PDGF (A) or serum (B) treatment, as assessed by immunoblotting with anti-GHRcyt-AL37, in multiple experiments. In each experiment the amount of receptor present in vehicle-treated samples was considered 100%. The mean ± SEM are shown for two (left panel) or three (right panel) independent determinations for each. P values are indicated. C, Calf serum-induced GHBP shedding in CHO-rbGHR cells. Serum-starved cells (one 90% confluent 100 x 20-mm dish in 4 ml serum-free medium/sample) were treated with vehicle (-) or calf serum (10%, vol/vol; +) at 37 C for 60 min in the presence or absence of the metalloprotease inhibitor, IC3 (50 µM added 30 min previously). Conditioned medium (1 ml) from each sample was incubated with [125I]hGH, and the fraction of GH bound to high affinity GHBP was determined as described in Fig. 2BGo. Data are plotted as the mean ± SEM for three independent determinations.

 
CHO-rbGHR cells did not respond to PDGF stimulation, probably because they do not express PDGF receptors (data not shown). However, GHR proteolysis (not shown) and GHBP shedding (Fig. 4CGo) in serum-starved CHO-rbGHR cells were easily detected in response to treatment for 60 min with 10% calf serum. The serum-induced GHBP shedding was inhibited 80% by inclusion of IC3 during the treatment period. These data provide the first evidence of which we are aware that a nonpharmacological stimulus can elicit GHBP formation by proteolysis.

GHR proteolysis contributes to PMA- and growth factor-induced down-regulation of GHR signaling
Down-regulation of GHR signaling by PMA and PDGF has been described previously (25, 47, 48) and, as indicated above, has been hypothesized to be accounted for by loss of GHRs through degradative or redistributive mechanisms. We tested whether the induced proteolysis of the GHR that we observed in response to PMA, PDGF, or calf serum affected GH-induced signaling. 3T3-F442A cells (Fig. 5AGo) and CHO-rbGHR cells (Fig. 5BGo) were pretreated with PMA, PMA plus IC3, or vehicle alone for 15 min before treatment with either GH alone (50 ng/ml) or its vehicle for an additional 15 min. GH-induced JAK2 tyrosine phosphorylation was then assessed by anti-JAK2AL33 immunoprecipitation and antiphosphotyrosine immunoblotting (Fig. 5Go, A and B, upper panels). JAK2 abundance in each sample was normalized by reprobing of the blots with anti-JAK2AL33 (middle panels). A ratio of tyrosine-phosphorylated JAK2/total JAK2 was determined for each condition in several such experiments (lower panels). GH-induced JAK2 activation was reduced significantly (by >74% in both cell types) by pretreatment with PMA (Fig. 5Go, A and B, lanes 1–3). Notably, inclusion of IC3 with PMA nearly completely reversed PMA’s inhibition of GH-induced JAK2 tyrosine phosphorylation (lane 4 vs. lanes 2 and 3). These results strongly support the conclusion that PMA down-regulation of GHR abundance by metalloprotease-mediated proteolysis results in inhibition of GH- induced JAK2 activation.



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  		Figure 5. PMA-, PDGF-, and calf-serum-induced metalloprotease-mediated down-regulation of GH-induced JAK2 tyrosine phosphorylation. A and B, PMA-induced down-regulation. Serum-starved cells (A, 3T3-F442A cells; B, CHO-rbGHR cells; one 90% confluent 100 x 25-mm dish/sample) were pretreated with or without PMA (30 min) and the metalloprotease inhibitor, IC3 (added 15 min before PMA), as indicated, before stimulation with or without GH (50 ng/ml) for 10 min. Anti-JAK2AL33 immunoprecipitates were resolved by SDS-PAGE and sequentially immunoblotted with APT (upper panels) and anti-JAK2AL33 (middle panels). Relative specific JAK2 tyrosine phosphorylation, determined as described in Materials and Methods for several experiments (three in A; five in B), is plotted for each condition in the lower panels. P values are indicated. C and D, PDGF- and calf serum-induced down-regulation. Serum-starved 3T3-F442A cells, as described in A, were pretreated with or without PDGF (C) or calf serum (20%, vol/vol; D; 30 min) and the metalloprotease inhibitor, IC3 (added 15 min before PDGF or serum), as indicated, before stimulation with or without GH (50 ng/ml) for 10 min. Anti-JAK2AL33 immunoprecipitates were processed and analyzed as described in A and B. Relative specific JAK2 tyrosine phosphorylation, determined for several experiments (five in C; three in D), is plotted for each condition in the lower panels. P values are indicated.

 
Similar inhibitory effects on GH-induced JAK2 tyrosine phosphorylation seen for PMA were also observed after treatment with PDGF (Fig. 5CGo) or calf serum (Fig. 5DGo). In each instance, growth factor pretreatment of 3T3-F442A cells significantly reduced GH-induced JAK2 activation (lane 3 vs. lane 2; approximately 50% reduction for PDGF and 37% reduction for calf serum). Inclusion of IC3 nearly completely inhibited PDGF- and serum-induced down-regulation of GH stimulation of JAK2 tyrosine phosphorylation (Fig. 5Go, C and D, lane 4 vs. lanes 2 and 3), just as seen for PMA-, PDGF-, and serum-induced down-regulation of GHR abundance and for PMA-induced down-regulation of GH-induced JAK2 activation. Neither PDGF nor calf serum alone had any effect on JAK2 tyrosine phosphorylation (not shown). These data are important in that they suggest that certain growth factors can down-regulate both GHR abundance and GHR signaling by a metalloprotease-mediated proteolytic mechanism.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In principle, regulated proteolysis of a cell surface molecule with release of its extracellular domain could subserve any of several possible functions, including 1) shedding of the extracellular domain to function as a soluble cytokine (which then exerts local or distant effects) or as a circulating receptor or ligand-binding protein; 2) modulation (e.g. down-regulation) of the surface concentration of the full-length molecule, thereby altering the effects the molecule exerts in the cell; or 3) generation of the transmembrane/cytoplasmic domain remnant of the molecule, which could then have signaling or regulatory functions independent of those of the full-length molecule (38). Such proteolysis is a common fate for numerous surface molecules (38, 39).

Proteolysis of the GHR has largely been considered in the context of being the principal mechanism accounting for the generation by shedding of circulating GHBP in humans and rabbits (7, 49). This is in contrast to mice and rats, in which the vast majority of circulating GHBP is believed to be derived from an alternatively spliced GHR that lacks the transmembrane and cytoplasmic domains and, by virtue of replacement of the transmembrane domain with a hydrophilic peptide, is thus secreted (2, 3, 4). Our findings that 3T3-F442A, a GH-responsive murine cell line, can be induced to proteolyze its GHR and release a GHBP (albeit at very low abundance) are notable in this regard. Others (50) have recently observed that the monkey GHR, when overexpressed transiently, spontaneously releases GHBP (presumably by proteolytic shedding) into the medium, but that, in addition to a full-length GHR mRNA, monkey tissues express a spliced mRNA that encodes a GHBP like that found in rodents. These findings were taken as evidence for coexistence within a single species of the two mechanisms for GHBP generation.

Although we were able to detect shed murine GHBP in concert with biochemical evidence for GHR proteolysis (loss of GHR, accumulation of GHR remnant) without overexpressing the GHR, we interpret our results cautiously regarding their implications for mechanisms of GHBP generation. PMA-induced GHBP concentrations in murine cell medium are at least 50-fold lower than those in medium from CHO transfectants or IM-9 cells. Our findings therefore do not constitute a refutation of the view that rodent circulating GHBP is largely derived from the product of the alternatively spliced mRNA referred to above, nor do they indicate that shedding contributes significantly to the circulating GHBP pool in the rodent under usual conditions. However, our findings using both 3T3-F442A and CHO-rbGHR cells in concert with our previous results (12) are important in that they indicate that GHRs in various species are susceptible to processing by proteolytic machinery with some common features. Assessment of the relative degree to which rodent and nonrodent GHRs can serve as targets of this machinery will await studies that carefully compare the expression of each receptor in various cell lines.

Our current results provide insights into the pathway(s) involved in the regulation of GHR proteolysis and GHBP shedding and the physiological roles of this receptor processing. Using two independent cell lines of different species (mouse and hamster), we found that a process that culminates in IC3-inhibitable GHR proteolysis can be activated by PMA via a pathway dependent on PKC activation and at least partially dependent on MEK1 activation (that is, it is partially inhibited by two distinct MEK1 inhibitors). This finding of a susceptibility of GHR proteolysis to MEK1 inhibitors is in concert with the recent findings of Gechtman et al. (44), Desdouits-Magnen et al. (45), and Fan and Derynck (46) for the shedding of heparin-binding epidermal growth factor (44), the secretion of the soluble Alzheimer amyloid precursor protein (45), and the shedding of transforming growth factor-{alpha} and some other surface proteins (46). In those studies, using both inhibitors and dominant-negative approaches, the MAPK pathway was also implicated in stimulation of metalloprotease-mediated proteolysis and shedding. Our observation that PMA-induced GHR proteolysis is partially, rather than completely, sensitive to MEK1 inhibition raises the interesting possibility that pathways downstream of both PKC and MEK1 may each contribute to PMA-induced receptor proteolysis to some degree and may therefore be points of differential regulation of GHR processing. This is a worthwhile area for future investigation.

Our findings that NEM can induce GHR proteolysis in 3T3-F442A cells and CHO-rbGHR cells (not shown) complement our previously reported results with IM-9 cells (12). In each system, NEM-induced GHR proteolysis and/or GHBP shedding, although sensitive to IC3, is insensitive to inhibitors of PKC and MEK1. This strongly suggests that the pathway(s) by which PMA leads to metalloprotease-dependent GHR processing is different from that engaged by NEM. Future studies will be required to determine which protease(s) is being regulated by each stimulus and whether both types of stimuli are acting at a point of convergence of proteolysis-promoting pathways or, for example, whether one stimulus (e.g. PMA) is acting as a metalloprotease activator and the other (e.g. NEM) is working to make the substrate (GHR) more susceptible to cleavage. Although we cannot yet decipher this situation, our findings argue that studies examining GHR proteolysis and GHBP shedding using only NEM as an inducer should be interpreted with caution with respect to their generality.

A major implication of our current work relates to mechanisms of GHR down-regulation and modulation of GH signaling on the basis of receptor availability. Although PMA- and PDGF-mediated down-regulation of GHR abundance and signaling have been reported, the work described herein is the first to establish that a substantial fraction of such down-regulation can be attributed to GHR proteolysis. We also observe that serum itself, when applied to serum-starved cells in culture, can down-regulate GH signaling and diminish receptor abundance via metalloprotease-mediated proteolysis. Further, we establish in CHO-rbGHR cells that GHBP shedding can be experimentally detected in response to serum stimulation. These observations that PDGF and serum stimulation can elicit GHR proteolysis and GHBP shedding will serve as the framework to explore the degree to which other physiologically relevant stimuli can regulate these processes and similarly affect GHR signaling. It will be interesting and important to determine whether it is the generation of the shed GHBP, the decrease in GHR abundance, and/or the generation of the GHR transmembrane/cytoplasmic domain remnant that account for the diminished capacity of GH to elicit JAK2 tyrosine phosphorylation after receptor proteolysis occurs. Such studies will require identification of the enzyme(s) that catalyzes GHR cleavage, determination of the cleavage site, and isolation of model systems in which cleavage can be prevented so as to test which component (increase in soluble GHBP, receptor loss, or remnant accumulation) is responsible for the effects on full-length receptor signaling.


   Acknowledgments
 
The authors appreciate helpful conversations with Drs. J. Kudlow, A. Paterson, E. Chin, L. Liang, S.-O. Kim, C. Carter-Su, and J. Messina and generous provision of reagents by those named in the text.


   Footnotes
 
1 This work was supported by a V.A. Merit Review award (to S.J.F.), grants from the NSF and the Northwestern Memorial Foundation (to 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, Toronto, Ontario, Canada, 2000. Back

2 Estimates of molar concentrations of GHBP in conditioned medium after PMA treatment, based on measurements of dissociation rates during gel filtration (8 51 ) and calculation of bound GH fractions based on the law of mass action using a Kd of 10-9 (52 53 ), yield mean values of 5.6 pM for 3T3-F442A medium and 360 pM for CHO-rbGHR medium, an approximately 65-fold difference. The mean GHBP concentration in IM-9 cell-conditioned medium after PMA treatment (12 ) is 550 pM, 100-fold greater than that in 3T3-F442A medium. Back

Received October 6, 2000.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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