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Endocrinology, doi:10.1210/en.2005-1484
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Endocrinology Vol. 147, No. 6 2839-2849
Copyright © 2006 by The Endocrine Society

Janus Kinase 2 Influences Growth Hormone Receptor Metalloproteolysis

Kimberly Loesch, Luqin Deng, Jon W. Cowan, Xiangdong Wang, Kai He, Jing Jiang, Roy A. Black and Stuart J. Frank

Endocrinology Section, Medical Service, Veterans Affairs Medical Center (S.J.F.), Birmingham, Alabama 35233; Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Alabama (X.W., K.H., J.J., S.J.F.), and Department of Cell Biology (K.L., L.D., J.W.C., S.J.F.), University of Alabama, Birmingham, Alabama 35294-0012; and Amgen, Inc. (R.A.B.), Seattle, Washington 98119-3105

Address all correspondence and requests for reprints to: Dr. Stuart J. Frank, University of Alabama, 1530 3rd Avenue South, BDB 861, Birmingham, Alabama 35294-0012. E-mail: sjfrank{at}uab.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GH signals through the GH receptor (GHR), a cytokine receptor superfamily member that couples to the cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2). In addition to its role in signaling, we recently implicated JAK2 in the regulation of cell surface GHR abundance by modulation of GHR trafficking and mature GHR stability. GHR is a target for constitutive and inducible metalloprotease-mediated cleavage that alters surface GHR levels and can modulate GH signaling. We previously found that metalloprotease cleavage of GHR is dramatically lessened in fibroblasts derived from mice with targeted deletion of the zinc-binding domain of TNF-{alpha}-cleaving enzyme [TACE; ADAM17 (a disintegrin and metalloprotease)], implicating this transmembrane ectoenzyme as a GHR metalloprotease. In this study we used a human fibrosarcoma reconstitution system to compare the effects of RNA interference-mediated knockdown of TACE vs. a related metalloprotease, ADAM10. We found that TACE knockdown dramatically reduced both the pace and the degree of inducible GHR proteolysis and augmented the abundance of mature GHR, suggesting a role for TACE in constitutive receptor proteolysis in this system as well. Notably, ADAM10 knockdown also reduced inducible GHR proteolysis, although to a lesser degree than TACE knockdown, suggesting a contribution from this metalloprotease also. To determine whether JAK2 affects GHR proteolysis, we compared JAK2-deficient vs. JAK2-replete cells and found that phorbol 12-methyl 13-acetate-induced GHR proteolysis was significantly diminished in cells that lacked JAK2. Reconstitution with a GHR mutant that lacks the box 1 region (which mediates JAK2 association) resulted in phorbol 12-methyl 13-acetate-induced proteolysis similar in degree to that of the wild-type GHR in JAK2-deficient cells. Introduction of JAK2 did not affect the proteolysis of this box 1-deleted GHR, suggesting GHR-JAK2 association is required for JAK2 to affect GHR proteolysis. Additionally, the inhibitory effect of anti-GHRext-mAb, a conformation-sensitive GHR antibody, on receptor proteolysis was lost in cells that lacked JAK2. Our data indicate that the susceptibility of GHR to proteolysis is substantially affected by JAK2, suggesting yet another role for this kinase in determining GH sensitivity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH IS A key regulator of postnatal growth and metabolism that exerts its pleiotropic effects by interaction with the GH receptor (GHR) on the surface of target cells (1, 2). GHR is a widely displayed type I glycoprotein member of the cytokine receptor superfamily (2, 3). GH binding to the dimerized GHR forms a 1:2 GH:GHR assemblage and results in changes in receptor conformation/orientation that trigger activation of the GHR-associated tyrosine kinase, Janus kinase 2 (JAK2), and intracellular signaling (4, 5, 6, 7, 8, 9, 10, 11, 12, 13). The level of GHR on the cell surface is an important determinant of cellular sensitivity to GH. Interestingly, JAK2, in addition to its role in GH signal transduction, may contribute to regulation of surface GHR abundance by influencing the stability of the surface receptor (14, 15).

The GHR, like some other surface proteins, is a target for regulated proteolytic processing. Proteolysis of GHRs from several species is constitutive, but can be additionally induced in various cell types by treatment with a protein kinase C activator [the phorbol ester, phorbol 12-methyl 13-acetate (PMA)], platelet-derived growth factor, or serum (9, 16, 17, 18, 19, 20, 21, 22). Pretreatment with hydroxamate-based metalloprotease inhibitors completely prevents constitutive and inducible GHR proteolysis, implicating metalloprotease activity in this processing (17, 18, 23). We previously used a genetic reconstitution strategy in mouse cells to identify a transmembrane metalloprotease member of the metzincin family, TNF-{alpha} converting enzyme [TACE; ADAM (a disintegrin and metalloprotease)] (17), as capable of mediating GHR proteolysis (23). Metalloproteolysis of GHR occurs in the proximal extracellular domain stem region of the receptor (20, 21) and results in loss of the full-length receptor, appearance of a cell-associated cytoplasmic domain-containing GHR fragment (the remnant protein), and a soluble GHR extracellular domain [referred to as the GH-binding protein (GHBP), in correspondence with the high affinity GHBP found in the circulation of many species, including humans] (24). Although some species (such as rats and mice) derive the bulk of GHBP by means other than GHR proteolysis (24), inducible metalloprotease-mediated cleavage is detected in GHR from rodent, rabbit, and human and is probably associated with regulation of cellular GH sensitivity (25); GH-induced signaling is dampened after exposing cells to stimuli that promote receptor cleavage, but not if metalloprotease inhibitors are present or if noncleavable receptor mutants are expressed (18, 21, 25).

TACE has numerous other substrates, including pro-TNF-{alpha}, pro-TGF-{alpha}, amyloid precursor protein, Notch, L-selectin, IL-1 receptor, heparin-binding epidermal growth factor, and amphiregulin (26, 27, 28, 29, 30, 31). Specificity for these substrates appears to reside in conformational and spatial factors rather than with specific amino acid sequences. Indeed, inducible GHR proteolysis is blocked by previous treatment with GH, but not by a GH antagonist, even though both bind GHR (19). This suggests that adoption of a GH-induced conformation renders the GHR less susceptible to cleavage. Consistent with this, pretreatment with a conformation-sensitive monoclonal antibody to the receptor extracellular domain also inhibits inducible GHR proteolysis (9). Interestingly, ADAM-10, another metzincin with structural similarities to TACE, can also cleave some TACE substrates (e.g. amyloid precursor protein and heparin-binding epidermal growth factor), suggesting enhanced complexity in the regulation of shedding of these surface proteins (26, 27, 28, 29, 30, 31).

In this study we explore the relative contributions of TACE and ADAM-10 to GHR proteolysis, using as our model system a well-characterized, GHR-expressing human fibrosarcoma cell line and small interfering RNA (siRNA)-mediated knockdown of each endogenous protease. We found that TACE knockdown in human cells profoundly affects inducible GHR proteolysis, as suggested by our previous studies in murine cells; however, we also uncover a role for ADAM-10 in this human system. We examined the impact of Janus kinase 2 (JAK2) on GHR proteolysis by comparing cells that express or lack JAK2. These studies reveal an unanticipated role for JAK2 in dampening basal GHR proteolysis and enhancing inducible proteolysis, which may relate to JAK2-dependent alterations of the conformation of the mature GHR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
PMA and routine reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO) unless otherwise noted. Recombinant human GH was provided by Eli Lilly & Co. (Indianapolis, IN). Zeocin, G418, and hygromycin B were purchased from Invitrogen Life Technologies, Inc. (Carlsbad, CA). Fetal bovine serum, gentamicin sulfate, penicillin, and streptomycin were purchased from BioFluids (Rockville, MD).

Antibodies
ADAM17/TACE and ADAM10 antibodies were purchased from Chemicon International (Temecula, CA). Anti-JAK2AL-33 (directed at residues 746-1129 of murine JAK2) polyclonal serum has been described previously (32). The rabbit polyclonal antiserum, anti-GHRcytAL-47, was raised against a bacterially expressed N-terminally histidine-tagged fusion protein incorporating human GHR residues 271–620 (the entire cytoplasmic domain) and has been previously described (19). Anti-GHRext-mAb is a mouse monoclonal antibody directed against a bacterially expressed glutathione-S-transferase fusion protein incorporating rabbit GHR residues 1–246; its generation and purification have been described previously (9, 17, 33, 34).

Cells and cell culture
{gamma}2A is a JAK2-deficient human fibrosarcoma cell line provided by Dr. G. Stark (Cleveland Clinic Foundation, Cleveland, OH) (35). A stable {gamma}2A cell line expressing rabbit GHR ({gamma}2A-GHR) has been described previously (19). {gamma}2A-GHR cells were maintained in DMEM (1 g/liter glucose) (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum, 50 µg/ml gentamicin sulfate, 100 U/ml penicillin, 100 µg/ml streptomycin, 200 µg/ml G418, and 100 µg/ml hygromycin B. A stable {gamma}2A cell line expressing rabbit GHR and mouse JAK2 (C14) was achieved by stable transfection of {gamma}2A-GHR with murine JAK2 as previously described (15), and was maintained in the above medium supplemented with 100 µg/ml zeocin. A stable {gamma}2A cell line expressing only murine JAK2 was achieved by introducing pcDNA3.1+-JAK2-zeocin into cells using Lipofectamine (Invitrogen Life Technologies, Inc.) according to the manufacturer’s protocol. Cells were selected in DMEM growth medium supplemented with 400 µg/ml zeocin and screened for JAK2 expression by blotting with anti-JAK2AL-33. Stable transfection of {gamma}2A-GHR{Delta}Box1 and {gamma}2A-GHR{Delta}Box1-JAK2 was achieved by introducing pSX-rbGHR{Delta}278–292 (36) and pSX-hygromycin-hemagglutinin, using Lipofectamine, into {gamma}2A and {gamma}2A-JAK2 cells, respectively. Each cell line was selected in DMEM growth medium (with ({gamma}2A-JAK2) or without ({gamma}2A) zeocin) supplemented with 200 µg/ml hygromycin and screened for GHR{Delta}Box1 expression by blotting with anti-GHRcytAL-47. Table 1Go lists each cell type and the features of their expressed GHR and JAK2 molecules.


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  		TABLE 1. Cell type and features of their expressed GHR and JAK2 molecules

 
RNA interference (RNAi)
TACE and ADAM10 siRNA duplexes were custom synthesized by Ambion, Inc. (Austin, TX). The 21-nucleotide sequences, located in the prodomain regions for each, are CAUAGAGCCACUUUGGAGAdTdT (TACE) (37) and GGAUUAUCUUACAAUGUGGdTdT (ADAM10; custom designed by Ambion). Silencer negative control 3 siRNA was purchased from Ambion, Inc., and used as a negative control. C14 cells were subjected to siRNA knockdown of TACE or ADAM10 for either 3 d (short-term knockdown) or 6 d (long-term knockdown). For short-term experiments, the cells were plated on six-well plates at a low density (30–50% confluence) and transfected with siRNA using Oligofectamine (Invitrogen Life Technologies, Inc.) following the manufacturer’s protocol. Briefly, cells were washed twice with PBS and transfected with 3.25 µg/well control, TACE, or ADAM10 siRNA in serum- and antibiotic-free OptiMEM (Invitrogen, Carlsbad, CA) for 4–5 h, after which serum was added back to the medium to a final 10% concentration. Twenty-four hours later, the medium was changed to serum starvation medium, substituting 0.5% (wt/vol) BSA for serum, for an overnight period of 18–24 h before PMA stimulation.

For long-term experiments, cells from a 10-cm plate (~50% confluent) were trypsinized, split into three equal fractions, and mixed with control, TACE, or ADAM10 siRNA plus Oligofectamine in serum- and antibiotic-free OptiMEM. Each fraction was split equally into four wells of a six-well plate (3.25 µg/well siRNA). For cells to adhere to the plate, 0.5 ml DMEM (1 g/liter glucose; Mediatech, Inc.) supplemented with 10% fetal bovine serum was added to each well (final serum concentration, 3%). The next day the medium was changed to complete medium (see above). On d 3, the plated cells were transfected again with each respective siRNA, as described above for short-term knockdown. The next day the medium was changed to DMEM (1 g/liter glucose; Mediatech, Inc.) supplemented with 5% fetal bovine serum. On d 5, the medium was changed to serum starvation medium for an overnight period of 18–24 h before PMA stimulation.

Cell stimulation, protein extraction, electrophoresis, and immunoblotting
Serum starvation of all cell lines was accomplished by substitution of 0.5% (wt/vol) BSA (fraction V; Roche, Indianapolis, IN) for serum in their respective culture media for 18–24 h before experiments. Stimulations were performed at 37 C. For the siRNA experiments, adherent cells were stimulated with vehicle control or PMA (0.1 µg/ml) for 0, 15, 30, and 60 min in DMEM (low glucose) with 0.5% (wt/vol) BSA. For antibody-blocking experiments, adherent cells were pretreated with either vehicle control or anti-GHRext-mAb (12 µg/ml) for 30 min before PMA (1 µg/ml) stimulation for 0 or 45 min in DMEM (low glucose) with 0.5% (wt/vol) BSA. Stimulations were terminated by washing cells three times with ice-cold PBS in the presence of 0.4 mM sodium orthovanadate (PBS-vanadate). For protein extraction, cells still attached to the six-well plate were solubilized in lysis buffer [1% (wt/vol) Triton X-100, 150 mM NaCl, 10% (vol/vol) glycerol, 50 mM Tris-HCl (pH 7.4), 50 mM sodium fluoride, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium orthovanadate, 10 mM 1,10-phenanthroline, 1 µg/ml leupeptin, and 10 µg/ml aprotinin] on ice for 20 min. The detergent extracts were collected and centrifuged at 20,000 x g for 15 min at 4 C, then electrophoresed under reducing conditions by addition of Laemmli SDS-PAGE sample buffer and resolved by SDS-PAGE (8% acrylamide, unless otherwise noted). Immunoprecipitation, Western transfer of proteins, and blocking of Hybond-ECL (Amersham Biosciences, Arlington Heights, IL) with 2% BSA were performed as previously described (14). Immunoblotting with anti-TACE (1:5000), anti-ADAM10 (1:3,000), anti-GHRcytAL-47 (1:2,000), and anti-JAKAL-33 (1:1,000) antibodies with horseradish peroxidase-conjugated antirabbit (1:50,000) and detection reagents (SuperSignal West Pico chemiluminescent substrate; all from Pierce Chemical Co., Rockford, IL) and stripping and reprobing of blots were accomplished according to the manufacturer’s suggestions.

Densitometric analysis
Densitometric quantitation of ECL immunoblots was performed using a high resolution scanner and the ImageJ 1.30 program (developed by W. S. Rasband, Research Services Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of TACE RNAi on GHR proteolysis
We previously showed that fibroblasts isolated from mice with targeted disruption of the TACE catalytic domain were severely impaired in inducible GHR proteolysis (23). To broaden our characterization of the impact of TACE on GHR proteolysis, we tested the effects of knockdown of TACE in our previously described C14 cell line (9, 14, 15). C14 is a stable transfectant resulting from reconstitution of the JAK2-deficient human fibrosarcoma cell, {gamma}2A (35), with both murine JAK2 and rabbit GHR (15). C14 cells respond robustly to GH stimulation and exhibit GHR proteolysis in response to PMA treatment (9). In addition, {gamma}2A and its derivatives, like their parent cell line, HT1080, are efficiently transfectable (38).

As is characteristic of ADAMs, TACE is a transmembrane protein that exists in two forms in cells. The 120-kDa pro-TACE form includes, at the N terminus of its extracellular domain, the so-called prodomain, which serves a negative regulatory function by inhibiting the activity of the nearby catalytic domain (27, 28, 30). The 110-kDa mature form of TACE lacks the prodomain, which is cleaved after biosynthesis; this is the active form and is generally the only form found on the cell surface (39, 40). Kinetic studies indicate that the mature form is substantially longer lived than the pro form (41, 42, 43). Thus, to pursue TACE knockdown, we transfected a well-validated siRNA sequence (37) into C14 cells according to two different schemes: short-term and long-term treatments (Fig. 1Go), as described in Materials and Methods. This was compared with long-term transfection of a control siRNA (silencer negative control 3 siRNA from Ambion, Inc.) as a negative control (short- and long-term control siRNA transfections produced indistinguishable results; data not shown). Both transfection regimens yielded nearly complete loss of pro-TACE, as indicated by anti-TACE immunoblotting and quantitation by densitometry (Fig. 1Go, A and B). Consistent with the longer half-life of mature TACE, its reduction was more dramatic with long-term than with short-term siRNA treatment (Fig. 1Go, A and B), with approximately 80% reduction achieved with long-term treatment. Thus, graded TACE knockdown was achieved by manipulation of the treatment regimen. Notably, blotting with anti-JAK2 serum (Fig. 1CGo) revealed no substantial change in JAK2 levels with TACE siRNA treatment, indicating the relative specificity of the knockdown.


Figure 1
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  		FIG. 1. Knockdown of TACE in C14 cells by RNAi. A and B, Reduction of pro-TACE and mature TACE by short- and long-term siRNA treatment. The two treatment protocols are detailed in Materials and Methods. A, Equal amounts of protein in extracts from treated, serum-starved cells were resolved by SDS-PAGE and immunoblotted with anti-TACE. The positions of the pro-TACE and mature TACE forms are indicated. NS, Nonspecific band. B, Densitometric quantitation of several independent experiments as described in A. In each experiment, the abundance of each form in cells treated with a scrambled siRNA sequence (control) is considered 100% and is compared with the abundance of each after the indicated treatment. Data are plotted as the mean ± SE (n = 4). a, P < 0.001 compared with untreated; b, P < 0.05 compared with untreated; c, P < 0.001 compared with untreated; d, P < 0.05 compared with short-term treatment. C, TACE RNAi effects are specific. Extracts from a representative experiment, described in A, were resolved by SDS-PAGE and immunoblotted with anti-JAK2AL33.

 
The effects of TACE knockdown on GHR proteolysis were examined by immunoblotting with anti-GHRcyt-AL47, a polyclonal serum directed at the GHR cytoplasmic domain (19) (Fig. 2Go). C14 cells subjected to long-term TACE siRNA treatments or control treatment were serum starved overnight, and aliquots of equal protein content were compared. In contrast to the lack of effect on JAK2 levels, long-term knockdown of active TACE was accompanied by increased (~28%) steady-state GHR abundance (Fig. 2Go, A and B). Because both JAK2 and GHR are driven by eukaryotic expression vectors in these cells, these results suggest that the effect of TACE reduction on GHR levels is probably explained by a reduction in GHR proteolytic processing, rather than an effect on GHR gene expression. As we have previously inferred from experiments with chemical metalloprotease inhibitors (17, 20, 21), these results support a role for TACE activity in constitutive GHR proteolysis.


Figure 2
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  		FIG. 2. Effect of TACE RNAi on constitutive and inducible GHR proteolysis in C14 cells. A and B, Effect on basal GHR abundance. A, Cells treated as described in Fig. 1Go were extracted, and equal protein contents were compared by immunoblotting with anti-GHRcyt-AL47. The position of the mature GHR is indicated. B, Densitometric quantitation of several independent experiments as described in A. In each experiment, the abundance of each form in cells treated with a scrambled siRNA sequence (control) was considered 100% and compared with the abundance of each after siRNA treatment. Data are plotted as the mean ± SE (n = 6). C and D, Effect on inducible GHR proteolysis. C, Cells treated with TACE siRNA as described in Figs. 1Go and 2AGo were serum starved and treated with PMA (0.1 µg/ml) for the indicated durations. Extracted proteins were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. The positions of the mature GHR, the GHR remnant protein, and a nonspecific band in the region of the remnant are indicated. D. Densitometric quantitation of several independent experiments as described in C. In each experiment, the abundance within each treatment group of mature GHR in cells treated with vehicle was considered 100% and compared with its abundance after PMA stimulation. Data are plotted as the mean ± SE (n = 4). a, P < 0.05 compared with control; b, P < 0.05 compared with control; c, P < 0.05 compared with short-term treatment. Note the progressive inhibition of GHR proteolysis with increasing degrees of TACE knockdown.

 
To examine inducible GHR proteolysis, C14 cells subjected to short- or long-term TACE siRNA vs. control treatment were serum starved, then exposed to PMA for 15, 30, or 60 min and compared with cells exposed to vehicle for 60 min (Fig. 2Go, C and D). Cell extracts were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47. As shown in the representative immunoblot in Fig. 2CGo, control cells (lanes 1–4) exhibited dramatic PMA-induced loss of GHR (bracket) and accumulation of the GHR remnant (arrow), as expected in these cells (9). Densitometric quantitation of the change in receptor abundance in multiple such experiments (Fig. 2DGo) revealed approximately 90% loss of GHR after 30-min exposure to PMA. Short-term TACE siRNA treatment (lanes 5–8) lessened both the pace and the degree of GHR loss achieved over the 60-min PMA treatment; PMA exposure for 15, 30, and 60 min induced approximately 29%, 62%, and 71% loss of GHR, on the average, respectively. Thus, compared with control cells, short-term TACE knockdown (which reduced active TACE by roughly one half) blunted PMA-induced GHR loss by approximately 30% at 30 min. Additional TACE knockdown, accomplished by long-term TACE siRNA treatment (lanes 9–12), further reduced PMA-induced GHR loss, with a 30-min treatment resulting in more than 75% less loss of receptor compared with the same treatment of control cells. These data confirm that TACE abundance is an important determinant of the susceptibility of GHR to inducible proteolysis in human cells.

Effects of ADAM10 RNAi on GHR proteolysis
TACE and ADAM10 constitute a subfamily within the ADAMs, based on their primary structure. Both have been associated with constitutive and inducible proteolysis of numerous membrane proteins, and for some substrates, they can have overlapping activities (26, 27, 28, 29, 30, 31, 44, 45). We used ADAM10 RNAi to test the influence of ADAM10 on GHR proteolysis. We designed an ADAM10 siRNA targeting the pro domain-encoding sequence of human ADAM10, which has no homology to the region in TACE that was targeted as described above. As with TACE siRNA transfection, we found that long-term treatment with ADAM10 siRNA markedly reduced both the pro and mature forms of ADAM10, as determined by immunoblotting (Fig. 3AGo). Importantly, ADAM10 siRNA did not reduce TACE abundance (Fig. 3BGo), and TACE siRNA did not affect ADAM10 abundance (Fig. 3CGo). In contrast to TACE knockdown (Fig. 2Go, A and B), long-term ADAM10 knockdown had no substantial impact on basal GHR abundance (Fig. 3DGo and data not shown).


Figure 3
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  		FIG. 3. Effect of ADAM10 RNAi on GHR proteolysis in C14 cells. A–C, Effect and specificity of ADAM10 RNAi. A, Equal amounts of protein in extracts from long-term ADAM10 siRNA-treated, serum-starved cells were immunoprecipitated with anti-ADAM10 or anti-TACE. Precipitated proteins were resolved by SDS-PAGE and immunoblotted with anti-ADAM10. The positions of the pro-ADAM10 and mature ADAM10 forms are indicated. B, Comparison of long-term TACE vs. ADAM10 siRNA treatment on TACE abundance. Extracts from treated cells were immunoblotted with anti-TACE. Note that ADAM10siRNA did not reduce TACE abundance. C, Comparison of long-term TACE vs. ADAM10 siRNA treatment on ADAM10 abundance. Extracts from treated cells were immunoblotted with anti-ADAM10. a, P < 0.05 compared with control. Note that TACE siRNA did not reduce ADAM10 abundance. D and E, Effect on inducible GHR proteolysis. D, Cells treated long term with ADAM10 siRNA, as described in A–C, were serum starved and treated with PMA (0.1 µg/ml) for the indicated durations. Extracted proteins were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. The positions of the mature GHR, the GHR remnant protein, and a nonspecific band in the region of the remnant are indicated. E, Densitometric quantitation of several independent experiments as described in D. In each experiment, the abundance within each treatment group of mature GHR in cells treated with vehicle is considered 100% and compared with its abundance after PMA stimulation. Data are plotted as the mean ± SE (n = 5). Note the modest inhibition of GHR proteolysis with long-term ADAM10 knockdown.

 
To examine effects on inducible GHR proteolysis, C14 cells subjected to long-term ADAM10 siRNA vs. control treatment were serum starved and treated with PMA over a 60-min time course. Anti-GHRcyt-AL47 immunoblotting (Fig. 3DGo) revealed that PMA-induced receptor loss and remnant accumulation were somewhat blunted in cells treated with ADAM10 siRNA, particularly early in the PMA treatment. Densitometric quantitation (Fig. 3EGo) indicated an approximately 55% reduction in PMA-induced GHR loss after 15 min in ADAM10 siRNA-treated vs. control cells. Although substantial, the effect of ADAM10 siRNA treatment was much less dramatic than similar treatment with TACE siRNA, which resulted in a more than 85% reduction in PMA-induced GHR loss vs. the control value at the same 15 min point (Fig. 2DGo). These data suggest that ADAM10 can contribute to inducible GHR proteolysis in these human fibroblasts, but that its effect is modest compared with that of TACE.

Presence of JAK2 affects GHR proteolysis
Emerging evidence suggests that JAK2, in addition to allowing GH-induced signaling, affects surface GHR stability (14). We explored whether JAK2 affects GHR metalloproteolysis by comparing {gamma}2A-GHR cells (which lack JAK2) with C14 cells (which were derived by stable transfection of {gamma}2A-GHR with JAK2; Fig. 4AGo) (15). We first examined PMA-induced GHR proteolysis and its sensitivity to the metalloprotease inhibitor, IC3 (Immunex Compound 3) (Fig. 4BGo). Serum-starved cells were pretreated with either IC3 or its vehicle, then exposed to PMA or its vehicle for 30 min. Anti-GHRcyt-AL47 immunoblotting revealed that untreated cells differed in the forms of GHR recognized in each. Consistent with our previous reports (14, 15), JAK2-deficient {gamma}2A-GHR cells manifested a greater ratio of precursor:mature GHR than did C14 cells. [Precursor is the endoglycosidase H-sensitive high-mannose GHR form that has yet to reach the cell surface; mature GHR has achieved endoglycosidase H resistance and has traversed the Golgi to populate the surface (14, 15, 21, 46, 47, 48).] Additionally, {gamma}2A-GHR cells displayed substantial remnant, even before PMA stimulation, whereas C14 cells had little remnant basally. PMA treatment for 30 min caused loss of mature receptor in both cell types and the appearance of ample remnant in C14 cells with little change in remnant abundance in {gamma}2A-GHR cells. Each of these PMA-induced changes in GHR and remnant were prevented by IC3, indicating that, as in C14, metalloprotease activity was responsible for GHR processing in {gamma}2A-GHR cells. Notably, however, IC3 treatment of {gamma}2A-GHR cells did not reduce the abundance of the basal remnant. Indeed, treatment with IC3 for as long as 96 h had little effect on basal remnant abundance in these cells (data not shown). Thus, we cannot be certain that the basal remnant in {gamma}2A-GHR cells is derived by metalloprotease activity in the same fashion as is the inducible production of remnant in C14 cells.


Figure 4
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  		FIG. 4. Comparison of GHR metalloproteolysis in JAK2-deficient vs. JAK2-replete cells. A, Lack of JAK2 in {gamma}2A-GHR cells. Equal amounts of protein in extracts from C14 and {gamma}2A-GHR cells were immunoprecipitated with anti-JAK2AL33. Precipitated proteins were resolved by SDS-PAGE and immunoblotted with anti-JAK2AL33. Note the absence of JAK2 in {gamma}2A-GHR cells. B, Effect of metalloprotease inhibitor on inducible GHR proteolysis in JAK2 in {gamma}2A-GHR vs. C14 cells. Serum-starved cells were pretreated with IC3 (50 µM) or vehicle for 15 min before treatment with PMA (1 µg/ml) or vehicle for 30 min. Extracted proteins were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. The positions of the mature GHR, the GHR remnant protein, and a nonspecific band in the region of the remnant are indicated. Note the PMA-induced GHR loss in both cells that is blocked by IC3, the IC3-inhibited PMA-induced remnant accumulation in C14 cells, and the ample basal remnant in {gamma}2A-GHR cells, which is relatively unaffected by either PMA or IC3. C and D, Comparison of inducible GHR proteolysis in {gamma}2A-GHR vs. C14 cells. C, Cells were serum starved and treated with PMA (0.1 µg/ml) for the indicated durations. Extracted proteins were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. The positions of the mature GHR, the GHR remnant protein, and a nonspecific band in the region of the remnant are indicated. D, Densitometric quantitation of several independent experiments as described in C. In each experiment, the abundance within each cell type of mature GHR in cells treated with vehicle is considered 100% and is compared with its abundance after PMA stimulation. Data are plotted as the mean ± SE (n = 3). a, P < 0.05, comparison between cells at the indicated time points. E, Comparison of TACE and ADAM10 levels in {gamma}2A-GHR vs. C14 cells. Equal amounts of proteins in extracts from {gamma}2A-GHR and C14 cells were resolved by SDS-PAGE and immunoblotted with anti-TACE or anti-ADAM10 as indicated. Note that there was no substantial difference in the level of each mature protein between the two cell types.

 
PMA time-course experiments were then performed with each cell type (Fig. 4CGo). Immunoblotting confirmed that the high basal level of remnant in {gamma}2A-GHR cells was not substantially increased by PMA over a 60-min period. Furthermore, densitometric quantitation (Fig. 4DGo) indicated a marked difference in the kinetics of PMA-induced receptor loss between the two cell types. GHR loss in {gamma}2A-GHR cells was much less rapid and robust than that in C14 cells. PMA treatment for 15 and 60 min resulted in approximately 55% and 40% reductions, respectively, in GHR loss in {gamma}2A-GHR cells compared with C14 cells. These results suggest that the degree of inducible GHR metalloproteolysis is diminished in JAK2-deficient cells compared with JAK2-replete cells. Notably, these differences in proteolysis were not accompanied by differences in the abundance of TACE or ADAM10 between the two cells (Fig. 4EGo).

To exert its influence on GH signaling, JAK2 must interact with GHR; this association requires the presence in the receptor’s membrane-proximal cytoplasmic domain of a proline-rich region, referred to as Box1 (36, 49, 50). To determine whether GHR-JAK2 association is related to the effects of JAK2 on GHR proteolysis, we prepared two cell lines. {gamma}2A-GHR{Delta}Box1 resulted from stable transfection of {gamma}2A cells with a GHR mutant in which 14 residues, including Box 1, are internally deleted (36). In {gamma}2A-GHR{Delta}Box1-JAK2, both the Box 1-deletent GHR and JAK2 were stably expressed in {gamma}2A cells. GHR{Delta}Box1 is expressed on the cell surface and binds GH with normal affinity; however, it is unable to transduce GH-induced activation of signaling even when expressed in cells that possess JAK2 (36). We examined the effects of PMA on mature GHR abundance in these two cell lines in the experiments summarized in Fig. 5AGo. Both cells were treated with PMA for 15 min, and GHR abundance was determined by anti-GHRcyt-AL47 immunoblotting and densitometry. The remaining GHR was expressed as a fraction of GHR present before PMA treatment; this fraction, determined in several independent experiments, was compared with the remaining GHR after PMA treatment of {gamma}2A-GHR and C14 cells. This analysis revealed that PMA-induced GHR loss was very similar for {gamma}2A-GHR{Delta}Box1 and {gamma}2A-GHR cells, and both significantly differed from that in C14 cells. Notably, PMA treatment caused no additional GHR loss in {gamma}2A-GHR{Delta}Box1-JAK2 compared with {gamma}2A-GHR{Delta}Box1 cells. Thus, the presence of JAK2 at similar levels (Fig. 5BGo) did not confer the degree of PMA-induced GHR loss in cells harboring the Box1 mutant GHR ({gamma}2A-GHR{Delta}Box1-JAK2 cells) that it did in cells that expressed wild-type GHR (C14 cells). These data suggest that GHR-JAK2 association is required for JAK2-mediated enhancement of inducible GHR proteolysis.


Figure 5
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  		FIG. 5. JAK2 does not influence PMA-induced GHR loss in cells that express GHR{Delta}Box1. A, {gamma}2A-GHR{Delta}Box1 and {gamma}2A-GHR{Delta}Box1-JAK2 cells were treated with PMA for 15 min and processed for anti-GHRcyt-AL47 immunoblotting and densitometry as described in Fig. 4Go, C and D. In each experiment the abundance within each cell type of mature GHR in cells treated with vehicle is considered 100% and is compared with its abundance after PMA stimulation. Data are plotted as the mean ± SE (n = 4 for {gamma}2A-GHR, n = 5 for {gamma}2A-GHR{Delta}Box1, n = 6 for {gamma}2A-GHR{Delta}Box1-JAK2, and n = 6 for C14 cells). a, P < 0.01, comparison between C14 and each other cell type. All others were not significantly different from each other. B, {gamma}2A-GHR{Delta}Box1-JAK2 and C14 cells express similar levels of JAK2. Equal amounts of protein extract from the two cells were resolved by SDS-PAGE and immunoblotted with anti-JAK2AL33.

 
Differential effects of anti-GHRext-mAb on GHR proteolysis in JAK2-deficient vs. JAK2-replete cells
Although both TACE and ADAM10 contribute to GHR proteolysis, our data suggest that altered abundance of these metalloproteases probably does not explain the effects of JAK2 on receptor processing. This raises the possibility that JAK2 could be exerting its effects by virtue of influencing the GHR’s susceptibility to proteolysis. To examine this possibility, we employed anti-GHRext-mAb, our monoclonal antibody directed at the receptor extracellular domain (9, 17, 33). Anti-GHRext-mAb immunoreacts with extracellular subdomain 2, a region that has within it the so-called dimerization interface, and engages the receptor on the surface of intact cells (9). Anti-GHRext-mAb is a conformationally sensitive antibody in that GH-induced conformational changes in the receptor dramatically reduce its immunoreactivity; furthermore, pretreatment of cells with this monoclonal antibody block GH-induced signaling, an effect not explained by prevention of GH binding (9). Notably, we have also observed that PMA-inducible GHR proteolysis in C14 cells is blocked by pretreatment with anti-GHRext-mAb, even though the antibody does not react with the perimembraneous stem region of the receptor that harbors the actual cleavage site (9, 20, 21).

We compared the effects of pretreatment with anti-GHRext-mAb on inducible receptor proteolysis in C14 vs. {gamma}2A-GHR cells (Fig. 6AGo). Serum-starved cells were pretreated with or without anti-GHRext-mAb (12 µg/ml) for 30 min before treatment with vehicle or PMA for 45 min. Immunoblotting of cell extracts with anti-GHRcyt-AL47 confirmed our previous findings that PMA-induced GHR loss and remnant accumulation were completely blocked by anti-GHRext-mAb pretreatment in C14 cells. Notably, however, PMA-induced loss of the mature GHR in {gamma}2A-GHR cells was unaffected by anti-GHRext-mAb pretreatment. Densitometric quantitation of several such experiments is shown in Fig. 6BGo. This interesting difference in receptor proteolytic sensitivity could, in principle, result from inability of the antibody to interact with the mature receptor on the surface of {gamma}2A-GHR cells. Because the lack of JAK2 in these cells precludes GH-induced signaling, we cannot verify productive anti-GHRext-mAb-GHR interaction by monitoring effects on GH-induced intracellular tyrosine phosphorylation in {gamma}2A-GHR as we have previously done in C14 cells (9). However, we previously showed that GHR dimers on the cell surface undergo interchain disulfide linkage via an unpaired cysteine residue (cysteine 241) in the extracellular stem region upon stimulation with GH and that this disulfide linkage reflects the GH-induced receptor conformational changes required for signaling (19, 33, 51). GH-induced GHR disulfide linkage occurs even in the absence of JAK2, and in C14 cells is blocked by anti-GHRext-mAb pretreatment (9, 19). To determine whether GHR on the surface of {gamma}2A-GHR cells is accessible to this antibody, we compared the ability of anti-GHRext-mAb pretreatment to block GH-induced GHR disulfide linkage in {gamma}2A-GHR vs. C14 cells (Fig. 6CGo). Serum-starved cells were pretreated with or without anti-GHRext-mAb before treatment with GH or vehicle for 10 min. Cells were extracted, and proteins were resolved under nonreducing conditions and immunoblotted with anti-GHRcyt-AL47. As expected, GH caused the appearance of the high-molecular mass, disulfide-linked form of the GHR in both cell types, and anti-GHRext-mAb blocked this disulfide linkage in C14 cells. Importantly, antibody pretreatment also blocked GH-induced GHR disulfide linkage in {gamma}2A-GHR cells, indicating that the receptor in those JAK2-deficient cells was accessible to anti-GHRext-mAb. Thus, the inability of the antibody to block inducible GHR proteolysis in {gamma}2A-GHR cells cannot be explained by a lack of interaction with the receptor.


Figure 6
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  		FIG. 6. The effect of anti-GHRext-mAb pretreatment on inducible GHR proteolysis depends on JAK2 expression status. A and B, Inducible GHR proteolysis in anti-GHRext-mAb-pretreated {gamma}2A-GHR vs. C14 cells. A, Serum-starved cells were pretreated with anti-GHRext-mAb (12 µg/ml) or vehicle for 30 min before treatment with PMA (1 µg/ml) or vehicle for 45 min. Extracted proteins were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. The positions of the mature GHR, the GHR remnant protein, and a nonspecific band in the region of the remnant are indicated. B, Densitometric quantitation of several independent experiments as described in A. In each experiment, the abundance within each pretreatment group of mature GHR in cells not treated with PMA was considered 100% and was compared with its abundance after PMA stimulation. Data are plotted as the mean ± SE (n = 3). Note the marked difference in inhibition of inducible GHR proteolysis by anti-GHRext-mAb between the two cells. C, Anti-GHRext-mAb pretreatment inhibited GH-induced GHR disulfide linkage in both {gamma}2A-GHR and C14 cells. Serum-starved cells were pretreated with anti-GHRext-mAb (12 µg/ml) or vehicle for 30 min before treatment with GH (500 ng/ml) or vehicle for 10 min. Extracted proteins were resolved by SDS-PAGE under nonreducing conditions and immunoblotted with anti-GHRcyt-AL47. The positions of the nondisulfide-linked (non-dsl) and disulfide-linked (dsl) GHRs are indicated. D, Inducible GHR proteolysis in GH-pretreated {gamma}2A-GHR vs. C14 cells. Serum-starved cells were pretreated with GH (500 ng/ml) or vehicle for 10 min before treatment with PMA or vehicle for 15 min. Extracted proteins were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. The positions of the mature GHR, the GHR remnant protein, and a nonspecific band in the region of the remnant are indicated. Densitometrically determined mature GHR abundance, in each instance compared with non-PMA-treated cells, is listed for each lane. Note that GH, unlike anti-GHRext-mAb, substantially inhibited PMA-induced GHR proteolysis in both cells.

 
Like anti-GHRext-mAb pretreatment, exposure of cells to GH can affect the sensitivity of GHR to proteolysis (19). We compared {gamma}2A-GHR and C14 cells with regard to this effect of GH in the experiment shown in Fig. 6DGo. As previously reported in other JAK2-expressing cells, pretreatment of serum-starved C14 cells with GH for 30 min markedly reduced GHR proteolysis induced by subsequent PMA treatment for 15 min. This is believed to indicate that GH-induced GHR conformational changes render the receptor less susceptible to cleavage. Notably, in contrast to anti-GHRext-mAb pretreatment, exposure of {gamma}2A-GHR cells to GH inhibited subsequent PMA-induced receptor proteolysis. Thus, inducible GHR proteolysis in cells that lack JAK2 was inhibited by the receptor’s engagement by GH, but not by anti-GHRext-mAb, whereas both GH and anti-GHRext-mAb inhibited inducible GHR proteolysis in JAK2-replete cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Regulated ADAM-mediated proteolytic processing is common to a large and diverse group of cell surface proteins. Targets include, among others, proteins involved in development (e.g. Notch), inflammation (e.g. pro-TNF-{alpha} and chemokines), extracellular matrix (e.g. collagen XVII), adhesion (e.g. L-selectin), growth factor signaling (e.g. proheparin-binding epidermal growth factor and pro-TGF-{alpha}), neuropathology (e.g. amyloid precursor protein), oncogenesis (e.g. ErbB-4), and receptor signaling (e.g. IL-6 receptor, nerve growth factor receptor, and GHR) (29, 30, 52, 53). Multiple functions are ascribed to these cleavage events. For humans, rabbits, and other species, metalloprotease-mediated cleavage of GHR in the perimembraneous stem region liberates the receptor extracellular domain, which can then circulate as a high-affinity GHBP and potentially impact GH action (17, 21, 24, 54). In some species (such as rats and mice), however, GHBP arises principally from alternative splicing of a common GHR precursor mRNA to yield a secreted protein encoding the extracellular domain and a short hydrophilic tail that replaces the transmembrane and cytoplasmic domains (55, 56, 57). Yet, we have shown that the mouse GHR is also susceptible to inducible metalloproteolysis, albeit at a reduced level compared with the rabbit GHR (18, 20). Furthermore, inducible proteolysis of rodent or rabbit GHR desensitizes cells to GH, making us consider that such dynamic regulation of surface GHR may be a highly conserved feature of this processing (18, 21). We also discovered that metalloprotease cleavage of GHR renders the remaining transmembrane/cytoplasmic domain remnant susceptible to additional cleavage by a {gamma}-secretase activity, which liberates the cytoplasmic domain and allows its accumulation in the nucleus (22). Although its functions there are as yet unknown, this two-step processing of GHR is analogous to that seen for the amyloid precursor protein, Notch, and others; in those systems, the nuclear-localized cytoplasmic fragments affect gene expression (58). With these diverse potential functions in mind, we consider it important to understand the mechanism(s) of GHR proteolysis in detail.

In this study we first examined the relative contributions of two related, but distinct, metalloproteases, TACE (ADAM17) and ADAM10. These two have been shown to have overlapping substrate specificities in some instances, and interestingly, in some circumstances, one confers constitutive proteolysis and the other inducible proteolysis to the same substrate (26, 27, 28, 29, 30, 31, 44, 45). Our previous study of fibroblasts from the TACE knockout mouse reconstituted with rabbit GHR with or without TACE indicated that TACE was required in that system for constitutive and inducible GHR proteolysis and GHBP shedding (23). In the current work we used an RNAi approach to examine GHR proteolysis in human cells. We found that specific knockdown of ADAM10 partially inhibited inducible GHR proteolysis in C14 cells, but had little apparent effect on constitutive proteolysis. Moreover, the effect of TACE knockdown on both constitutive and inducible receptor proteolysis was far more dramatic than that of ADAM10. Furthermore, we exploited the long half-life of the mature form of TACE in varying conditions of TACE knockdown to reduce mature TACE in a graded fashion. This allowed us to detect a rough dose dependency between TACE abundance and the degree of inducible GHR proteolysis. In concert with our previous findings in mouse fibroblasts, these results strongly suggest that TACE is the principal ADAM used for proteolysis of rabbit GHR. Given the high homology between rabbit and human GHR, we tentatively infer the same conclusion for the human receptor. With the emerging design of drugs that may be selective for TACE vs. ADAM10 (44) and the potentially important role of GHR proteolysis in GH action, we view our findings as relevant for future studies in which specific inhibition of one or the other metalloprotease is pursued. We note that recent studies suggest that ADAM9 and -10 can collaborate in cleavage of cellular prion, and in that system, ADAM9 may contribute its effects indirectly by altering the ability of ADAM10 to carry out the cleavage (59). Whether our findings that both TACE and ADAM10 may exert effects on GHR cleavage reflect such indirect effects by one or the other is a topic worthy of additional study.

We also pursued the potential effects of JAK2 on GHR proteolysis. We embarked on these studies because of recent work in our laboratory and others indicating that JAKs may have roles in hormone and cytokine signaling in addition to their roles as the tyrosine kinases necessary for cytokine receptor family signal propagation (14, 15, 60, 61, 62, 63, 64). JAK2 fosters enhanced cell surface GHR abundance in at least two ways (14). First, it modestly stimulates GHR maturation during biosynthesis. Second, the mature cell surface GHR is markedly stabilized in cells that express JAK2 compared with those that are JAK2 deficient. The mechanisms underlying this stabilization are not entirely clear, but probably involve either diminished constitutive GHR endocytosis and/or enhanced recycling of constitutively endocytosed receptors. Notably, short-term (several hours) treatment with metalloprotease inhibitors did not extend the half-life of GHRs in JAK2-deficient cells (14).

Nonetheless, in the current study we observed marked differences in GHR proteolysis in JAK2-deficient vs. JAK2-replete cells. In {gamma}2A-GHR cells, a high basal level of remnant protein was detected, but it is unlikely to originate largely from metalloproteolysis of the GHR, because its abundance was unaltered by IC3 treatment. Rather, it may arise by an as yet uncharacterized, IC3-resistant GHR protease activity that is potentiated in the absence of JAK2. In contrast, PMA-inducible GHR loss in both cell types and remnant accumulation in C14 cells were metalloprotease mediated, and there were significant differences in the pace and degree of these inducible metalloproteolytic events between C14 and {gamma}2A-GHR cells. These findings suggest important influences of JAK2 on receptor processing such that the presence of JAK2 facilitates inducible GHR metalloproteolysis.

This is emphasized by our studies with anti-GHRext-mAb. In C14 cells, this conformation-specific antibody blocks inducible GHR proteolysis (9) and GH-induced signaling (9), but only partially inhibits GH binding (9). By assessing GH-induced GHR disulfide linkage, we also found that anti-GHRext-mAb binds GHR on the surface of both C14 (9) and {gamma}2A-GHR (this study) cells. Yet, interestingly, anti-GHRext-mAb did not block inducible GHR proteolysis in {gamma}2A-GHR cells. Thus, JAK2 renders mature GHR more sensitive to inducible proteolysis, but also allows anti-GHRext-mAb pretreatment to block such processing. This suggests that in addition to its effects on GHR signaling, trafficking, and surface stability, JAK2 has a role in regulating inducible receptor metalloproteolysis. How could JAK2 exert this effect? Several testable possibilities might be considered. First, JAK2 interacts with GHR, even in the absence of ligand engagement (4, 11, 36, 49), and cells harboring a GHR mutated in the Box1 JAK2-binding region manifested significantly diminished PMA-induced GHR loss regardless of whether JAK2 was coexpressed. Thus, the ability of JAK2 to enhance inducible GHR proteolysis appears to depend at least in part on its association with the receptor. Perhaps JAK2 interaction with GHR allows it to adopt a conformation in which cleavage is optimal. Second, Schantl et al. (65) detected an association between GHR and TACE. JAK2 may enhance the likelihood of this GHR-TACE interaction, thus fostering catalysis of GHR cleavage. Finally, it is conceivable that JAK2 could directly interact with TACE, thereby promoting the formation of a GHR-JAK2-TACE ternary complex. Future studies will address these potential mechanisms and will additionally exploit anti-GHRext-mAb’s ability to serve as a specific probe of subtle aspects of GHR conformational change that may be important in these effects.


   Acknowledgments
 
We appreciate helpful conversations with Drs. J. Collawn, E. Benveniste, G. Fuller, G. Johnson, C. Chang, R. Serra, J. Messina, Y. Huang, and N. Yang, and the generous provision of reagents by those named in the text.


   Footnotes
 
This work was supported by a Veterans Affairs Merit Review Award (to S.J.F.) and in part by National Institutes of Health Grant DK-58259 (to S.J.F.), a GAANN fellowship (to K.L.), and National Institutes of Health Training Grant T-32-GM-08111 (to J.D.C.).

K.L., L.D., J.W.C., X.W., K.H., J.J., and S.J.F. have nothing to declare. R.A.B. is employed by and has equity in Amgen, Inc.

First Published Online February 23, 2006

Abbreviations: ADAM, A disintegrin and metalloprotease; GHBP, GH-binding protein; GHR, GH receptor; IC3, Immunex Compound 3; JAK2, Janus kinase 2; PMA, phorbol 12-methyl 13-acetate; RNAi, RNA interference; siRNA, small interfering RNA; TACE, TNF-{alpha}-cleaving enzyme.

Received November 22, 2005.

Accepted for publication February 15, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Isaksson OG, Eden S, Jansson JO 1985 Mode of action of pituitary growth hormone on target cells. Annu Rev Physiol 47:483–499[CrossRef][Medline]
  2. Frank SJ, Messina JL 2002 Growth hormone receptor. In: Oppenheim JJ, Feldman M, eds. Cytokine reference on-line. London: Academic Press, Harcourt; 1–21
  3. Bazan JF 1990 Structural design and molecular evolution of a cytokine receptor superfamily. Proc Natl Acad Sci USA 87:6934–6938[Abstract/Free Full Text]
  4. Gent J, van Kerkhof P, Roza M, Bu G, Strous GJ 2002 Ligand-independent growth hormone receptor dimerization occurs in the endoplasmic reticulum and is required for ubiquitin system-dependent endocytosis. Proc Natl Acad Sci USA 99:9858–9863[Abstract/Free Full Text]
  5. Frank SJ 2002 Receptor dimerization in GH and erythropoietin action: it takes two to tango, but how? Endocrinology 143:2–10[Abstract/Free Full Text]
  6. Rowlinson SW, Barnard R, Bastiras S, Robins AJ, Brinkworth R, Waters MJ 1995 A growth hormone agonist produced by targeted mutagenesis at binding site 1. Evidence that site 1 regulates bioactivity. J Biol Chem 270:16833–16839[Abstract/Free Full Text]
  7. Rowlinson SW, Behncken SN, Rowland JE, Clarkson RW, Strasburger CJ, Wu Z, Baumbach W, Waters MJ 1998 Activation of chimeric and full-length growth hormone receptors by growth hormone receptor monoclonal antibodies. A specific conformational change may be required for full-length receptor signaling. J Biol Chem 273:5307–5314[Abstract/Free Full Text]
  8. Mellado M, Rodriguez-Frade JM, Kremer L, von Kobbe C, de Ana AM, Merida I, Martinez AC 1997 Conformational changes required in the human growth hormone receptor for growth hormone signaling. J Biol Chem 272:9189–9196[Abstract/Free Full Text]
  9. Jiang J, Wang X, He K, Li X, Chen C, Sayeski PP, Waters MJ, Frank SJ 2004 A conformationally-sensitive GHR (growth hormone (GH) receptor) antibody: impact on GH signaling and GHR proteolysis. Mol Endocrinol 18:2981–2996[Abstract/Free Full Text]
  10. de Vos AM, Ultsch M, Kossiakoff AA 1992 Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science 255:306–312[Abstract/Free Full Text]
  11. Argetsinger LS, Campbell GS, Yang X, Witthuhn BA, Silvennoinen O, Ihle JN, Carter-Su C 1993 Identification of JAK2 as a growth hormone receptor-associated tyrosine kinase. Cell 74:237–244[CrossRef][Medline]
  12. Carter Su C, Schwartz J, Smit LS 1996 Molecular mechanism of growth hormone action. Annu Rev Physiol 58:187–207[CrossRef][Medline]
  13. Brown RJ, Adams JJ, Pelekanos RA, Wan Y, McKinstry WJ, Palethorpe K, Seeber RM, Monks TA, Eidne KA, Parker MW, Waters MJ 2005 Model for growth hormone receptor activation based on subunit rotation within a receptor dimer. Nat Struct Mol Biol 12:814–821[CrossRef][Medline]
  14. He K, Loesch K, Cowan JW, Li X, Deng L, Wang X, Jiang J, Frank SJ 2005 JAK2 enhances the stability of the mature GH receptor. Endocrinology 145:4755–4765
  15. He K, Wang X, Jiang J, Guan R, Bernstein KE, Sayeski PP, Frank SJ 2003 Janus kinase 2 determinants for growth hormone receptor association, surface assembly, and signaling. Mol Endocrinol 17:2211–2227[Abstract/Free Full Text]
  16. Goldsmith JF, Lee SJ, Jiang J, Frank SJ 1997 Growth hormone induces detergent insolubility of GH receptors in IM-9 cells. Am J Physiol 273:E932–E941
  17. Alele J, Jiang J, Goldsmith JF, Yang X, Maheshwari HG, Black RA, Baumann G, Frank SJ 1998 Blockade of growth hormone receptor shedding by a metalloprotease inhibitor. Endocrinology 139:1927–1935[Abstract/Free Full Text]
  18. Guan R, Zhang Y, Jiang J, Baumann CA, Black RA, Baumann G, Frank SJ 2001 Phorbol ester- and growth factor-induced growth hormone (GH) receptor proteolysis and GH-binding protein shedding: relationship to GH receptor down-regulation. Endocrinology 142:1137–1147[Abstract/Free Full Text]
  19. Zhang Y, Guan R, Jiang J, Kopchick JJ, Black RA, Baumann G, Frank SJ 2001 Growth hormone (GH)-induced dimerization inhibits phorbol ester-stimulated GH receptor proteolysis. J Biol Chem 276:24565–24573[Abstract/Free Full Text]
  20. Wang X, He K, Gerhart M, Jiang J, Paxton RJ, Menon RK, Black RA, Baumann G, Frank SJ 2003 Reduced proteolysis of rabbit growth hormone (GH) receptor substituted with mouse GH receptor cleavage site. Mol Endocrinol 17:1931–1943[Abstract/Free Full Text]
  21. Wang X, He K, Gerhart M, Huang Y, Jiang J, Paxton RJ, Yang S, Lu C, Menon RK, Black RA, Baumann G, Frank SJ 2002 Metalloprotease-mediated GH receptor proteolysis and GHBP shedding. Determination of extracellular domain stem region cleavage site. J Biol Chem 277:50510–50519[Abstract/Free Full Text]
  22. Cowan JW, Wang X, Guan R, He K, Jiang J, Baumann G, Black RA, Wolfe MS, Frank SJ 2005 Growth hormone receptor is a target for presenilin-dependent {gamma}-secretase cleavage. J Biol Chem 280:19331–19342[Abstract/Free Full Text]
  23. Zhang Y, Jiang J, Black RA, Baumann G, Frank SJ 2000 TACE is a growth hormone binding protein sheddase: the metalloprotease TACE/ADAM-17 is critical for (PMA-induced) growth hormone receptor proteolysis and GHBP generation. Endocrinology 141:4324–4348
  24. Baumann G 2001 Growth hormone binding protein 2001. J Pediatr Endocrinol Metab 14:355–375[Medline]
  25. Baumann G, Frank SJ 2002 Metalloproteinases and the modulation of GH signaling. J Endocrinol 174:361–368[Abstract]
  26. Blobel CP 2005 ADAMs: key components in EGFR signalling and development. Nat Rev Mol Cell Biol 6:32–43[CrossRef][Medline]
  27. Black RA, White JM 1998 ADAMs: focus on the protease domain. Curr Opin Cell Biol 10:654–659[CrossRef][Medline]
  28. Becherer JD, Blobel CP 2003 Biochemical properties and functions of membrane-anchored metalloprotease-disintegrin proteins (ADAMs). Curr Top Dev Biol 54:101–123[Medline]
  29. Huovila AP, Turner AJ, Pelto-Huikko M, Karkkainen I, Ortiz RM 2005 Shedding light on ADAM metalloproteinases. Trends Biochem Sci 30:413–422[CrossRef][Medline]
  30. Seals DF, Courtneidge SA 2003 The ADAMs family of metalloproteases: multidomain proteins with multiple functions. Genes Dev 17:7–30[Free Full Text]
  31. Schlondorff J, Blobel CP 1999 Metalloprotease-disintegrins: modular proteins capable of promoting cell-cell interactions and triggering signals by protein-ectodomain shedding. J Cell Sci 112:3603–3617[Abstract]
  32. Jiang J, Liang L, Kim SO, Zhang Y, Mandler R, Frank SJ 1998 Growth hormone-dependent tyrosine phosphorylation of a GH receptor-associated high molecular weight protein immunologically related to JAK2. Biochem Biophys Res Commun 253:774–779[CrossRef][Medline]
  33. Zhang Y, Jiang J, Kopchick JJ, Frank SJ 1999 Disulfide linkage of growth hormone (GH) receptors (GHR) reflects GH-induced GHR dimerization. Association of JAK2 with the GHR is enhanced by receptor dimerization. J Biol Chem 274:33072–33084[Abstract/Free Full Text]
  34. Kim SO, Jiang J, Yi W, Feng GS, Frank SJ 1998 Involvement of the Src homology 2-containing tyrosine phosphatase SHP-2 in growth hormone signaling. J Biol Chem 273:2344–2354[Abstract/Free Full Text]
  35. Kohlhuber F, Rogers NC, Watling D, Feng J, Guschin D, Briscoe J, Witthuhn BA, Kotenko SV, Pestka S, Stark GR, Ihle JN, Kerr IM 1997 A JAK1/JAK2 chimera can sustain {alpha} and {gamma} interferon responses. Mol Cell Biol 17:695–706[Abstract]
  36. Frank SJ, Gilliland G, Kraft AS, Arnold CS 1994 Interaction of the growth hormone receptor cytoplasmic domain with the JAK2 tyrosine kinase. Endocrinology 135:2228–2239[Abstract]
  37. Gschwind A, Hart S, Fischer OM, Ullrich A 2003 TACE cleavage of proamphiregulin regulates GPCR-induced proliferation and motility of cancer cells. EMBO J 22:2411–2421[CrossRef][Medline]
  38. Watling D, Guschin D, Muller M, Silvennoinen O, Witthuhn BA, Quelle FW, Rogers NC, Schindler C, Stark GR, Ihle JN, Kerr IM 1993 Complementation by the protein tyrosine kinase JAK2 of a mutant cell line defective in the interferon-{gamma} signal transduction pathway. Nature 366:166–170[CrossRef][Medline]
  39. Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner BJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA, Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, Cerretti DP 1997 A metalloproteinase disintegrin that releases tumour-necrosis factor-{alpha} from cells. Nature 385:729–733[CrossRef][Medline]
  40. Moss ML, Jin SL, Milla ME, Bickett DM, Burkhart W, Carter HL, Chen WJ, Clay WC, Didsbury JR, Hassler D, Hoffman CR, Kost TA, Lambert MH, Leesnitzer MA, McCauley P, McGeehan G, Mitchell J, Moyer M, Pahel G, Rocque W, Overton LK, Schoenen F, Seaton T, Su JL, Warner J, Willard D, Becherer JD 1997 Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-{alpha}. Nature 385:733–736[CrossRef][Medline]
  41. Doedens JR, Black RA 2000 Stimulation-induced down-regulation of tumor necrosis factor-{alpha} converting enzyme. J Biol Chem 275:14598–14607[Abstract/Free Full Text]
  42. Borroto A, Ruiz-Paz S, de la Torre TV, Borrell-Pages M, Merlos-Suarez A, Pandiella A, Blobel CP, Baselga J, Arribas J 2003 Impaired trafficking and activation of tumor necrosis factor-{alpha}-converting enzyme in cell mutants defective in protein ectodomain shedding. J Biol Chem 278:25933–25939[Abstract/Free Full Text]
  43. Schlondorff J, Becherer JD, Blobel CP 2000 Intracellular maturation and localization of the tumour necrosis factor {alpha} convertase (TACE). Biochem J 347:131–138[CrossRef][Medline]
  44. Ludwig A, Hundhausen C, Lambert MH, Broadway N, Andrews RC, Bickett DM, Leesnitzer MA, Becherer JD 2005 Metalloproteinase inhibitors for the disintegrin-like metalloproteinases ADAM10 and ADAM17 that differentially block constitutive and phorbol ester-inducible shedding of cell surface molecules. Comb Chem High Throughput Screen 8:161–171[CrossRef][Medline]
  45. Maretzky T, Reiss K, Ludwig A, Buchholz J, Scholz F, Proksch E, de Strooper B, Hartmann D, Saftig P 2005 ADAM10 mediates E-cadherin shedding and regulates epithelial cell-cell adhesion, migration, and ß-catenin translocation. Proc Natl Acad Sci USA 102:9182–9187[Abstract/Free Full Text]
  46. Yi W, Kim SO, Jiang J, Park SH, Kraft AS, Waxman DJ, Frank SJ 1996 Growth hormone receptor cytoplasmic domain differentially promotes tyrosine phosphorylation of signal transducers and activators of transcription 5b and 3 by activated JAK2 kinase. Mol Endocrinol 10:1425–1443[Abstract/Free Full Text]
  47. Sotiropoulos A, Goujon L, Simonin G, Kelly PA, Postel-Vinay MC, Finidori J 1993 Evidence for generation of the growth hormone-binding protein through proteolysis of the growth hormone membrane receptor. Endocrinology 132:1863–1865[Abstract/Free Full Text]
  48. Lobie PE, Wood TJJ, Chen CM, Waters MJ, Norstedt G 1994 Nuclear translocation and anchorage of the growth hormone receptor. J Biol Chem 269:31735–31746[Abstract/Free Full Text]
  49. Vanderkuur JA, Wang X, Zhang L, Campbell GS, Allevato G, Billestrup N, Norstedt G, Carter-Su C 1994 Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase. J Biol Chem 269:21709–21717[Abstract/Free Full Text]
  50. Sotiropoulos A, Perrot-Applanat M, Dinerstein H, Pallier A, Postel-Vinay MC, Finidori J, Kelly PA 1994 Distinct cytoplasmic regions of the growth hormone receptor are required for activation of JAK2, mitogen-activated protein kinase, and transcription. Endocrinology 135:1292–1298[Abstract]
  51. Frank SJ, Gilliland G, Van Epps C 1994 Treatment of IM-9 cells with human growth hormone (GH) promotes rapid disulfide linkage of the GH receptor. Endocrinology 135:148–156[Abstract]
  52. White JM 2003 ADAMs: modulators of cell-cell and cell-matrix interactions. Curr Opin Cell Biol 15:598–606[CrossRef][Medline]
  53. Killar L, White J, Black R, Peschon J 1999 Adamalysins. A family of metzincins including TNF-{alpha} converting enzyme (TACE). Ann NY Acad Sci 878:442–452[CrossRef][Medline]
  54. Baumann G, Amburn K, Shaw MA 1988 The circulating growth hormone (GH)-binding protein complex: a major constituent of plasma GH in man. Endocrinology 122:976–984[Abstract/Free Full Text]
  55. Baumbach WR, Horner DL, Logan JS 1989 The growth hormone-binding protein in rat serum is an alternatively spliced form of the rat growth hormone receptor. Genes Dev 3:1199–1205[Abstract/Free Full Text]
  56. Sadeghi H, Wang BS, Lumanglas AL, Logan JS, Baumbach WR 1990 Identification of the origin of the growth hormone-binding protein in rat serum. Mol Endocrinol 4:1799–1805[Abstract/Free Full Text]
  57. Smith WC, Kuniyoshi J, Talamantes F 1989 Mouse serum growth hormone (GH) binding protein has GH receptor extracellular and substituted transmembrane domains. Mol Endocrinol 3:984–990[Abstract/Free Full Text]
  58. Kopan R, Ilagan MX 2004 {gamma}-Secretase: proteasome of the membrane? Nat Rev Mol Cell Biol 5:499–504[CrossRef][Medline]
  59. Cisse MA, Sunyach C, Lefranc-Jullien S, Postina R, Vincent B, Checler F 2005 The disintegrin ADAM9 indirectly contributes to the physiological processing of cellular prion by modulating ADAM10 activity. J Biol Chem 49:40624–40631
  60. Radtke S, Hermanns HM, Haan C, Schmitz-Van De Leur H, Gascan H, Heinrich PC, Behrmann I 2002 Novel role of Janus kinase 1 in the regulation of oncostatin M receptor surface expression. J Biol Chem 277:11297–11305[Abstract/Free Full Text]
  61. Ragimbeau J, Dondi E, Alcover A, Eid P, Uze G, Pellegrini S 2003 The tyrosine kinase Tyk2 controls IFNAR1 cell surface expression. EMBO J 22:537–547[CrossRef][Medline]
  62. Suzuki K, Nakajima H, Watanabe N, Kagami S, Suto A, Saito Y, Saito T, Iwamoto I 2000 Role of common cytokine receptor {gamma} chain ({gamma}c)- and Jak3-dependent signaling in the proliferation and survival of murine mast cells. Blood 96:2172–2180[Abstract/Free Full Text]
  63. Huang LJ, Constantinescu SN, Lodish HF 2001 The N-terminal domain of Janus kinase 2 is required for Golgi processing and cell surface expression of erythropoietin receptor. Mol Cell 8:1327–1338[CrossRef][Medline]
  64. Hofmann SR, Lam AQ, Frank S, Zhou YJ, Ramos HL, Kanno Y, Agnello D, Youle RJ, O’Shea JJ 2004 Jak3-independent trafficking of the common {gamma} chain receptor subunit: chaperone function of Jaks revisited. Mol Cell Biol 24:5039–5049[Abstract/Free Full Text]
  65. Schantl JA, Roza M, Van Kerkhof P, Strous GJ 2004 The growth hormone receptor interacts with its sheddase, the tumour necrosis factor-{alpha}-converting enzyme (TACE). Biochem J 377:379–384[CrossRef][Medline]



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