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Endocrinology, doi:10.1210/en.2007-0455
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Endocrinology Vol. 148, No. 12 5955-5965
Copyright © 2007 by The Endocrine Society

Endoplasmic Reticulum-Associated Degradation of Growth Hormone Receptor in Janus Kinase 2-Deficient Cells

Kimberly Loesch, Luqin Deng, Xiangdong Wang, Kai He, Jing Jiang and Stuart J. Frank

Department of Cell Biology (K.L., L.D., S.J.F.) and Department of Medicine (X.W., K.H., J.J., S.J.F.), Division of Endocrinology, Diabetes, and Metabolism, University of Alabama at Birmingham, Birmingham, Alabama 35294; and Endocrinology Section (S.J.F.), Medical Service, Veterans Affairs Medical Center, Birmingham, Alabama 35233

Address all correspondence and requests for reprints to: Stuart J. Frank, University of Alabama at Birmingham, 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
 
A key factor governing cellular sensitivity to GH is cell surface GH receptor (GHR) abundance, which is affected transcriptionally and posttranscriptionally. Mature cell surface GHR abundance is regulated by constitutive and inducible metalloproteolysis and constitutive endosomal/lysosomal degradation. We previously found that Janus kinase 2 (JAK2)-deficient GHR-expressing cells have a greater precursor/mature GHR ratio, exhibit diminished inducible metalloproteolysis, and have a cytoplasmic domain-containing GHR fragment called the basal remnant (by virtue of comigration on SDS-PAGE with the inducible, metalloprotease-generated remnant). Herein we examined the mechanism of generation of basal remnant in JAK2-deficient cells, asking whether it originates from precursor vs. mature receptor and which protease(s) catalyzes its appearance. Prolonged metalloprotease inhibitor treatment or small interfering RNA knockdown of TNF-{alpha} converting enzyme (TACE) and a disintegrin and metalloprotease-10 (ADAM10) (both implicated in inducible GHR proteolysis) did not reduce basal remnant, indicating its generation is not metalloprotease dependent. However, a mutant GHR resistant to metalloprotease cleavage did not yield basal remnant when expressed in JAK2-deficient cells, suggesting common structural determinants for generation of the inducible remnant and the non-metalloprotease-generated basal remnant seen in JAK2-deficient cells. Treatment of JAK2-deficient cells with a proteasome inhibitor, but not two separate lysosome inhibitors, dramatically decreased basal remnant, accompanied by decreased precursor GHR and increased mature GHR abundance. Disruption of endoplasmic reticulum-to-Golgi transport with brefeldin A (BFA) also reduced basal remnant, and washout of BFA allowed regeneration of basal remnant along with GHR precursor. Notably, BFA washout in the presence of cycloheximide blocked both basal remnant and precursor GHR reappearance, but BFA washout in the presence of lactacystin blocked only basal remnant reappearance, suggesting that basal remnant is generated proteasome dependently from precursor GHR. Collectively, our data suggest that JAK2, by association with GHR in the secretory pathway, blunts proteasome activity-dependent discrete GHR cleavage and endoplasmic reticulum-dependent degradation of the precursor receptor. In so doing, JAK2 enables efficient processing of precursor receptor to mature GHR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH IS AN ANTERIOR pituitary-derived hormone that is crucial for normal postnatal growth and metabolism in humans and other vertebrates and exerts its effects by interacting with the cell surface GH receptor (GHR) on target cells (1, 2). The GHR is a broadly expressed type I glycoprotein member of the cytokine receptor superfamily (2, 3). Existing evidence suggests that GHR homodimers form in the endoplasmic reticulum (ER) and that binding of GH at the cell surface results in receptor conformation/orientation changes that activate the GHR-associated tyrosine kinase, Janus kinase 2 (JAK2) and intracellular signaling (4, 5, 6, 7, 8, 9, 10, 11, 12, 13). In addition to its role in GH signaling, JAK2 enhances the abundance of cell surface GHR by influencing its stability (14, 15). This property of JAK2 was unmasked by comparing the effect of protein synthesis inhibition with cycloheximide (CHX) on the fate of the mature GHR in cells that either did or did not express JAK2. The results indicated a substantial prolongation of the half-life of the mature GHR in cells that expressed JAK2 and suggested that this enhanced stability of the mature GHR is in large measure accounted for its reduced constitutive endosomal/lysosomal degradation.

GHR surface abundance is a key determinant of cellular GH sensitivity. In addition to constitutive down-regulation, mature surface GHR availability is also affected by proteolysis and ectodomain shedding. The GHR is a target for cleavage in the proximal extracellular domain, mediated by the metalloprotease TNF-{alpha} converting enzyme [TACE, or a disintegrin and metalloprotease (ADAM)-17] and, to a lesser extent, the structurally similar ADAM10, in response to activators of protein kinase C and some growth factors (16, 17, 18, 19, 20, 21). This inducible metalloproteolytic cleavage results in loss of GHR, 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), in correspondence with the high-affinity GH-binding protein found in the circulation of many species, including humans; the net effect is desensitization to GH (18, 20). Interestingly, cells that lack JAK2 exhibit diminished inducible GHR proteolysis when compared with JAK2-replete cells (19). Knockdown of metalloprotease activity yields increased abundance of mature GHR in cells that express JAK2, suggesting that constitutive surface GHR metalloproteolysis also plays a role in regulating GHR availability (19).

In principle, a third locus of regulation of mature surface GHR availability is receptor biogenesis. Indeed, cells that express JAK2 exhibit enhanced maturation of GHR in comparison with JAK2-deficient cells (14). After synthesis, precursor GHR is transported from the ER to the Golgi apparatus. During Golgi transit, the receptor undergoes maturation that is characterized by changes in the glycosylation pattern such that the mature glycosylated GHR lacks the high-mannose carbohydrates characteristic of the precursor. This transition can be tracked biochemically by determining the sensitivity of immunopurified GHR to in vitro deglycosylation; mature GHR is endoglycosidase H (endoH) resistant in contrast to the high-mannose GHR precursor (that has not yet traversed the Golgi en route to the cell surface), which is sensitive to deglycosylation by endoH (14, 15, 20, 22, 23).

In this study, we further explore the role of JAK2 in regulating GHR biogenesis. We observe in cells where the GHR and JAK2 cannot associate and in JAK2-deficient cells the presence of a cytoplasmic domain-containing GHR fragment that is indistinguishable by SDS-PAGE and immunoblotting from the inducible remnant formed in JAK2-replete cells by metalloprotease activity. We systematically test the potential roles of metalloprotease, proteasome, and lysosome activities in generation of this basal remnant and examine from which receptor form (precursor vs. mature) that this remnant emanates. Our data suggest that JAK2, by association with the GHR in the secretory pathway, blunts a proteasome activity-dependent discrete cleavage of the GHR that is linked to ER-associated degradation (ERAD) of the precursor receptor. In so doing, JAK2 enables efficient processing of the precursor receptor to mature GHR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Phorbol myristate acetate (PMA), CHX, clasto-lactacystin ß-lactone (referred to as lactacystin), ammonium chloride, bafilomycin A1, and routine reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO) unless otherwise noted. Brefeldin A (BFA) was purchased from Calbiochem, Inc. (San Deigo, CA). 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). Immunex Compound 3 (IC3) was kindly provided by Dr. Roy Black of Amgen Corp. (Seattle, WA). The {gamma}-secretase inhibitor, N-(N-3,5)-difluorophenacetyl-L-alanyl)-S-phenylglycine-t-butyl ester (DAPT) was used, as described (24).

Antibodies
The ADAM17/TACE and ADAM10 antibodies were purchased from Chemicon International (Temecula, CA). The rabbit polyclonal antisera, anti-GHRcytAL-47, was raised against a bacterially expressed N-terminally His-tagged fusion protein incorporating human GHR residues 271–620 (the entire cytoplasmic domain) and has been previously described (25). Anti-GHRcyt-mAb is a mouse monoclonal antibody (IgG2b{kappa}) directed against a bacterially expressed glutathione S-transferase fusion protein incorporating human GHR residues 271–620 and has been previously described (26).

Cells and cell culture
{gamma}2A is a JAK2-deficient human fibrosarcoma cell line kindly provided by Dr. G. Stark (Cleveland Clinic Foundation, Cleveland, OH) (27). A stable {gamma}2A cell line expressing the rabbit GHR ({gamma}2A-GHR) has been described (25). {gamma}2A-GHR and {gamma}2A-GHR{Delta}Box1 cells were maintained in DMEM (1 g/liter glucose) (Cellgro, Inc., 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 (called C14) was achieved by stable transfection of {gamma}2A-GHR with murine JAK2 as described (15) and was maintained in the above medium supplemented with 100 µg/ml Zeocin. Generation of the stable {gamma}2A-GHR{Delta}Box1 and {gamma}2A-GHR{Delta}Box1-JAK2 cell lines has been described (19). {gamma}2A-GHR{Delta}Box1-JAK2 cells were maintained in the same media as C14 cells. A stable cell line expressing rabbit GHR and murine JAK21–511 was achieved by introducing pcDNA3.1+- JAK21–511-HA-Zeocin (15) into {gamma}2A-GHR cells using Lipofectamine (Invitrogen), 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 3F10 (anti-HA). {gamma}2A-GHR-JAK21–511 cells were maintained in the same medium as C14 cells. Preparation of adenoviruses and adenoviral infection of {gamma}2A cells was achieved using methods previously described (20, 21, 24).

RNA interference
TACE and ADAM10 small interfering RNA (siRNA) duplexes were custom synthesized by Ambion (Austin, TX). The 21-nucleotide sequences, located in the prodomain regions for each, are CAUAGAGCCACUUUGGAGAdTdT (TACE) (28) and GGAUUAUCUUACAAUGUGGdTdT (ADAM10; custom designed by Ambion). Silencer Negative Control #3 siRNA was purchased from Ambion for a negative control. C14 cells were subjected to siRNA knockdown of TACE or ADAM10 for 6 d (19). 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 Opti-MEM. 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., Herndon, VA) 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. Briefly, cells were washed twice with PBS and transfected with 3.25 µg/well of control, TACE, or ADAM10 siRNA in serum- and antibiotic-free Opti-MEM (Invitrogen) for 4–5 h, after which serum was added back to the medium to a final 10% concentration. The next day, the medium was changed to DMEM (1 g/liter glucose; Mediatech) 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 Applied Science, 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. 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 plate (six-well) 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 NaF, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 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 and 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 antibodies anti-TACE (1:5000), anti-ADAM10 (1:3000), and anti-GHRcytAL-47 (1:2000) and with horseradish peroxidase-conjugated antirabbit (1:50,000) and detection reagents (SuperSignal West Pico chemiluminescent substrate) (all from Pierce, Rockford, IL) and stripping and reprobing of blots were accomplished according to the manufacturers’ suggestions.

Blockade of protein synthesis and inhibition of metalloprotease activity
{gamma}2A-GHR cells were grown to 80% confluence in six-well dishes and serum starved overnight. Cells were then incubated with CHX (20 µg/ml) for 4 h. For metalloprotease inhibition experiments, IC3 (50 µM) was included in the serum starvation medium. Cell lysates were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47.

Effects of proteasome, lysosome, and {gamma}-secretase inhibition on {gamma}2A-GHR basal remnant
{gamma}2A-GHR cells were grown to 80% confluence in six-well dishes and serum starved overnight. Cells were pretreated with or without {gamma}-secretase inhibitor for 30 min before addition of lactacystin (5 µM) or vehicle for 5 h. Individual treatments with the various inhibitors are as detailed in the figure legends. Cell extracts were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47.

Effects of BFA on {gamma}2A-GHR basal remnant
{gamma}2A-GHR cells were grown to 80% confluence in six-well dishes and serum starved overnight. Cells were then incubated with BFA (2 µg/ml) for 0–5 h. For BFA washout experiments, serum-starved cells were treated with BFA or vehicle (durations indicated in figure legends) followed by a 3-h washout period in which cells were washed three times with PBS at the conclusion of BFA treatment, and serum starvation medium was added back for a 3-h chase period. Cell extracts were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47.

For enzymatic deglycosylation of BFA-treated cells, extracts were immunoprecipitated with anti-GHRcyt-mAb. Immunoprecipitated proteins were digested with endoH (New England Biolabs, Beverly, MA; 500 U) or N-glycosidase F (New England Biolabs; 500 U) and neuraminidase (New England Biolabs; 50 U) (combination referred to as F/N) in deglycosylation buffer at 37 C overnight according to the manufacturer’s suggestions. Samples were resolved by SDS-PAGE and immunoblotted as above.

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, Bethesda, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GHR-JAK2 association prevents basal remnant generation
Previous findings indicate that the association of GHR with JAK2 is necessary for GH signaling (29, 30, 31, 32, 33) and that inducible GHR metalloproteolysis is influenced by GHR-JAK2 association (19). To study the role of JAK2 in GHR trafficking and signaling, we previously developed a reconstitution system in which JAK2-deficient human fibrosarcoma cells ({gamma}2A) (27) were stably transfected with the GHR alone or with the GHR and JAK2 (the stable clones are referred to as {gamma}2A-GHR and C14 cells, respectively) (14, 15, 19). As previously noted (14, 19), immunoblotting of detergent extracts from serum-starved unstimulated {gamma}2A-GHR and C14 cells with a polyclonal antibody to the GHR cytoplasmic domain revealed three main forms of the receptor (Fig. 1AGo). In both the JAK2-deficient and JAK2-replete cells, the mature and precursor GHRs were detected, as indicated by the bracket and arrowhead, respectively, but, consistent with previous data (14, 15), the ratio of precursor/mature GHR forms was markedly increased in {gamma}2A-GHR cells. [Also indicated with an asterisk is the position of a likely nonglycosylated precursor form (see Discussion).] In addition, a GHR cytoplasmic domain-containing protein of roughly 60 kDa (arrow) was detected in {gamma}2A-GHR cells; this protein migrated in SDS-PAGE indistinguishably from the GHR remnant we previously described as induced in C14 cells in response to PMA by metalloprotease activity (9, 19) and is thus referred to as the basal remnant protein. The basal remnant was markedly more abundant in {gamma}2A-GHR cells than in C14 cells, leading us to hypothesize that this GHR fragment’s abundance is regulated by JAK2.


Figure 1
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  		FIG. 1. Lack of JAK2 association with the GHR results in a basal GHR remnant. A, Difference in pattern of GHR forms in {gamma}2A-GHR vs. C14 cells. Cell extracts from serum-starved {gamma}2A-GHR and C14 cells were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. The positions of the mature GHR, precursor GHR, basal GHR remnant, and a nonspecific (NS) band in the region of the remnant are indicated, as are the positions of the 118- and 83-kDa molecular mass markers (these same markers are indicated on the left side of blots in the remaining figures). Note the higher precursor/mature ratio in {gamma}2A-GHR vs. C14 cells and the presence of a basal GHR remnant. Also indicated with an asterisk is the position of a likely nonglycosylated precursor form (see Discussion). B, Comparison of the pattern of the various GHR forms in C14, {gamma}2A-GHR{Delta}Box1, {gamma}2A-GHR{Delta}Box1-JAK2, and {gamma}2A-GHR cells. Protein extracts from serum-starved cells were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. Note the abundance of basal remnant in cells expressing GHR harboring deletion of Box1 regardless of JAK2 expression. The data shown are representative of three such experiments. C, Comparison of the pattern of the various GHR forms in {gamma}2A-GHR-JAK21–511 and {gamma}2A-GHR cells. Protein extracts from serum-starved cells were prepared as in A and immunoblotted with anti-GHRcyt-AL47. Note the lack of basal remnant in {gamma}2A-GHR-JAK21–511. The data shown are representative of three such experiments.

 
The GHR-JAK2 association requires the proline-rich Box1 region in the GHR proximal cytoplasmic domain and the amino-terminal FERM (Band 4.1, ezrin, radixin, moesin) domain region of JAK2 (15, 29, 31, 33). We previously prepared stably transfected clones that expressed the GHR mutant with an internal deletion of Box1, GHR{Delta}Box1, either alone ({gamma}2A-GHR{Delta}Box1) or with JAK2 ({gamma}2A-GHR{Delta}Box1-JAK2) (19). In Fig. 1BGo, serum-starved {gamma}2A-GHR{Delta}Box1 and {gamma}2A-GHR{Delta}Box1-JAK2 cells were compared with {gamma}2A-GHR and C14 cells. Anti-GHR immunoblotting of detergent extracts revealed the presence of basal remnant in {gamma}2A-GHR, but not C14 cells, as anticipated. Expression of the Box1 mutant GHR in the absence of JAK2 ({gamma}2A-GHR{Delta}Box1) also resulted in basal remnant generation; notably, the introduction of JAK2 in cells harboring the Box1 mutant receptor ({gamma}2A-GHR{Delta}Box1-JAK2) did not prevent basal remnant generation, unlike cells that express wild-type GHR. This suggests that the Box1 element in GHR, which allows JAK2 association, also is required for protection of the receptor from basal remnant-generating proteolysis. In Fig. 1CGo, we compared in the same fashion {gamma}2A-GHR cells with {gamma}2A-GHR-JAK21–511 cells, in which are expressed the wild-type GHR and a JAK2 mutant that includes residues 1–511 (which includes the FERM domain, but not the pseudokinase or kinase domains) (15). Interestingly, JAK21–511, which retains the capacity to associate with GHR but cannot function as a kinase, prevented basal remnant generation. These data suggest that the ability of JAK2 to associate with GHR is critical for prevention of GHR basal remnant generation.

Basal GHR remnant generation is prevented by mutation of the inducible cleavage site
Metalloprotease cleavage of GHR occurs in the membrane-proximal extracellular domain stem region, and deletion of three residues (237–239) of the rabbit GHR prevents PMA-induced receptor cleavage (20, 21). We tested whether expression of a GHR with deletion of these residues in {gamma}2A cells (which lack both GHR and JAK2) would affect the level of basal GHR remnant (Fig. 2Go). Wild-type GHR (GHR-WT) and mutant GHR (GHR{Delta}237–239) (20) were cloned into vectors that directed expression of adenoviruses encoding each and cells were infected with either Ad-GHR-WT or Ad-GHR{Delta}237–239. Serum-starved cells were lysed, and detergent extracts were resolved by SDS-PAGE and blotted for GHR (Fig. 2AGo). Mature and precursor GHR was detected in both cells. Notably, however, basal remnant was detected in cells expressing WT receptor but not GHR{Delta}237–239. Treatment of Ad-GHR-WT cells with PMA for 15 min resulted in mature GHR loss and a small increase in remnant (Fig. 2BGo), confirming that the adenoviral expression system behaved similarly to the stable transfection system, as expected. Thus, deletion of the same residues that prevents inducible receptor cleavage also prevented basal remnant generation.


Figure 2
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  		FIG. 2. A metalloprotease cleavage-defective GHR mutant (GHR{Delta}237–239) does not generate basal remnant in JAK2-deficient cells. A and B, GHR-WT and GHR{Delta}237–239 were cloned into vectors directing expression of adenoviruses encoding each. {gamma}2A cells, which lack both GHR and JAK2, were infected with adenovirus encoding either GHR-WT or GHR{Delta}237–239 for 48 h. The positions of the mature GHR, precursor GHR, basal GHR remnant, and a nonspecific (NS) band in the region of the remnant are indicated. A, Twenty-four hours after infection, cells were serum starved overnight, and protein extracts were compared by immunoblotting with anti-GHRcyt-AL47. B, Serum-starved cells that express GHR-WT were treated with vehicle or PMA (0.1 µg/ml) for 15 min, followed by anti-GHRcyt-AL47 immunoblotting. Note the presence of basal remnant and the inducible loss of mature GHR in Ad-GHR-WT-expressing cells but the absence of basal remnant in cells expressing Ad-GHR{Delta}237–239. The data shown are representative of three such experiments.

 
Basal GHR remnant protein in JAK2-deficient cells is not a product of metalloprotease cleavage of the receptor
Because a cleavage-defective mutant GHR does not produce basal remnant and the basal remnant in JAK2-deficient cells comigrates with the GHR remnant produced by metalloprotease-dependent receptor cleavage, we reasoned that the basal remnant might also be formed in a metalloprotease-dependent fashion. To approach this, we first investigated the relative stability of the basal remnant. Whereas the stability of the mature GHR is markedly enhanced by JAK2, treatment of JAK2-deficient cells with the protein synthesis inhibitor CHX results in rapid loss of anti-GHR-detectable mature receptor; furthermore, CHX treatment of either JAK2-deficient or JAK2-replete cells results in rapid loss of precursor GHR (14). Notably, CHX treatment of {gamma}2A-GHR cells for 3 h or more resulted in nearly complete loss of the basal remnant (see Fig. 7DGo, and data not shown), indicating that, like the inducible remnant (24), the basal remnant found in JAK2-deficient cells is relatively labile.


Figure 7
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  		FIG. 7. Basal remnant is generated from GHR in the secretory pathway. A, Effects of BFA on mature, precursor, and basal remnant GHR. Serum-starved {gamma}2A-GHR cells were treated with BFA (2 µg/ml) for 1–5 h or vehicle for 5 h. Equal amounts of extracted protein were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. Note the loss of basal remnant and conversion of precursor GHR to endoH-resistant mature GHR. The data shown are representative of four such experiments. The positions of the mature GHR, precursor GHR, basal GHR remnant, and a nonspecific (NS) band in the region of the remnant are indicated. Also indicated with an asterisk is the position of a likely nonglycosylated precursor form (see Discussion). B, BFA treatment causes newly synthesized GHR to become glycosylated, Serum-starved {gamma}2A-GHR were treated with BFA (2 µg/ml) for 2 and 5 h or vehicle for 5 h. Cell lysates were immunoprecipitated with anti-GHRcyt-mAb. The precipitated protein was split into three equal fractions and treated as indicated with endoH, F/N, or vehicle, as described in Materials and Methods. Proteins were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. The positions of endoH-resistant and endoH-sensitive forms are indicated. The data shown are representative of two such experiments. C, BFA washout allows regeneration of basal remnant and reappearance of mature and precursor GHR. Serum-starved cells were treated as described in B followed by a 3-h BFA washout period. Cell lysates were immunoprecipitated (IP) with anti-GHRcyt-mAb. Proteins were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. Note the reappearance of the basal remnant upon removal of BFA. The data shown are representative of six such experiments. D, Generation of basal remnant derives from precursor GHR. Serum-starved cells were treated with BFA for 5 h followed by a 3-h BFA washout period in the presence of either CHX or lactacystin (lacta). The 5-h CHX and lactacystin treatments were begun after the initial 3 h of BFA treatment; both were added back for the duration of the 3-h washout period. The 3-h CHX and lactacystin treatments were begun at the start of the BFA washout period. Cell lysates were immunoprecipitated with anti-GHRcyt-mAb, resolved by SDS-PAGE, and immunoblotted with anti-GHRcyt-AL47. The data shown are representative of three such experiments.

 
To directly test whether or not the basal remnant is a product of metalloprotease cleavage, we treated {gamma}2A-GHR cells with the potent hydroxamate-based metalloprotease inhibitor IC3, which completely inhibits PMA- and platelet-derived growth factor-induced GHR metalloproteolysis in a variety of cell types, including {gamma}2A-GHR (16, 18, 19). Notably, neither short-term (15 min) treatment (19) nor treatment with IC3 for 24 h (Fig. 3Go) greatly affected the abundance of the basal remnant. To control for the effects of IC3, JAK2-replete cells (C14) were pretreated with IC3 for 30 min and then stimulated with PMA (Fig. 3Go, lanes 3–5). In accordance with previous reports (16, 19, 20), IC3 pretreatment inhibits the PMA-induced loss of GHR and accumulation of the metalloprotease derived remnant. Because the basal remnant is relatively unstable, the inability of IC3 to affect remnant abundance suggests that metalloprotease activity does not substantially contribute to basal remnant generation.


Figure 3
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  		FIG. 3. Metalloprotease inhibition does not affect generation of basal remnant. {gamma}2A-GHR cells were serum starved for 24 h in the presence of IC3 (50 µM) or vehicle. Serum-starved C14 cells were pretreated with IC3 (50 µM) or vehicle for 30 min before PMA (0.1 µg/ml) stimulation for 30 min. Equal amounts of protein were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. Note that the relative basal GHR remnant is not lessened by prolonged IC3 treatment. The positions of the mature GHR, precursor GHR, basal GHR remnant, and a nonspecific (NS) band in the region of the remnant are indicated. Also indicated with an asterisk is the position of a likely nonglycosylated precursor form (see Discussion).

 
We further addressed this issue by testing the effect of knockdown of relevant metalloproteases on basal remnant abundance. TACE and ADAM10 are two related transmembrane metalloproteases that cleave their substrates in the extracellular domains. We previously identified TACE and, to a lesser extent, ADAM10, as important in PMA-induced GHR proteolysis and GH-binding protein shedding in cells that express JAK2 (17, 19). As in C14 cells (19), treatment of {gamma}2A-GHR cells with TACE siRNA dramatically reduced the level of both the pro-form (which contains the inhibitory pro-domain) and the mature form of TACE protein (Fig. 4Go, A and B). Despite this reduction in TACE levels, the TACE siRNA-treated {gamma}2A-GHR cells displayed a very similar level of basal remnant as did the cells treated with control siRNA (Fig. 4CGo). As an additional control for the efficacy of TACE knockdown, {gamma}2A-GHR cells treated with control vs. TACE siRNA were subjected to PMA treatment for 0–60 min, and detergent extracts were evaluated by anti-GHR immunoblotting (data not shown). Densitometric analysis of several such experiments indicated that PMA-induced GHR loss after 15 min was substantially blunted in cells treated with TACE siRNA (15% loss) compared with control siRNA (38% loss), as found previously in C14 cells (19). Thus, the lack of effect of TACE knockdown on basal remnant abundance in {gamma}2A-GHR cells cannot be accounted for by a lack of knockdown of TACE activity. Similar to the findings for TACE knockdown, treatment of {gamma}2A-GHR cells with ADAM10 siRNA achieved specific reduction of ADAM10 protein and lessened PMA-induced GHR loss but did not affect the level of basal remnant (data not shown). This supports the conclusion that metalloproteases that mediate inducible GHR proteolysis and impact the basal level of mature GHR (19) do not regulate the abundance of the basal remnant seen in the absence of JAK2. Collectively, these data indicate that metalloprotease activity appears not to contribute to basal remnant abundance. In light of the observation that basal remnant is not generated in cells expressing the metalloprotease-resistant mutant GHR, our findings further suggest that a determinant in the GHR extracellular domain required for metalloprotease-mediated GHR cleavage may be shared or similar to that important for the constitutive non-metalloproteolytic cleavage that yields the basal remnant and is protected against by JAK2.


Figure 4
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  		FIG. 4. Knockdown of TACE does not affect basal remnant abundance in JAK2-deficient cells. A and B, Reduction in pro-TACE and mature TACE by siRNA treatment. Cells were subjected to the treatment protocol as detailed in Materials and Methods. A, Equal amounts of protein from serum-starved {gamma}2A-GHR cells treated with a scrambled (control) or TACE siRNA were resolved by SDS-PAGE and immunoblotted with anti-TACE. The positions of pro-TACE and mature TACE are indicated. B, Densitometric quantitation of several independent experiments as described in A. In each experiment, the abundance of each form in cells treated with scrambled siRNA sequence (control) is considered 100% and is compared with the abundance of each after TACE siRNA treatment (white bar, pro-form; black bar, mature form). Data are plotted as the mean ± SE (n = 4). a, P < 0.05 compared with control. Note that efficient knockdown of TACE was achieved. C, Effects of TACE RNAi on basal remnant abundance and inducible GHR proteolysis in {gamma}2A-GHR cells. Cells treated as in A were serum starved, and equal amounts of protein were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. Note that knockdown of TACE by RNAi did not reduce the basal GHR remnant abundance. The positions of the mature GHR, precursor GHR, basal GHR remnant, and a nonspecific (NS) band in the region of the remnant are indicated. Also indicated with an asterisk is the position of a likely nonglycosylated precursor form (see Discussion). The data shown are representative of seven such experiments.

 
Basal remnant appearance is proteasome activity dependent but not blocked by lysosome inhibitors
Constitutive down-regulation of the mature GHR is markedly reduced by chemical inhibitors of the lysosome (14). We therefore tested whether basal remnant generation reflected such lysosomal degradation of the GHR by treating {gamma}2A-GHR cells with two different lysosome inhibitors (Fig. 5Go). Serum-starved cells were treated with bafilomycin A1 (10 nM), ammonium chloride (50 mM), or vehicle for 5 h, and detergent-extracted proteins were resolved by SDS-PAGE and immunoblotted with anti-GHR. As expected, lysosome inhibitor treatment resulted in increased abundance of mature GHR, but basal remnant abundance did not change. These findings suggest that lysosome activity is not involved in generation of the basal remnant in JAK2-deficient cells.


Figure 5
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  		FIG. 5. Basal remnant appearance is not prevented by lysosome inhibitors. Serum-starved {gamma}2A-GHR cells were treated with bafilomycin A1 (10 nM), ammonium chloride (50 mM), or their vehicle for 5 h. Equal amounts of extracted protein were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. The positions of the mature GHR, precursor GHR, basal GHR remnant, and a nonspecific (NS) band in the region of the remnant are indicated. The data shown are representative of three such experiments.

 
Proteasome activity has been implicated in several aspects of the GHR life cycle. Strous and colleagues (34, 35, 36) have shown that GH-induced GHR down-regulation is impaired at a nonpermissive temperature in cells that harbor a temperature-sensitive mutation that inactivates the ubiquitin-proteasome system and have identified a cytoplasmic domain motif (the UbE motif) believed to be required for ubiquitin-dependent endocytosis. Our studies have revealed the generation of the so-called GHR stub that arises from intramembranous cleavage of the metalloprotease-derived GHR remnant by {gamma}-secretase activity; the stability of this stub is greatly enhanced in the presence of lactacystin, a highly specific proteasome inhibitor (24). Furthermore, we previously showed that the enhanced lability of the mature GHR in JAK2-deficient cells is reversed in the presence of lactacystin (14). Given these diverse roles for proteasome activity in GHR dynamics, we tested the effects of proteasome inhibition on basal GHR remnant abundance in JAK2-deficient cells.

Serum-starved {gamma}2A-GHR cells were treated for 5 h with lactacystin or vehicle and detergent extracts were evaluated by anti-GHR immunoblotting. Notably, lactacystin treatment significantly changed the abundance of all three forms of the receptor (mature, precursor, and basal remnant) in these JAK2-deficient cells (Fig. 6AGo, first two lanes). As seen in the immunoblot and densitometrically estimated in several experiments (Fig. 6BGo), lactacystin treatment increased the abundance of mature GHR by roughly 35% and decreased precursor GHR by approximately 30%. The decrease in basal remnant abundance was even more dramatic, measuring roughly 43%. In other experiments (not shown), qualitatively similar changes, although of lesser magnitude, were observed with as little as 3 h lactacystin treatment. Of interest, these changes in the GHR in the JAK2-deficient {gamma}2A-GHR cells yielded relative patterns of the three receptor forms more similar to those seen in JAK2-replete C14 cells, a GHR immunoblot of which is shown for comparison in Fig. 6AGo, right lane. Collectively, the data in Fig. 6Go indicate that, in contrast to lysosome inhibition, proteasome inhibition reduces the non-metalloprotease-generated basal remnant and enhances GHR maturation.


Figure 6
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  		FIG. 6. Basal remnant appearance is proteasome dependent. A and B, Proteasome inhibition alters the abundance of mature, precursor, and basal remnant GHR in {gamma}2A-GHR cells. A, Serum-starved {gamma}2A-GHR cells were treated with lactacystin (5 µM) or vehicle for 5 h. Equal amounts of extracted protein were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. For comparison, C14 cell extract resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47 is included. Note the increase in mature GHR and the loss of precursor and basal remnant in {gamma}2A-GHR cells resulting in a pattern of receptor forms more similar to C14. The positions of the mature GHR, precursor GHR, basal GHR remnant, and a nonspecific (NS) band in the region of the remnant are indicated. Also indicated with an asterisk is the position of a likely nonglycosylated precursor form (see Discussion). B, Densitometric quantitation of several independent experiments as described in A. In each experiment, the abundance of each GHR form in cells treated with vehicle is considered 100% and is compared with the abundance of each after lactacystin treatment. Data are plotted as the mean ± SE (n = 5). a, P < 0.05 compared with control; b, P < 0.005 compared with control.

 
Basal remnant is generated from GHR in the secretory pathway
Because proteasome inhibition decreased the abundance of both precursor GHR and basal remnant in {gamma}2A-GHR cells, we hypothesized that the maturation state of the receptor affects its susceptibility to cleavage and formation of the basal remnant. To test this hypothesis, we used the fungal metabolite BFA to inhibit ER to Golgi transport of newly synthesized GHR. BFA treatment causes the Golgi to disassemble and collapse into the ER, thereby inhibiting transport of proteins destined for secretion or plasma membrane insertion (37). As a consequence, secretory and membrane proteins are retained in the ER, as are Golgi processing enzymes, which results in newly synthesized proteins becoming glycosylated and acquiring endoH resistance in the ER (38).

In the experiment shown in Fig. 7AGo, serum-starved {gamma}2A-GHR cells were treated with BFA for 1–5 h and compared with control cells treated with vehicle for 5 h. Anti-GHR immunoblotting of detergent extracts revealed a BFA-induced loss of basal remnant, most notable after two or more hours of treatment. Mature GHR abundance was dramatically reduced initially (after 1 h) by BFA and precursor GHR compensatorily increased. With longer BFA incubation, precursor GHR lessened and/or appeared to change migration such that its SDS-PAGE mobility decreased and its migration took on an appearance more similar to mature GHR. We reasoned that the initial buildup of precursor might correspond to interruption of ER to Golgi transport and that the corresponding loss of mature GHR is consistent with the short half-life of mature GHR we previously documented in these JAK2-deficient cells (14). Furthermore, we hypothesized that the accumulation of mature GHR over the 2- to 5-h BFA treatment period that corresponded to the loss of basal remnant might be attributable to glycosylation of ER-retained precursor GHR by Golgi enzymes residing in the ER. To test this, we treated cells with BFA for 2 and 5 h or with vehicle for 5 h and then subjected detergent extracts to immunoprecipitation with a monoclonal antibody to the GHR cytoplasmic domain, anti-GHRcyt-mAb (9, 20, 26) (Fig. 7BGo). The precipitated proteins were split into three equal portions and treated with endoH, its vehicle control, or a combination of N-glycosidase F and neuraminidase (F/N). F/N removes carbohydrate chains independent of their content and is thus a control to indicate the pattern obtained by full deglycosylation. As previously reported, precursor GHR was sensitive to endoH deglycosylation (and thus manifests a markedly increased mobility in SDS-PAGE), whereas the mature form is not (lanes 1–3). After 2 h of BFA treatment, the mature form of the GHR (presumably the post-Golgi-trafficked receptor before BFA treatment) was largely absent, replaced by an endoH-resistant form termed mature GHR to distinguish it from mature receptor present before BFA (lanes 4–6). BFA treatment for 5 h resulted in markedly reduced endoH-sensitive GHR with most of the receptor being endoH resistant (lanes 7–9). Complete deglycosylation with F/N caused all immunodetectable GHR to migrate rapidly, independent of BFA treatment, as expected.

The effects of BFA can be reversed by removing the drug and allowing the secretory pathway to reassemble (37, 38, 39). To test the effects of BFA washout on basal remnant generation, {gamma}2A-GHR cells treated with BFA for 2 and 5 h or vehicle for 5 h were then incubated in the absence of BFA for a chase period of 3 h (Fig. 7CGo). GHR was isolated by anti-GHRcyt-mAb precipitation, and eluates were resolved by SDS-PAGE and immunoblotted with anti-GHR. As in Fig. 7AGo, BFA caused progressive loss of basal remnant after 2 and 5 h, coupled with buildup of the endoH-resistant mature GHR (lanes 1–3). Interestingly, however, we observed reaccumulation of the basal remnant over the 3-h BFA washout period along with reappearance of the mature and precursor GHR (lanes 4–6). These data suggest that the basal remnant in {gamma}2A-GHR cells might emanate from GHR precursor newly synthesized during the BFA washout period.

To further investigate the mechanism by which the {gamma}2A-GHR basal remnant is generated, we examined the effects of CHX and lactacystin on BFA-treated cells (Fig. 7DGo). Serum-starved {gamma}2A-GHR cells were treated with BFA alone (5 h) or in combination with CHX (3 and 5 h) or lactacystin (3 and 5 h), followed by a 3-h washout period. The 5-h treatment of CHX and lactacystin was begun 3 h after BFA treatment was initiated; the 5-h BFA exposure was followed by a washout period in which both CHX and lactacystin, respectively, were added back to the chase media. The 3-h treatments with CHX and lactacystin corresponded to the duration of BFA washout. Immunoprecipitated GHR from each sample was evaluated by anti-GHR immunoblotting. As expected and consistent with the data presented above, BFA induced loss of basal remnant (lane 2 vs. 1) and CHX treatment alone for either 3 h (lane 7) or 5 h (lane 4) caused loss of mature and precursor GHR and the basal remnant. Also as anticipated, washout of BFA allowed reappearance of the basal remnant (lane 3 vs. 2). However, the inclusion of CHX during the BFA washout (whether or not it was also present for the final 2 h of BFA treatment) strongly inhibited the reappearance of the basal remnant and of the precursor GHR (lanes 5 and 6 vs. 2). Notably, lactacystin also prevented the reappearance of the basal remnant during BFA washout (lanes 9 and 10 vs. 3). In contrast to CHX treatment, lactacystin did not, however, prevent reappearance of the precursor GHR (lanes 9 and 10 vs. 5 and 6). Collectively, although not absolute proof, these data strongly support the conclusion that the basal remnant present in JAK2-deficient cells arises as the result of proteolysis of the precursor GHR and that this proteolysis is proteasome activity dependent.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The GHR is required for GH to exert its actions, which are diverse and relate to aspects of physiology and aging (1, 40). Although intracellular GHR has been observed and may play important roles (24, 41, 42, 43), the level of receptor at the cell surface is important and likely highly regulated. In addition to regulation at the level of GHR gene expression, emerging evidence suggests that GHR cell surface abundance is modulated at the posttranslational level by various mechanisms (44, 45). The study of these mechanisms is critical for more detailed understanding of GH action; however, this line of research also promises to reveal more general aspects of how cells process surface receptors of the cytokine receptor superfamily to which GHR belongs (3).

In our recent studies, we have found that the tyrosine kinase JAK2, in addition to being a key GH signal transducer, has a role in several processes that regulate cell surface GHR abundance. Inducible metalloproteolysis modulates GHR surface availability (18), and the sensitivity of GHR to cleavage is decreased in cells that lack JAK2 or in which JAK2 cannot associate with GHR (19). Stability of the surface GHR is also regulated by JAK2 in a non-metalloprotease-dependent fashion (14). The half-life of the mature GHR is significantly increased in JAK2-replete vs. JAK2-deficient cells, and the rapid loss of mature GHR in JAK2-deficient cells after blockade of new protein synthesis with CHX is inhibited by proteasome and lysosome inhibitors but not by the metalloprotease inhibitor IC3. Additionally, pulse-chase experiments suggest that JAK2 also has a role in enhancing GHR biogenesis (14).

In the current study, we address the effects of JAK2 on GHR processing. In cells that lack JAK2, we observe the presence of a basal GHR remnant, so called because it comigrates in SDS-PAGE with and is indistinguishable by immunoblotting from the PMA-induced GHR remnant derived from metalloprotease activity in (JAK2-replete) C14 cells. Because it resembles the inducible remnant, we first reasoned that the high level of basal remnant in JAK2-deficient cells might be due to constitutive metalloprotease activity. However, metalloprotease inhibition by IC3 failed to reduce the abundance of basal remnant; similarly, RNA-interference (RNAi)-mediated knockdown of TACE and ADAM10, the metalloproteases that have been implicated in GHR proteolysis, also did not affect basal remnant abundance. Thus, the basal remnant in JAK2-deficient cells does not appear to be generated to any major degree by metalloprotease activity. Yet, interestingly, the same deletion in the extracellular domain stem region (residues 237–239; GHR{Delta}237–239) that abrogates inducible metalloproteolytic GHR cleavage (20) also inhibits basal remnant generation. This suggests either that the same site is used to achieve both metalloproteolytic (in JAK2-replete cells) and non-metalloproteolytic (in JAK2-deficient cells) receptor cleavage or that intactness of this region is required for basal remnant generation by cleavage at an alternative site. We previously mapped the metalloproteolytic cleavage to between residues 238 and 239 (20); we have yet to map the cleavage site that generates the basal remnant. Knowledge of this site will help discriminate between the two possibilities for why the same stem region deletion affects both cleavage events.

We found that treatment of JAK2-deficient cells with two lysosome inhibitors did not affect basal remnant abundance, but treatment with lactacystin, a powerful and specific proteasome inhibitor, coordinately increased the abundance of mature GHR and decreased the abundance of both the precursor GHR and the basal remnant, in effect making the pattern of relative abundance of these receptor forms more like that observed in JAK2-replete cells. Indeed, in JAK2-deficient cells, acute PMA treatment after lactacystin pretreatment caused significantly more loss of mature GHR compared with that seen in the absence of lactacystin (75% loss vs. 50% loss, P < 0.05; n = 3; and data not shown), resulting in a loss of GHR similar to that seen in JAK2-replete cells. In light of our previous data mentioned above concerning the effects of proteasome inhibition on mature GHR stability (14), we interpret our current data as indicating that inhibition of the proteasome in JAK2-deficient cells allows enhanced maturation of the precursor into the mature GHR and, at the same time, inhibits constitutive degradation of the mature cell surface receptor presumably by different mechanisms. As has been suggested for other cytokine receptors [such as the IFNAR1 subunit of the type I interferon receptor and thrombopoietin receptor, and their cognate JAK family members (46, 47)], mature GHR at the cell surface undergoes constitutive down-regulation, likely via endocytosis and lysosomal degradation. The rate and/or degree of the GHR’s constitutive down-regulation is blunted by proteasome inhibitors and dampened by its association with JAK2. In contrast to this inhibitory effect of JAK2 on constitutive GHR down-regulation, our current data on the maturation of precursor to mature GHR indicate that JAK2 fosters this transition, as does proteasome inhibition in cells that lack JAK2.

The reduction in both the precursor and non-metalloproteolytically derived basal remnant in JAK2-deficient cells by proteasome inhibition led us to hypothesize that the maturation state of the GHR determines its susceptibility to cleavage and generation of the basal remnant. We tested this in the experiments using BFA to block ER to Golgi transport of newly synthesized GHR precursor in JAK2-deficient cells. BFA treatment led to time-dependent changes in the mature and precursor GHR. Acutely, BFA caused rapid loss of mature GHR, consistent with our earlier observations of its lability in the absence of JAK2, and precursor (endoH-sensitive glycosylated) GHR increased in abundance, as its production (synthesis) was unaffected and its conversion was blocked. With longer (≥2 h) BFA exposure, precursor abundance decreased as it transitioned into a more mature (endoH-resistant) receptor form, presumably resulting from actions of the Golgi-processing enzymes that relocated into the ER with BFA treatment (38). Importantly, this BFA-induced decline in precursor and increase in endoH-resistant GHR was accompanied by a loss of basal remnant, similar to the effects of lactacystin on precursor, mature GHR, and basal remnant. This suggested that basal remnant emanates from precursor, an interpretation supported by the results of the BFA washout experiments. Removal of BFA and washout for 3 h (allowing reconstitution of the secretory pathway) fostered reappearance of the precursor and change in the mature GHR to a pattern of migration consistent with the surface form; correspondingly, the BFA washout caused reappearance of the basal remnant. Notably, blockade of protein synthesis (with CHX) during the BFA washout period (Fig. 7DGo) blocked reappearance of both the precursor and the basal remnant, as would be anticipated if the remnant derived from newly synthesized precursor. [Interestingly, the mature GHR loss observed in the presence of CHX (Fig. 7DGo, lanes 4 and 7, and Ref. 14) in these JAK2-deficient cells was strongly blunted in cells subjected to BFA treatment and washout (Fig. 7DGo, lanes 5 and 6). This may reflect incomplete presentation of the mature glycosylated GHR to the cell surface after release from BFA and suggests that the locus of JAK2’s protection of the mature GHR is at the cell surface or the constitutive endocytosis/degradative trafficking that emanates from the surface (14).] We note also that blockade of proteasome action with lactacystin during the period of BFA washout also prevented basal remnant reappearance, despite the reappearance of precursor under those conditions. This is a powerful result that supports the conclusion that basal remnant arises from precursor in a proteasome-dependent fashion.

Together, the data obtained with proteasome inhibitors and BFA suggest that GHR in cells that lack JAK2 is a target for ERAD. Proteins, either soluble or membrane anchored, that enter the ER after translation and fail to fold properly or are otherwise deemed as abnormal by various quality control mechanisms are targeted for degradation by ERAD (48, 49, 50). Typically, such degradation depends on retrotranslocation of the target protein back into the cytosol for complete degradation by the proteasome; however, emerging data suggest the existence of alternative ERAD pathways (50). Our data support the notion that the absence of JAK2 promotes ERAD (or ERAD-like)-mediated loss of the precursor GHR, which is glycosylated, but has not achieved endoH resistance (and thus has not traversed the Golgi). Notably, this proteasome-dependent degradation of the GHR results in a discrete cleavage product, the basal remnant, rather than in the rapid and complete degradation of the receptor. Proteasomal degradation is typically understood to involve the threading of target proteins via their termini through the gated substrate channel of the proteasome complex; only recently has there been described an example of incomplete cleavage of nuclear factor-{kappa}B p105 yielding the p50 subunit (51, 52). In this case, the generation of a discrete product is mediated by a glycine-rich region in the middle of the molecule that serves as a processing stop signal. Another recent study demonstrated that some substrates may be cleaved endoproteolytically by the proteasome; in that study, fusion proteins were used as targets (53). We do not yet have evidence that the precursor GHR in JAK2-deficient cells is being cleaved directly by the proteasome (rather than by a proteasome-dependent protease). However, future studies will be directed to determining the answer to this question and whether the formation of the basal remnant reflects a novel mechanism of ERAD-mediated cleavage.

A chaperone effect of a JAK-family member has been implicated for the trafficking of several other cytokine receptors to the cell surface, including the erythropoietin receptor and the oncostatin M receptor (54, 55, 56). However, ERAD of these receptors in the absence of the JAK protein has not been characterized and generation of a basal remnant equivalent has not been described. Very recently, van den Eijnden et al. (57) reported that transient overexpression of GHR in cells that express JAK2 resulted in production of the mature and precursor GHR as well as a nonglycosylated form of the receptor. In that study, evidence is provided suggesting that this nonglycosylated GHR, but not the precursor GHR, undergoes ERAD. However, as acknowledged by the authors, the vast overexpression in this system led to unusual features; for example, the half-lives of the mature and precursor receptors were quite long. The nonglycosylated GHR form described in that study likely corresponds to the band identified in our figures with an asterisk, which comigrates with the GHR formed from endo F/N deglycosylation. We note that in our stable transfection system, this form, which is detected at much lower levels than the precursor, behaves in all instances as does the precursor GHR in both JAK2-deficient and JAK2-replete cells. This leads us to conclude that the nonglycosylated form and the precursor undergo the same processing events in our system, as might be predicted if the nonglycosylated form is the nascent receptor that enters the secretory pathway and rapidly becomes the precursor. We believe that the data presented herein are therefore the first example of ERAD-associated cleavage of a cytokine receptor that results from lack of expression of its cognate JAK molecule.

Although we observe some surface GHR even in the absence of JAK2, we see the logic in cells evolving mechanisms to limit such expression if there is not JAK2 that can couple to the receptor for signaling. Our data suggest that the mechanisms by which JAK2 prevents ERAD of GHR depends on the ability of the JAK2 to associate with the receptor, rather than to possess a kinase domain. In fact, expression of wild-type JAK2 in cells that express a GHR that cannot couple to JAK2 does not prevent basal remnant generation. How GHR-JAK2 association protects the receptor during processing is not yet known. One possibility is that a region of the GHR, perhaps in the cytoplasmic domain, that is recognized by the ER quality control apparatus as unfolded or defective is masked upon binding of JAK2 to the receptor. Another intriguing possibility is that JAK2 binding to the GHR changes receptor conformation at another region and thereby allows escape from quality control. In support of the latter possibility, we note that susceptibility of the GHR to inducible metalloproteolysis (which occurs in the extracellular domain stem region) is reduced when GHR and JAK2 cannot interact; likewise, the ability of a conformation-sensitive extracellular domain antibody to inhibit metalloproteolysis is lost when GHR-JAK2 interaction is prevented (19). As the non-metalloproteolytic generation of basal remnant is prevented by mutation of the same stem region that abrogates inducible metalloproteolysis, this possibility is worth pursuing in future studies.


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


   Footnotes
 
This work was supported by National Institutes of Health (NIH) Grant DK58259 and a Veterans Affairs Merit Review Award (both to S.J.F.), a Graduate Assistance in Areas of National Need fellowship (to K.L.), and NIH T32AR053458 and NIH P30AR050948 (University of Alabama Department of Dermatology, to K.L.).

Disclosure Summary: K.L., L.D., X.W., K.H., J.J., and S.J.F. have nothing to declare.

First Published Online August 30, 2007

Abbreviations: ADAM, A disintegrin and metalloprotease; BFA, brefeldin A; CHX, cycloheximide; endoH, endoglycosidase H; ER, endoplasmic reticulum; ERAD, ER-associated degradation; F/N, N-glycosidase F and neuraminidase; GHR, GH receptor; IC3, Immunex Compound 3; JAK2, Janus kinase 2; PMA, phorbol myristate acetate; RNAi, RNA-interference; siRNA, small interfering RNA; TACE, TNF-{alpha} converting enzyme; WT, wild type.

Received April 9, 2007.

Accepted for publication August 21, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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X. Wang, J. Jiang, J. Warram, G. Baumann, Y. Gan, R. K. Menon, L. A. Denson, K. R. Zinn, and S. J. Frank
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