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Endocrinology, doi:10.1210/en.2005-0514
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Endocrinology Vol. 146, No. 11 4755-4765
Copyright © 2005 by The Endocrine Society

Janus Kinase 2 Enhances the Stability of the Mature Growth Hormone Receptor

Kai He, Kimberly Loesch, Jon W. Cowan, Xin Li, Luqin Deng, Xiangdong Wang, Jing Jiang and Stuart J. Frank

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

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
 
The abundance of surface GH receptor (GHR) is an important determinant of cellular GH sensitivity and is regulated at both transcriptional and posttranscriptional levels. In previous studies of GHR-expressing Janus kinase 2 (JAK2)-deficient human fibrosarcoma cells ({gamma}2A-GHR), we demonstrated that stable transfection with JAK2 resulted in increased steady-state levels of mature GHR (endoH-resistant; relative molecular mass, 115–140 kDa) relative to precursor GHR (endoH-sensitive; relative molecular mass, 100 kDa). We now examine further the effects of JAK2 on GHR trafficking by comparing {gamma}2A-GHR to {gamma}2A-GHR cells stably reconstituted with JAK2 (C14 cells). In the presence of JAK2, GHR surface expression was increased, as assessed by surface biotinylation, 125I-labeled human GH cell surface binding, and immunofluorescence microscopy assays. Although the absence of JAK2 precluded GH-stimulated signaling, GH-induced GHR disulfide linkage (a proxy for the GH-induced conformational changes in the GHR dimer) proceeded independent of JAK2 expression, indicating that the earliest steps in GH-induced GHR triggering are not prevented by the absence of JAK2. RNA interference-mediated knockdown of JAK2 in C14 cells resulted in a decreased mature to precursor ratio, supporting a primary role for JAK2 either in enhancing GHR biogenesis or dampening mature GHR degradation. To address these potential mechanisms, metabolic pulse-chase labeling experiments and experiments in which the fate of previously synthesized GHR was followed by anti-GHR immunoblotting after cycloheximide treatment (cycloheximide chase experiments) were performed. These indicated that the presence of JAK2 conferred modest enhancement (1.3- to 1.5-fold) in GHR maturation but substantially prolonged the t1/2 of the mature GHR, suggesting a predominant effect on mature GHR stability. Cycloheximide chase experiments with metalloprotease, proteasome, and lysosome inhibitors indicated that the enhanced stability of mature GHR conferred by JAK2 is not related to effects on constitutive receptor metalloproteolysis but rather is a result of reduced constitutive endosomal/lysosomal degradation of the mature GHR. These results are discussed in the context of emerging information on how JAK-family members modulate surface expression of other cytokine receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH IS AN ANTERIOR pituitary-derived peptide hormone that is a prime regulator of growth and metabolism in humans and other vertebrates (1). GH exerts its effects by interacting with cell surface GH receptors (GHRs), which are widely displayed in tissues throughout the body (2). The GHR is a heavily glycosylated transmembrane protein that shares features with a large family of cytokine receptors including those for erythropoietin, interleukins, and interferons (3), all of which initiate ligand-stimulated signaling by coupling to nonreceptor cytoplasmic tyrosine kinases of the Janus kinase (JAK) family. GH-induced signaling is characterized by activation of several pathways, including the signal transducer and activator of transcription-5 (STAT-5), ERK, and phosphatidylinositol-3 kinase pathways, that all require GH-induced triggering of JAK2 kinase activity (2, 4). The GHR exists on the cell surface as a homodimer in the absence of GH; GH stimulation is believed to induce (as yet incompletely characterized) conformational changes in the receptor dimer that achieve a 1:2 GH:GHR stoichiometry and render JAK2 activated (5, 6, 7, 8, 9, 10, 11).

The relative abundance of surface GHR is a key determinant of cellular GH sensitivity; thus, a thorough appreciation of factors governing receptor biogenesis and stability is critical for understanding GH action. GHR is synthesized in the endoplasmic reticulum (ER) and transported to the plasma membrane via the secretory pathway, undergoing characteristic carbohydrate processing during Golgi transport. GHR precursor possesses high-mannose carbohydrates that are sensitive to deglycosylation with endoH; in contrast, transport through the Golgi to the cell surface renders the mature receptor resistant to endoH (12, 13, 14). Regulation of GHR gene expression and the pace of maturation of newly synthesized GHR (GHR biogenesis) are thus important determinants of cell surface GHR availability (15, 16). In the absence of GH, mature GHRs are cleared from the cell surface constitutively by either endoctyosis or proteolytic shedding (17). Constitutive (and GH induced) GHR endocytosis requires an intact cellular ubiquitin-proteasome system and the GHR is ubiquitinated; however, a GHR mutant that cannot be ubiquitinated is still endocytosed (15, 18, 19). Thus, although a well-conserved 10-residue stretch, the ubiquitin-dependent endocytosis motif, in the proximal one third of the cytoplasmic domain is required for efficient GHR endocytosis, GHR ubiquitination per se is not required for receptor endocytosis, and GHR endocytosis results in lysosomal, rather than proteasomal, degradation (20, 21, 22, 23).

Like other JAKs [JAK1, JAK3, and tyrosine kinase 2 (TYK2)], JAK2 is a large cytoplasmic protein with a tyrosine kinase domain at its C terminus. Just N-terminal to the kinase domain is a kinase-like (pseudokinase) domain, which is catalytically inactive and may negatively regulate the activity of the kinase domain (24, 25, 26, 27). In addition to their roles in signaling, JAKs have been suggested as influential in determining the levels of their associated cytokine receptors, although there is variability in the mechanisms involved for different cytokine receptor-JAK combinations (28, 29, 30, 31, 32). In particular, there is uncertainty as to whether the JAKs exert these effects on cell surface cytokine receptor abundance by chaperoning the receptor through the secretory pathway to the surface, regulating the rate of receptor internalization, or in some other fashion selectively stabilizing the surface form of the receptor. In previous work, we used JAK2-deficient human fibrosarcoma cells to demonstrate that although JAK2 expression is not required for surface GHR expression, stable reconstitution with JAK2 dramatically enhances the fraction of GHRs that achieve a mature glycosylation pattern (endoH resistance) (33, 34).

In this report, we explore the mechanisms by which JAK2 fosters enhanced surface GHR abundance. We compare JAK2-deficient and JAK2-replete cells with regard to GHR stability. In contrast to reported effects of JAK2 on erythropoietin receptor (EpoR) biogenesis (29), our data point to dramatic effects of JAK2 on stability of the mature GHR and suggest that JAK2 regulates the susceptibility of the receptor to constitutive endosomal-lysosomal pathway-mediated down-regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant human GH (hGH) was kindly provided by Eli Lilly Co. (Indianapolis, IN). Phorbol 12-myristate 13-acetate (PMA), cycloheximide (CHX), ammonium chloride, chloroquine, clasto-lactacystin ß-lactone (referred to as lactacystin), and other routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. Zeocin was purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum, gentamicin sulfate, penicillin, and streptomycin were purchased from Biofluids (Rockville, MD). Sulfo-NHS-LC-biotin and streptavidin-horseradish peroxidase (streptavidin-HRP) were purchased from Pierce (Rockford, IL). Immunex Compound 3 (IC3) was kindly provided by Dr. Roy Black (Amgen, Inc., Thousand Oaks, CA). The plasmid encoding the serine protease inhibitor (Spi) 2.1 GAS-like element (GLE)-luciferase (35) was provided by Dr. William Lowe, Northwestern University, Chicago, IL.

Antibodies
The 4G10 monoclonal antiphosphotyrosine antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-JAK2AL33 (directed at residues 746-1129 of murine JAK2) polyclonal serum has been described (36). The rabbit polyclonal antiserum, anti-GHRcyt-AL47, 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 (33). Anti-GHRcyt-mAb is a mouse monoclonal antibody directed against a bacterially expressed glutathione-S-transferase fusion protein incorporating human GHR residues 271–620 (37). Anti-GHRext-mAb is a mouse monoclonal antibody directed against a bacterially expressed glutathione-S-transferase fusion protein incorporating rabbit GHR residues 1–246 (10, 37, 38, 39).

Cells, cell culture, and transfection
{gamma}2A is a JAK2-deficient human fibrosarcoma cell line (kindly provided by Dr. G. Stark, Cleveland Clinic Foundation, Cleveland, OH) (40). A stable {gamma}2A cell line expressing rabbit GHR ({gamma}2A-GHR) has been described (33). {gamma}2A-GHR 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 (clone 14 or C14 cells) was achieved by stable transfection of {gamma}2A-GHR with murine JAK2, as described (34), and was maintained in the above medium supplemented with 100 µg/ml Zeocin. Transient transfection was achieved using LipofectAMINE Plus (Invitrogen) according to the manufacturer’s instructions.

Cell stimulation, protein extraction, immunoprecipitation, electrophoresis, and immunoblotting
Serum starvation of cells was accomplished by substitution of 0.5% (wt/vol) BSA (fraction V; Roche Molecular Biochemicals, Indianapolis, IN) for serum in their respective culture media for 16–20 h before experiments. Unless otherwise noted, stimulations were performed at 37 C. Details of the hGH (500 ng/ml unless otherwise noted) and PMA (1 µM) treatment protocols have been described (10, 14, 33, 34). Briefly, adherent cells were stimulated in binding buffer [consisting of 25 mM Tris-HCl (pH 7.4), 120 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 0.1% (wt/vol) BSA, and 1 mM dextrose] or DMEM (low glucose) with 0.5% (wt/vol) BSA. Stimulations were terminated by washing the cells once with ice-cold PBS in the presence of 0.4 mM sodium orthovanadate (PBS-vanadate). Cells were harvested by scraping in ice-cold PBS-vanadate, and pelleted cells were collected by brief centrifugation. For protein extraction, pelleted cells were solubilized for 30 min at 4 C in lysis buffer [1% (vol/vol) Triton X-100, 150 mM NaCl, 10% (vol/vol) glycerol, 50 mM Tris-HCl (pH 8.0), 100 mM NaF, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 mM benzamidine, and 10 µg/ml aprotinin], as indicated. After centrifugation at 15,000 x g for 15 min at 4 C, the detergent extracts were electrophoresed under reducing conditions or subjected to immunoprecipitations, as indicated.

For immunoprecipitation with the rabbit anti-JAK2AL33 and anti-GHRcyt-AL47, 3 µl of antiserum was used per precipitation. Protein-A Sepharose (Amersham Biosciences, Piscataway, NJ) was used to adsorb immune complexes, and after extensive washing with lysis buffer, SDS sample buffer eluates were resolved by SDS-PAGE and immunoblotted as indicated. Resolution of proteins by SDS-PAGE, Western transfer of proteins, and blocking of Hybond-ECL (Amersham Biosciences) with 2% BSA were performed as previously described (10, 33, 34, 37). Immunoblotting with antibodies 4G10 (1:2000), anti-GHRcyt-AL47 (1:1000), or anti-JAK2AL33 (1:1000), with HRP-conjugated antimouse or antirabbit secondary antibodies (1:5000) and ECL detection reagents (all from Amersham Biosciences) and stripping and reprobing of blots were accomplished according to the manufacturer’s suggestions.

Transactivation assay
{gamma}2A-GHR and C14 cells (one 70% confluent 100 x 20 mm dish per transfection) were transfected with Spi-GLE-luc reporter plasmid using LipofectAMINE Plus (Invitrogen) for 6 h. The cells were trypsinized and seeded into six-well plates at 4.5 x 105 cells per well, allowed to attach overnight in serum-containing medium, and then washed and placed into serum-free medium for 14 h. Cells were stimulated with GH (500 ng/ml) for another 18 h. Stimulations (performed in triplicate) were terminated by aspiration of the medium and the addition of luciferase lysis buffer; luciferase activity was assayed as has been described previously (34). Data are displayed as the GH-induced fold increases of activity (mean ± SE).

Cell surface biotinylation
Surface biotinylation of {gamma}2A-GHR and C14 cells was achieved using Sulfo-NHS-LC-biotin, according to the manufacturer’s protocol (Pierce). Cells were grown to 80% confluence and serum starved overnight. They were washed three times with ice-cold PBS, followed by 30 min incubation at 4 C on a rotatory shaker with 0.5 mg/ml Sulfo-NHS-SS-biotin. The cells were then washed twice with ice-cold PBS and solubilized for 15 min at 4 C as above. Detergent extracts were subjected to immunoprecipitation with anti-GHRcyt-AL47 and then adsorbed with protein A-Sepharose (Amersham Biosciences) at 4 C. SDS sample buffer eluates were resolved by SDS-PAGE and blotted with streptavidin-HRP (Pierce), as indicated by the manufacturer.

RNA interference
A 21-nucleotide small interfering RNA (siRNA) duplex targeting mouse JAK2 gene was custom synthesized by Ambion (Austin, TX). The sequence used was GGAGAGUAUCUGAAGUUUCtt, corresponding to nucleotides 247–265 and the N-terminal region of the JAK2 protein (amino acids 52–58). Transfection of siRNA duplexes was carried out using Oligofectamine (Invitrogen) and 4.2 µg siRNA duplex per well of a six-well plate, according to the manufacturer’s protocol and previously described methods (41). Two days after transfection, C14 cells were serum starved overnight and then solubilized as indicated above. Detergent extracts were resolved by SDS-PAGE and immunoblotted with anti-GHRAL47 and anti-JAK2AL33.

Immunofluorescence microscopy
{gamma}2A-GHR and C14 cells were grown on coverslips and serum starved overnight. Cells were first fixed with formaldehyde (Tousimis Research Corp., Rockville, MD) (4% vol/vol) for 5 min with (for anti-GHRcyt-mAb) or without (for anti-GHRext-mAb) treatment with 0.1% Triton X-100 for 15 min. Fixed cells were labeled with anti-GHRext-mAb or anti-GHRcyt-mAb (both at 25 µg/ml), which target intracellular and extracellular regions of GHR, respectively (as described above), for 1 h at room temperature. After extensive washing with PBS, cells were labeled with Alexa594-coupled antimouse secondary antibodies (1:100) (Molecular Probes, Eugene, OR) for 1 h at room temperature. After washing, coverslips were mounted onto glass and sealed with clear fingernail polish. Confocal laser scanning microscopy (as in Ref.42) was performed with a Leica TCS SP unit equipped with a UV laser (Coherent Laser Group Enterprise, Santa Clara, CA).

Competitive [125I]hGH binding assay
{gamma}2A-GHR and C14 cells were equally divided into multiple wells of a six-well plate and serum starved for 16 h, as described above. After washing, cells were incubated with a constant amount of [125I]hGH [50,000 cpm (~25 pM) per well] either in the presence (to determine nonspecific binding) or absence of 2 µg/ml (~91 nM) unlabeled hGH for 1 h at room temperature with gentle agitation. After incubation, cells were washed three times with cold PBS and solubilized in a buffer of 1% SDS, 0.1 N NaOH. The lysate was subjected to {gamma}-counting. Data within each experiment were expressed as specific [125I]GH binding in C14 cells relative to that measured in {gamma}2A-GHR cells (considered 100%) and displayed as pooled data from several different experiments, as noted in the figure legend.

Metabolic labeling
{gamma}2A-GHR and C14 cells were grown to 80% confluence in 60-mm dishes and serum starved overnight. The cells were washed twice with PBS and incubated in methionine- and cysteine-free DMEM for 30 min in 37 C. [35S]Methionine and [35S]cysteine (100–150 µCi/ml Promix; Amersham, Costa Mesa, CA) was added to each plate. The incubation was continued for 15 min at 37 C in a CO2 incubator. The radioactivity was replaced with DMEM containing 0.5% (wt/vol) BSA and chased for 20–240 min. Cells were harvested and solubilized as indicated above. Detergent extracts were immunoprecipitated with anti-GHRcyt-mAb. SDS sample buffer eluates were resolved by SDS-PAGE and subjected to autoradiography. The maturation efficiency was considered as the intensity of the signal corresponding to mature receptor at the 60-min chase time (the point of maximal signal for both {gamma}2A-GHR and C14 cells) divided by the intensity of signal corresponding to the GHR precursor at the 0-min chase time.

Degradation of GHR after blockade of protein synthesis (CHX chase)
{gamma}2A-GHR and C14 cells were grown to 80% confluence in six-well dishes and serum starved overnight. Cells were then incubated with CHX (20 µg/ml) for 0–4 h. Cell lysates were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47.

Effects of metalloprotease, proteasome, and lysosome inhibitors on GHR stability
{gamma}2A-GHR cells were grown to 80% confluence in six-well dishes and serum starved overnight. After incubation with IC3 (50 µM), lactacystin (5 µM), ammonium chloride (100 mM), or chloroquine (200 µM) (duration for different inhibitor treatments is indicated in figure legends), CHX (20 µg/ml) or vehicle was added for 2 h. Cell lysates were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47.

Densitometric analysis
Densitometric quantitation of ECL immunoblots and autoradiograms 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
 
Stable expression of JAK2 in JAK2-deficient cells reconstitutes GH signaling and enhances surface GHR expression but is not required for GH-induced GHR conformational change
{gamma}2A-GHR is a JAK2-deficient human fibrosarcoma cell line stably expressing rabbit GHR (33). To study the effects of JAK2 on GHR, we stably transfected {gamma}2A-GHR with murine JAK2, producing a clonal line, {gamma}2A-GHR-JAK2 (herein referred to as C14 cells) (34). JAK2 expression status and its consequences on GH signaling were investigated in the experiments shown in Fig. 1Go. Serum-starved cells were treated with or without GH for 10 min, after which cellular proteins were extracted and subjected to precipitation with anti-JAK2AL33 serum (Fig. 1AGo). Sequential immunoblotting with anti-JAK2AL33 and antiphosphotyrosine antibodies confirmed that JAK2 present in C14 cells was inducibly tyrosine phosphorylated in response to GH and that no JAK2 was detected in {gamma}2A-GHR cells. Downstream signaling was assessed by measuring the ability of GH to cause transactivation of a STAT5-dependent luciferase reporter gene introduced by transient transfection of the Spi-GLE-luc plasmid (35) (Fig. 1BGo). GH-dependent luciferase activity was detected in C14 but not {gamma}2A-GHR cells, consistent with their differential expression of JAK2.



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  		FIG. 1. Characterization of GHR in {gamma}2A-GHR and C14 cells. A, GH-induced tyrosine phosphorylation of JAK2. {gamma}2A-GHR and C14 cells were serum starved and treated with (+) or without (–) GH for 10 min before detergent lysis and anti-JAK2AL33 immunoprecipitation. Precipitated proteins were resolved by SDS-PAGE and immunoblotted sequentially with anti-pY (top) and anti-JAK2AL33 (bottom). The data shown are representative of three such experiments. B, GH-induced reporter gene transactivation. {gamma}2A-rbGHR and C14 cells were transiently transfected with Spi-GLE-luc reporter plasmid. Samples were exposed to GH for 18 h in serum-free conditions before measurement of luciferase activity. Data are plotted as the GH-induced fold increase in luciferase activity relative to untreated samples (mean ± SE of three experiments). C and D, GH-induced GHR disulfide linkage. Serum-starved {gamma}2A-GHR and C14 cells were treated with (+) or without (–) GH for 10 min. Detergent extracts were resolved by SDS-PAGE under reducing (C) or nonreducing (D) conditions, and immunoblotted with anti-GHRcyt-AL47. The positions of the mature (bracket) and precursor (arrowhead) GHR forms are indicated in C. The disulfide-linked and non-disulfide-linked receptors are indicated in D. Note that GH induces disulfide linkage of GHR independent of JAK2 expression. The data shown are representative of three such experiments. WB, Western blot.

 
Because {gamma}2A-GHR is the parent cell for C14, GHR is detectable in both cells by anti-GHRcyt-AL47 immunoblotting (Fig. 1Go, C and D). When resolved by SDS-PAGE under reducing conditions, rabbit GHR is detected in two predominant forms, a broad 115- to 140-kDa form and an approximately 100-kDa form. These correspond to the mature (endoH-resistant) and precursor (endoH-sensitive) forms of GHR, respectively (13, 14, 34, 43, 44). Consistent with previous observations (34), C14 cells displayed more mature relative to precursor GHR than did {gamma}2A-GHR cells, and acute GH treatment caused a slight electrophoretic shift in the mature GHR in C14 cells, but not in {gamma}2A-GHR, consistent with GH-induced GHR tyrosine phosphorylation in the cells that express JAK2 (Fig. 1CGo). However, resolution of cell extracts under nonreducing conditions revealed that GH induced disulfide linkage of GHR in both the JAK2-deficient and JAK2-replete cells (Fig. 1DGo). Inducible disulfide linkage, mediated through an unpaired extracellular domain cysteine residue, reflects GH-induced conformational changes in the receptor required for signaling (33, 37, 45, 46). These findings indicate that, despite the lack of GH-induced signaling in {gamma}2A-GHR and differences in the fraction of receptors that are mature in {gamma}2A-GHR vs. C14, mature GHRs in both cells can undergo initial GH-induced conformational changes similarly.

We further characterized cell surface GHRs in {gamma}2A-GHR and C14 cells by two methods, surface biotinylation and radiolabeled GH binding (Fig. 2Go). Serum-starved cells were incubated with Sulfo-NHS-LC-biotin for 30 min at 4 C while shaking. Equal protein amounts of cell lysates were subjected to immunoprecipitation with anti-GHRcyt-AL47. Eluates were resolved by SDS-PAGE and sequentially blotted with streptavidin-HRP and anti-GHRcyt-AL47 (Fig. 2AGo, as indicated). As expected, the pattern revealed by anti-GHR precipitation and blotting was similar to that observed by blotting reduced cell extracts (Fig. 1CGo), with a diminished mature to precursor GHR ratio in {gamma}2A-GHR cells compared with C14 cells (Fig. 2AGo, third and fourth lanes). Streptavidin-HRP blotting (Fig. 2AGo, first and second lanes) revealed that surface biotinylated GHR was detected in both cells, migrating only in the position of the mature form detected by anti-GHRAL-47 blotting. Notably, none of the precursor GHR found in either cell was biotinylated, consistent with its lack of appearance at the cell surface. Surface radiolabeled GH binding was performed as a companion approach (Fig. 2BGo). Equal numbers of {gamma}2A-rbGHR and C14 cells were serum starved and incubated with [125I]hGH either in the presence (to determine nonspecific binding) or absence of excess unlabeled hGH for 1 h, washed, harvested, and subjected to {gamma}-counting. Although both cells exhibited specific binding, C14 cells displayed roughly 2.6 times greater GH binding capacity compared with {gamma}2A-rbGHR. Collectively, these data suggest JAK2 increases GHR cell surface presentation and enhances surface GH binding capacity.



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  		FIG. 2. JAK2 enhances GHR cell surface presentation and GH binding capacity. A, Surface biotinylation assay. Serum-starved {gamma}2A-GHR or C14 cells were incubated with Sulfo-NHS-LC-biotin for 30 min at 4 C while shaking. Cell lysates (equal protein abundance) were subjected to immunoprecipitation with anti-GHRcyt-AL47 and immunoblotted by streptavidin-HRP (left) and reprobed with anti-GHRcyt-AL47 (right). The positions of the mature (bracket) and precursor (arrowhead) GHR forms are indicated. The data shown are representative of two such experiments. B, [125I]hGH cell surface binding assay. Serum-starved {gamma}2A-GHR or C14 cells were incubated with [125I]hGH [50,000 cpm (~25 pM) per well] either in the presence or absence of 2 µg/ml (~91 nM) unlabeled hGH for 1 hr at room temperature, followed by washing, harvesting, and {gamma}-counting. The specific binding of {gamma}2A-GHR cells was set as 100% for comparison purposes (mean ± SE; n = 3 independent experiments). WB, Western blot.

 
Reduction of JAK2 levels in C14 cells by RNA interference lessens mature GHR abundance
In principle, differences in the ratio of mature to precursor GHR between {gamma}2A-GHR and C14 cells could relate to clonal variation rather than JAK2 levels per se. We thus sought to test the effect of lowering JAK2 abundance in C14 cells by RNA interference (Fig. 3Go). We designed an siRNA against JAK2 that recognizes an N-terminal region within murine (and, identically, in human) JAK2 that is without significant similarity to other JAK family members. The effect of transfection of this siRNA was monitored by immunoblotting with anti-JAK2AL33, a polyclonal serum against the C-terminal one third of murine JAK2 (36); this revealed marked loss of the stably expressed murine JAK2 in the transfected C14 cells (Fig. 3AGo, lane 3 vs. 2). Densitometric quantitation of several such experiments indicated approximately 75% knockdown of JAK2 (Fig. 3BGo). Notably, blotting with anti-GHRcyt-AL47 revealed that even though JAK2 knockdown was transient and incomplete, it resulted in reduction of mature GHR abundance and concomitant increase in detectable precursor GHR (Fig. 3CGo, lane 3 vs. 2). The mature to precursor GHR ratio decreased by 43.5 ± 6.8% (mean ± SE; n = 3) in JAK2 siRNA-treated C14 cells compared with cells treated with scrambled siRNA. These results strongly support the conclusion that the abundance of JAK2 influences the relative state of maturity of GHR within the same clonal cell line.



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  		FIG. 3. Reduction of JAK2 results in loss of mature GHR in C14 cells. A and B, JAK2 siRNA knocks down JAK2 protein level. A, C14 cells transiently transfected with scrambled siRNA (lane 2) or JAK2 siRNA (lane 3) or untransfected {gamma}2A-GHR cells (lane 1) were serum starved and detergent solubilized. Extracts containing equal amounts of total protein were resolved by SDS-PAGE and immunoblotted with anti-JAK2AL33. The positions of the JAK2 and a nonspecific band are indicated. The data shown are representative of three such experiments. B, Three immunoblots as shown in A (lanes 2 and 3) were subjected to densitometric analysis to derive estimates of the relative abundance of JAK2. Abundance of JAK2 in cells transfected with scrambled siRNA is set as 100% (mean ± SE; n = 3 independent experiments). C, Effect of JAK2 knockdown on mature GHR abundance. Detergent extracts from A were immunoblotted with anti-GHRcyt-AL47. The positions of the mature (bracket) and precursor (arrowhead) GHR forms are indicated. The data shown are representative of three such experiments. WB, Western blot.

 
JAK2 expression alters GHR cellular localization
We used {gamma}2A-GHR and C14 cells to examine effects of JAK2 on GHR localization within the cell. For these studies, we used two separate mouse monoclonal anti-GHR antibodies, which target different GHR regions. Anti-GHRext-mAb reacts with the rabbit and human GHR extracellular domain (34, 37, 38, 39), whereas anti-GHRcyt-mAb recognizes the rabbit and human GHR intracellular domain (34, 37). Cells were grown on coverslips, serum starved, and prepared for fluorescence confocal microscopy with each of these monoclonal antibodies, as in Materials and Methods (Fig. 4Go). Staining with anti-GHRext-mAb (Fig. 4Go, top) revealed both intracellular and surface signal in C14 cells. However, in {gamma}2A-GHR cells, signal was predominantly intracellular with much less at the cell surface. [Exposure to secondary antibody only revealed no significant staining of either cell (not shown), indicating that the signals with anti-GHRext-mAb were specific.] Similar findings were obtained when cells were stained with anti-GHRcyt-mAb (Fig. 4Go, bottom), with greater surface GHR detected in cells that express JAK2 (C14 cells) and relatively more intracellular GHR in the JAK2-deficient {gamma}2A-GHR cells. These microscopy findings suggest that the effects of JAK2 on the maturation status of GHR are reflected by its effects on surface vs. intracellular GHR distribution.



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  		FIG. 4. Stable expression of JAK2 alters the cellular localization of GHR. {gamma}2A-GHR (left panels) and C14 cells (right panels) were grown on coverslips and serum starved overnight. Cells were first fixed with formaldehyde with or without treatment with 0.1% Triton X-100. Fixed cells were labeled with the mouse monoclonal antibody anti-GHRext-mAb (upper panels) or anti-GHRcyt-mAb (lower panels). After extensive washing with PBS, cells were labeled with Alexa594-coupled antimouse secondary antibodies, counterstained with Hoechst, and analyzed by confocal laser scanning microscopy. The black arrow with white outline indicates surface GHR; the white arrow indicates intracellular GHR. Background (no primary antibody) was negative (not shown).

 
JAK2 enhances stability of mature GHR
Our findings in Figs. 1–4GoGoGoGo suggest that JAK2 augments the steady-state level of mature vs. precursor GHR and, concomitantly, the level of surface GHR available for interaction with GH. This important effect of JAK2 could be explained by influence either on GHR biogenesis (i.e. the process of biosynthesis and processing in the secretory pathway) or on stability of the mature receptor. To probe this issue further, we pursued two kinds of experiments to examine receptor biogenesis and stability. We first metabolically pulse labeled serum-starved {gamma}2A-GHR and C14 cells for 15 min with [35S]methionine/[35S]cysteine, followed by a chase in unlabeled methionine/cysteine for 0–240 min. At each time point, GHR was immunoprecipitated from detergent cell extracts with anti-GHRcyt-mAb, resolved by SDS-PAGE, and revealed by autoradiography. In both cells, GHR was initially synthesized as a lower Mr form (~110 kDa) that corresponds to the endoH-sensitive precursor form (15) and maximally converted by 60 min into a higher Mr (~115–140 kDa) mature GHR form. In two separate experiments, we densitometrically measured the GHR maturation efficiency (the mature GHR at 60 min divided by the precursor GHR at 0 min) for the two cells. In one experiment, the maturation efficiency was 17.7% for {gamma}2A-GHR cells and 23.0% for C14 cells; in a separate experiment, the maturation efficiency was 24.9% for {gamma}2A-GHR cells and 36.4% for C14 cells. Thus, JAK2 expression augmented GHR maturation efficiency by approximately 1.3- to 1.5-fold, suggesting that JAK2 modestly augments GHR biogenesis in these cells, perhaps by virtue of a chaperone effect like that postulated for JAK2 on EpoR (29).

To examine the effects of JAK2 on GHR stability, we performed experiments in which steady GHR levels were monitored after inhibiting new protein synthesis with CHX. Serum-starved {gamma}2A-GHR and C14 cells were treated with CHX for 0–4 h, harvested, and detergent extracted. Equal amounts of protein from each sample were resolved by SDS-PAGE, and GHR was detected by anti-GHRcyt-AL47 blotting (Fig. 5AGo). As expected, the abundance of GHR precursor relative to mature GHR was notably greater in {gamma}2A-GHR vs. C14 cells before CHX exposure. With increasing duration of CHX treatment, precursor abundance dropped precipitously and to a similar degree in both cells. In contrast to the precursor, inhibition of new protein synthesis with CHX resulted in disparate kinetics of mature GHR loss. Mature GHR disappeared much less rapidly and completely in C14 cells than in {gamma}2A-GHR cells. Reprobing with anti-epidermal growth factor receptor demonstrated that similar amounts of cellular protein were loaded in each lane. Quantitation (Fig. 5Go, B and C) confirmed that the rate of loss of the precursor is rapid in both cells and also made plain the difference in mature GHR t1/2 between the cells, with mature GHR dramatically protected by the presence of JAK2.



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  		FIG. 5. JAK2 prevents degradation of mature GHR; CHX chase assay. A, Serum-starved {gamma}2A-GHR and C14 cells were treated with CHX (20 µg/ml) for 0–4 h, as indicated. Detergent extracts were resolved by SDS-PAGE and blotted with anti-GHRAL47. The positions of the mature (bracket) and precursor (arrowhead) GHR forms are indicated. The data shown are representative of three such experiments. B and C, Densitometric quantitation of the anti-GHRAL47 blots. The precursor (B) and mature (C) GHR abundance at time 0 was considered 100%, respectively, for each (mean ± SE; n = 3 independent experiments). WB, Western blot.

 
Proteasome and lysosome inhibitors enhance stability of mature GHR in JAK2-deficient cells
To begin to understand how JAK2 might stabilize the mature GHR, we tested the effects on CHX-induced loss of mature GHR in {gamma}2A-GHR cells of drugs known to affect GHR fate. GHR is a target for metalloprotease-mediated cleavage in the juxtamembrane extracellular domain, which occurs constitutively and is accelerated by stimuli such as growth factors and phorbol ester (14, 38, 47, 48). We tested whether the metalloprotease inhibitor IC3 (which prevents both constitutive and inducible GHR proteolysis) (38) affected CHX-induced receptor loss in {gamma}2A-GHR cells (Fig. 6AGo). As expected, CHX treatment for 2 h resulted in complete disappearance of precursor GHR and marked loss of mature GHR in these cells that lack JAK2. Pretreatment with IC3 for 40 min before the CHX treatment did not change the degree of mature GHR loss revealed in the absence of protein synthesis (Fig. 6Go, A and B). As shown in Fig. 6CGo, pretreatment with IC3 completely blocked PMA-induced receptor loss, as previously reported (14, 38, 42, 47, 48, 49), verifying that the drug was active. These findings indicate that constitutive metalloprotease activity, at least over a 160-min period tested with IC3, does not account for the lability of the mature GHR seen in the absence of JAK2.



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  		FIG. 6. Metalloprotease inhibitor does not increase stability of mature GHR in JAK2-deficient cells. A, Serum-starved {gamma}2A-GHR cells were incubated with or without the metalloprotease inhibitor IC3 for 40 min, followed by treatment with or without CHX for 2 h. Detergent extracts were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47. The positions of the mature (bracket) and precursor (arrowhead) GHR forms are indicated. The data shown are representative of three such experiments. B, Densitometric quantitation of anti-GHRcyt-AL47 blots. Mature GHR abundance at time 0 was considered 100% (mean ± SE; n = 3 independent experiments). C, Serum-starved C14 cells were incubated with or without IC3 for 40 min, followed by treatment with or without PMA for 30 min. Detergent extracts were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47. The positions of the GHR (bracket), its remnant, and a nonspecific (NS) band (arrows) are indicated. Note that PMA-induced GHR proteolysis was inhibited by IC3. WB, Western blot.

 
Even though GHR ubiquitination itself is not necessary and GHR is not targeted to the proteasome, an intact ubiquitin-proteasome system is believed to be required for constitutive and ligand-induced GHR internalization and resultant lysosomal degradation (15, 19). We tested the effect of proteasome inhibition on the fate of mature GHR after CHX treatment (Fig. 7AGo). Pretreatment with a specific proteasome inhibitor, clasto-lactacystin ß-lactone, an active analog of lactacystin (referred to as lactacystin) did not block the CHX-induced loss of precursor. However, CHX-induced loss of mature GHR was substantially blunted by lactacystin pretreatment (~82% loss of mature GHR induced by CHX in the absence of lactacystin but only approximately 30% loss in the presence of lactacystin). Similar results were obtained when each of two lysosomal inhibitors, ammonium chloride (Fig. 7BGo) and chloroquine (Fig. 7CGo), was tested in the same fashion. These findings indicate that the increased lability of mature GHR in cells that lack JAK2 is reversed when either the ubiquitin-proteasome pathway or lysosomal degradation are inhibited. This suggests that JAK2 may exert its effect on mature GHR stability by blunting constitutive GHR entry into or progress through this endocytic pathway-mediated degradation. As expected, treatment of C14 cells with lysosomal inhibitors did not substantially affect the fate of GHR during a 2-h CHX treatment, because very little receptor loss was observed under these conditions (Fig. 5Go and data not shown).



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  		FIG. 7. Proteasome and lysosome inhibitors enhance stability of mature GHR in JAK2-deficient cells. A–C, Serum-starved {gamma}2A-GHR cells were incubated with or without the proteasome inhibitor lactacystin for 5 h (A) or the lysosome inhibitors ammonium chloride (B) or chloroquine (C) for 2 h, followed by treatment with or without CHX for 2 h. Top, Detergent extracts were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47. The positions of the mature (bracket) and precursor (arrowhead) GHR forms are indicated. The data shown are representative of three such experiments. Bottom, Densitometric quantitation of anti-GHRcyt-AL47 blots. Mature GHR abundance at time 0 was considered 100% (mean ± SE; n = 3 independent experiments for each). WB, Western blot.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
GH responsiveness at target tissues may be governed by a complex interplay of factors, including the level and degree of pulsatility of GH secretion from the anterior pituitary, the level of GHR gene expression, posttranscriptional regulation of GHR abundance, and the presence and subcellular localization of downstream signaling molecules (2, 16, 50, 51, 52, 53, 54). In vivo and in vitro studies in which GHR is either absent or its abundance is altered suggest that the level of GHR can influence GH action (14, 48, 53, 55, 56, 57, 58, 59). The activity of JAK2 as a tyrosine kinase is clearly critical for GH responsiveness, because without JAK2, virtually all GH signaling is prevented (2, 60, 61). Additionally, other mechanisms, including the effects of suppressor-of-cytokine-signaling proteins on the stability of GHR and JAK2, have roles in governing GH sensitivity (62, 63, 64, 65). The current study focuses on the influence of JAK2 on GHR surface availability.

We previously demonstrated that after biosynthesis GHR could, to some degree, traverse the secretory pathway and achieve a mature glycosylation pattern, even in the absence of JAK2 (34). However, we also observed that expression of JAK2 in the JAK2-deficient {gamma}2A-GHR human fibrosarcoma cell line markedly affected the ratio of mature to precursor GHR. Comparison of multiple clones with varying JAK2 expression levels indicated that the mature to precursor GHR ratio was positively correlated with the level of JAK2 expressed. Our current results confirm and extend these observations by comparing cells that lack JAK2 ({gamma}2A-GHR) with cells that express GHR and JAK2 (C14 cells).

Although JAK2 was required for GH-induced signaling, it was not necessary for surface GHR to undergo disulfide linkage in response to GH. Because GH-induced GHR disulfide linkage reflects the receptor’s adoption of a conformation competent for signaling (37), this finding indicates that JAK2 is apparently not necessary for GH-induced GHR conformational change. However, the effect of JAK2 on GHR surface presentation was substantial. As assessed by both surface biotinylation and [125I]hGH surface binding, mature GHR present at the cell surface was markedly greater in JAK2-expressing cells than in those that lacked JAK2. This was further reinforced by confocal microscopy of {gamma}2A-GHR and C14 cells. Staining with monoclonal antibodies directed against both the extracellular and cytoplasmic domains revealed greatly enhanced surface GHR in C14 cells.

We approached potential mechanisms for these findings. First, short-term siRNA-mediated knockdown of JAK2 by approximately 75% in C14 cells reduced the mature to precursor GHR ratio, indicating that the observations between clones could be confirmed within C14 cells by reducing JAK2. These findings suggest that the diminished GHR surface abundance in {gamma}2A-GHR cells is rectified by JAK2 expression, favoring the likelihood that JAK2 deficiency is causative of the altered GHR distribution. We considered three main hypotheses as to how JAK2 expression might cause the differences in mature to precursor GHR ratio and associated surface GHR presentation in C14 vs. {gamma}2A-GHR cells: 1) JAK2 could enhance mature to precursor GHR by increasing the pace or efficiency of receptor biogenesis by chaperoning the receptor through the secretory pathway and/or masking a retention signal that would otherwise impede its progress; 2) JAK2 could affect the stability of the mature surface GHR by lessening its susceptibility to constitutive metalloproteolysis; and 3) JAK2 could affect the stability of the mature surface GHR by lessening its constitutive endocytosis and lysosomal degradation and/or enhancing recycling to the surface of constitutively endocytosed receptors.

The first hypothesis was addressed by pulse-chase metabolic labeling followed by anti-GHR immunoprecipitation and autoradiography. This revealed only a modest increase in GHR maturation efficiency conferred by the presence of JAK2. In these experiments, the kinetics of transition from precursor to mature GHR was very similar between C14 and {gamma}2A-GHR cells (not shown). Similarly, in the CHX chase experiments, the disappearance of immunoblottable precursor after blockade of new protein synthesis with CHX was equally rapid in both cells. These findings suggest that JAK2, which can interact with the precursor GHR in the endoplasmic reticulum early in the process of biogenesis (8), may exert part of its effect on GHR surface abundance by a chaperone effect. However, our data also suggest that the presence of JAK2 causes a marked difference in the t1/2 of the mature GHR between the two cells. In cells expressing JAK2, mature GHR was much more stable.

We approached mechanisms whereby JAK2 might stabilize the mature receptor (hypotheses 2 and 3 above) by testing the effects of metalloprotease, proteasome, and lysosomal enzyme inhibitors on the fate of mature GHR in {gamma}2A-GHR cells after exposure to CHX. The metalloprotease inhibitor IC3 had no effect on mature GHR stability, indicating that constitutive metalloprotease-mediated GHR cleavage at the cell surface is likely not the parameter affected by JAK2 to account for its stabilizing effect. This does not rule out important effects of constitutive GHR cleavage but indicates that, if present, they are not revealed by short-term IC3 inhibition. However, different conclusions are drawn from the proteasome and lysosome inhibitor experiments. In both, mature GHR stability in {gamma}2A-GHR cells was greatly enhanced. In light of Strous’s elegant work implicating the ubiquitin-proteasome pathway in mediating GHR entry into the endocytic pathway (15), these results strongly point to the locus of the effects of JAK2 on GHR availability being at the level of constitutive endocytosis and lysosomal degradation. Our data do not yet allow us to completely discriminate whether JAK2 is inhibiting entry into the endocytic pathway vs. encouraging recycling of already endocytosed receptors, but the proteasome inhibitor experiments favor the former over the latter possibility.

Our results are noteworthy in particular in the context of emerging information about the effects of JAKs on trafficking and stability of other cytokine receptors. EpoR, unlike the GHR, is significantly retained in the ER and undergoes ER degradation (18, 20, 21, 66). Huang et al. (29) reported that JAK2 associates with the precursor EpoR and that expression of JAK2 in JAK2-deficient cells augments the abundance of mature EpoR and detection of EpoR on the cell surface. Although pulse-chase experiments were not reported, these authors concluded that JAK2 served a chaperone role for EpoR, facilitating its carbohydrate processing maturation and efficient surface expression, an effect that was mapped to the N-terminal FERM (band 4.1, ezrin, radixin, moesin) domain of JAK2 and was independent of its kinase function (29). Similarly, Radtke et al. (28) showed that overexpressed oncostatin-M receptor (OSMR) accumulates mainly in the ER and the OSMR maturation status and cell surface localization were increased upon JAK1 coexpression. Although no direct assessment of the rate of precursor to mature receptor transition was offered, it was concluded that JAK1 masks a signal within the membrane-proximal cytoplasmic domain region of OSMR that prevents efficient surface expression. In studying the type I interferon-{alpha} receptor (IFNAR1), Ragimbeau et al. (30) found that in the absence of TYK2, the IFNAR1 localized in perinuclear endosomes, and coexpressed TYK2 markedly increased surface mature IFNAR1 levels. In contrast to the reports on EpoR/JAK2 and OSMR/JAK1, in which a chaperone effect of the JAK was inferred, these studies included biochemical estimates of IFNAR1 t1/2 by CHX chase experiments and found that TYK2 expression dramatically delayed IFNAR1 degradation in a proteasome and lysosome inhibitor-dependent fashion (30). In this system, it was concluded that TYK2, independent of its kinase activity, inhibits internalization and degradation of the mature IFNAR1. The other cytokine receptor/JAK pair recently studied in this fashion is the common {gamma}-chain ({gamma}c) and JAK3 (32). In these studies, JAK3 was not required for surface {gamma}c expression, but increased JAK3 levels boosted surface {gamma}c and no chaperone role for JAK3 could be detected.

Although we observed somewhat enhanced GHR maturation efficiency in cells expressing JAK2, our findings for GHR/JAK2 are most reminiscent of those for IFNAR1/TYK2, in that we observed a predominant effect of JAK2 on mature GHR stability. However, like the findings for {gamma}c/JAK3, we did not find an absolute requirement for JAK2 to allow some surface presentation of mature GHR. It is not understood why different mechanisms might apply to different cytokine receptor/JAK pairs in the effect of the JAKs on surface cytokine receptor levels. It is possible that multiple mechanisms are in play in each system, but there are technical limitations to observe all but the most dramatic effects. Similarly, it is not yet known whether the structural underpinnings of these effects among different receptor/JAK pairs are the same. Further investigation will include mapping of GHR and JAK2 regions required to confer the effect of enhancement of surface mature GHR seen with expression of JAK2. It is also intriguing to speculate that effects on mature GHR stability may have secondary influence on aspects of GHR biogenesis. More detailed subcellular localization studies of GHR in the presence and absence of JAK2 may be illustrative. Our findings in this study are limited to a single, albeit informative, in vitro system, the JAK2-deficient {gamma}2A-GHR cell with JAK2 reconstitution. Although our current and previous data strongly suggest that JAK2 deficiency, rather than another unrelated property of this system, causes differences in mature GHR surface availability, in vitro and in vivo testing in other systems will be required for validation of this conclusion. If validated, we believe that the effects of JAK2 on GHR surface abundance add to the complexity of control of GH responsiveness and suggest that a more complete understanding of the regulation of GH action will include additional studies of how JAK2 might affect GHR’s constitutive access to, and progression through, its endocytic down-regulation pathway.


   Acknowledgments
 
We appreciate helpful conversations with Drs. J. Collawn, E. Benveniste, G. Fuller, J. Kudlow, A. Theibert, J. Messina, Y. Huang, and N. Yang and the generous provision of reagents by those named in the text. We also acknowledge the expert assistance of Dr. Albert Tousson, University of Alabama at Birmingham Cell Biology Imaging Core Facility.


   Footnotes
 
This work was supported by National Institutes of Health (NIH) Grant DK58259 and in part by NIH Grant DK46395, a Veterans Affairs Merit Review Award (to S.J.F.), and NIH training grant T-32 GM08111 (to J.D.C.).

Parts of this work were presented at the 86th Annual Meeting of The Endocrine Society, New Orleans, LA, 2004.

First Published Online August 4, 2005

Abbreviations: {gamma}c, {gamma}-Chain; EpoR, erythropoietin receptor; ER, endoplasmic reticulum; GHR, GH receptor; hGH, human GH; HRP, horseradish peroxidase; IC3, Immunex Compound 3; IFNAR1, type I interferon-{alpha} receptor; JAK, Janus kinase; OSMR, oncostatin-M receptor; PMA, phorbol 12-myristate 13-acetate; siRNA, small interfering RNA; STAT, signal transducer and activator of transcription; TYK, tyrosine kinase.

Received April 29, 2005.

Accepted for publication July 29, 2005.


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 Introduction
 Materials and Methods
 Results
 Discussion
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