Janus Kinase 2 Determinants for Growth Hormone Receptor Association, Surface Assembly, and Signaling

Kai He, Xiangdong Wang, Jing Jiang, Ran Guan, Kenneth E. Bernstein, Peter P. Sayeski and Stuart J. Frank

Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, and Department of Cell Biology, University of Alabama (K.H., X.W., J.J., R.G., S.J.F.), Birmingham, Alabama 35294; Department of Pathology, Emory University School of Medicine (K.E.B.), Atlanta, Georgia 30322; Department of Physiology and Functional Genomics, University of Florida College of Medicine (P.P.S.), Gainesville, Florida 32610; and Endocrinology Section, Medical Service, Veterans Affairs Medical Center (S.J.F.), Birmingham, Alabama 35233

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GH signaling depends on functional interaction of the GH receptor (GHR) and the cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2), which possesses a C-terminal kinase domain, a catalytically inactive pseudokinase domain just N-terminal to the kinase domain, and an N-terminal half shown by us and others to harbor elements for GHR association. Computational analyses indicate that JAKs contain in their N termini (~450 residues) divergent FERM domains. FERM domains (or subdomains within them) in JAKS may be important for associations with cytokine receptors. For some cytokine receptors, JAK interaction may be required for receptor surface expression. We previously demonstrated that a JAK2 mutant devoid of its N-terminal 239 residues (JAK2-{Delta}1–239) did not associate with GHR and could not mediate GH- induced signaling. In this report we employ a JAK2-deficient cell line to further define N-terminal JAK2 regions required for physical and functional association with the GHR. We also examine whether JAK2 expression affects cell surface expression of the GHR. Our results suggest that FERM motifs play an important role in the interaction of GHR and JAK2. While JAK2 expression is not required for detectable surface GHR expression, an increased JAK2 level increases the fraction of GHRs that achieves resistance to deglycosylation by endoglycosidase H, suggesting that the GHR-JAK2 association may enhance either the receptor’s efficiency of maturation or its stability. Further, we report evidence for the existence of a novel GH-inducible functional interaction between JAK2 molecules that may be important in the mechanism of GH-triggered JAK2 signaling.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ENGAGEMENT of the GH receptor (GHR) by GH causes a variety of cellular and tissue responses necessary for normal growth and metabolism. Among these are regulation of target genes, such as IGF-I, c-fos, serine protease inhibitor 2.1 (Spi2.1), and others and modulation of sensitivity to insulin and other growth factors and hormones (reviewed in Ref.1). The GHR is a transmembrane glycoprotein member of the cytokine receptor superfamily that spans the plasma membrane once, binds GH in its extracellular domain, and initiates signaling by virtue of its intracellular domain’s regulated interactions with the cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2), and other signaling molecules (2, 3, 4, 5, 6). JAK2 is a member of a family of related molecules [JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2) in mammals] that are typified by having a C-terminal kinase domain, a catalytically inactive kinase-like (pseudokinase) domain just N-terminal to the kinase domain, and an N-terminal half that is believed to be involved with protein interactions, kinase regulation, and structural integrity (7, 8).

The active signaling conformation of the receptor is as a dimer bound to a monomeric GH molecule (9), and recent studies suggest that the unliganded GHR may exist as a preformed dimer that undergoes a GH-induced conformational change to achieve activation (10, 11, 12, 13, 14). Several intracellular signaling pathways are activated in response to GH. These include the activation of signal transducers and activators of transcription-1 (STAT1), STAT3, STAT5, MAPKs, and phosphoinositol 3-kinase (Refs. 1 ,6 , and 15 and references therein). Activation of JAK2 kinase activity is believed to be critical for the initiation of most, if not all, of these signaling pathways. Yet the mechanism(s) by which GH binding leads to JAK2 activation is as yet uncertain.

Previous studies suggest that, like several other cytokine receptor family members that associate with JAKs, the GHR’s physical and functional interaction with JAK2 requires a perimembranous proline-rich receptor cytoplasmic domain region known as Box 1, although other regions may also contribute to the interaction (16, 17, 18, 19). Reciprocal studies of the regions of JAK2 that confer productive association with the GHR have been few. Our findings (20) demonstrated that C-terminal deletion of the kinase domain abrogated GH-induced JAK2 activation, but did not affect association with the GHR, whereas deletion of the pseudokinase domain prevented neither GHR association nor GH-induced activation. Further, deletion of the N-terminal 240 residues of JAK2 prevented both physical association with the GHR and GH-induced activation. This suggested that an N-terminal region(s) (not delimited in that study) is required for JAK2’s interaction with the GHR and that physical association of the receptor and JAK2 was probably a prerequisite for their functional interaction. These findings were largely corroborated by Tanner et al. (21), who found that the first 551 residues of JAK2 (roughly the N-terminal half of the molecule) are sufficient for interaction with the GHR cytoplasmic domain, but again did not delimit the required regions at the N terminus. Interaction between the GHR and JAK2 can be detected even in the absence of GH stimulation and appears to be independent of tyrosine phosphorylation of either molecule (16). Yet treatment of cells with GH enhances GHR-JAK2 association (4, 5), suggesting that a GH-induced conformational change in the receptor changes either its affinity for JAK2 or the stoichiometry of the GHR-JAK2 assemblage.

Recent computational analyses have revealed that JAKs contain in their N termini (roughly 450 residues) divergent FERM domain motifs (22, 23). The FERM domain (named for the original proteins in which it was found: erythrocyte protein band 4.1, ezrin, radixin, moesin) may have a role in tethering proteins to the cytoplasmic tails of cell surface proteins (24). JAK FERM motifs include conserved interspersed blocks of hydrophobic residues. There is evidence that FERM domains (or subdomains within them) in JAKs may be important in their associations with cytokine receptors, e.g. for the interactions between JAK1 and glycoprotein 130 (25, 26), JAK2, and erythropoietin receptor (EpoR) (27), and JAK3 and the IL-2 receptor common {gamma}-chain ({gamma}c) (28). Interestingly, however, the determinants within JAKs that mediate their interactions with receptors may vary for different receptors. For example, in contrast to its association with the GHR, interaction of JAK2 with the serpentine angiotensin II receptor (AT1R) is dependent on an intact JAK2 kinase domain and is ligand- (AT-II) and JAK2 tyrosine phosphorylation-dependent (29, 30). Physical interaction of JAK2 with the AT1R has been mapped to a discrete N-terminal JAK2 motif, 231YRFRR (31). Further, the interactions of a particular JAK (JAK1) with different cytokine receptor cytoplasmic domains has recently been shown to use varying N-terminal subregions of the JAK for different receptors (32).

In this report we employ a JAK2-deficient cell line to further define N-terminal JAK2 regions required for physical and functional association with the GHR. We also examine whether JAK2 expression affects cell surface expression of the GHR. Our results suggest that FERM motifs play an important role in the interaction of GHR and JAK2. Although JAK2 expression is not required for detectable surface GHR expression, increased JAK2 levels increases the fraction of GHRs that achieves resistance to deglycosylation by endoglycosidase H, suggesting that the GHR-JAK2 association may enhance either the receptor’s efficiency of maturation or its stability. Further, we report evidence for the existence of a novel GH-inducible functional interaction between JAK2 molecules that may be important in the mechanism of GH-triggered JAK2 signaling.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effects of JAK2 Expression on Surface GHR Assembly and Signaling in Transiently and Stably Transfected {gamma}2A Cells
Recent studies suggest that JAKs, in addition to being required for signaling, may regulate cytokine receptor expression at the cell surface. In particular, findings for JAK1 and the oncostatin-M receptor (33) and for JAK2 and the EpoR (27) suggest that these JAKs perform a chaperone role in mediating cytokine receptor expression at the cell surface. Ragimbeau et al. (34) reported that TYK2 regulates cell surface interferon-2 receptor-1 (IFNAR1) expression, but in that case by lessening the rate of receptor internalization and thereby stabilizing it from degradation. In contrast, Suzuki, et al. (35) found no requirement for JAK3 in mediating or potentiating the surface expression of the IL-2R {gamma}c chain. In COS-7 cells, coexpression of GHR and JAK2 enables GH-dependent JAK2 activation (20), but we have yet to rigorously determine the influence of JAK2 on GHR cell surface expression. To facilitate studies of JAK2’s role in GHR surface assembly and mapping of the JAK2-GHR interaction, we used the JAK2-deficient human fibrosarcoma cell line, {gamma}2A (36).

We first examined the effects of transient expression of GHR alone vs. coexpression of GHR and JAK2 on GHR glycosylation status. The GHR undergoes characteristic processing events after synthesis in the endoplasmic reticulum and during Golgi transport to the cell surface. Immature (high mannose form) GHRs have yet to traverse the trans-Golgi, while the mature carbohydrate pattern is present in those receptors that have exited the Golgi. These GHR pools can be distinguished by their sensitivity to deglycosylation with endoglycosidase H (endoH) (37). Immature forms are sensitive to deglycosylation with endoH, whereas endoH resistance characterizes mature GHRs that populate the cell surface. {gamma}2A cells were transiently transfected with an expression vector encoding rabbit GH receptor (rbGHR) or with vector alone (as a negative control; Fig. 1AGo). rbGHRs were immunoprecipitated from detergent cell extracts of each transfected pool with a GHR cytoplasmic domain antiserum (anti- GHRcyt-AL37) (5). Precipitates were divided into three 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. Eluates of the precipitates from each treatment were resolved by SDS-PAGE and immunoblotted with anti-GHRAL47, a separate receptor cytoplasmic domain antiserum (38).



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Fig. 1. Transient Expression of rbGHR and JAK2 in {gamma}2A Cells

A, Endoglycosidase sensitivity of transiently expressed rbGHR. {gamma}2A cells were transiently transfected with the cDNA expression plasmid encoding the rbGHR (lanes 4–6) or the empty vector control (lanes 1–3) as indicated. Anti-GHRcyt-AL37 immunoprecipitates from detergent extracts were treated as indicated with endoH, F/N, or vehicle, as described in Materials and Methods. Proteins were then resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. The positions of the endoH-resistant, endoH-sensitive, and F/N-sensitive (Endo H-sensitive degly) forms of the rbGHR are indicated, as is the 112-kDa molecular mass standard. The data shown are representative of three such experiments. B and C, Effect of transient JAK2 expression on rbGHR glycosylation status. B, {gamma}2A cells were transiently transfected with the rbGHR cDNA expression plasmid plus either empty vector (lanes 1 and 2) or a murine JAK2 expression plasmid (lanes 3 and 4), as indicated. Anti-GHRcyt-AL37 immunoprecipitates were subjected to endo H treatment, SDS-PAGE, and immunoblotting with anti-GHRcyt-AL47 as described in A. Note the similarity of the pattern of deglycosylation independent of JAK2 expression. C, Aliquots of the transfected pools of cells from B were stimulated with GH or vehicle, as indicated, for 10 min at 37 C before detergent extraction and anti-JAK2AL33 immunoprecipitation. Eluates were resolved by SDS-PAGE and immunoblotted sequentially with anti-pY (upper panel) or anti-JAK2AL33 (lower panel) antibodies. The data shown in B and C are representative of three such experiments. D and E, Effect of transient JAK2 expression on rbGHR surface [125I]hGH cross-linking. D, {gamma}2A cells transiently expressing rbGHR alone or rbGHR plus JAK2 were treated as indicated with [125I]hGH in the presence or absence of excess unlabeled hGH before surface cross-linking as described in Materials and Methods. Extracted proteins were resolved by SDS-PAGE, and the surface cross-linked rbGHR-[125I]hGH complex (bracket) was identified by autoradiography. E, Aliquots of the transfected pools of cells from D were subjected to anti-GHRcyt-AL37 immunoprecipitation and immunoblotting with anti-GHRcyt-AL47 (upper panel) or anti-JAK2AL33 (lower panel) to verify the presence of the transfected proteins. The data shown in D and E are representative of two such experiments.

 
The immunoprecipitated transfected rbGHR was detected as two forms: a broad set of bands with a molecular mass of roughly 115–140 kDa and a doublet at roughly 100 kDa (Fig. 1AGo, lane 4). As we and others have previously observed for rbGHR (37, 39, 40, 41), endoH treatment caused the sharper bands to migrate more rapidly, but did not affect migration of the broad 115- to 140-kDa receptor form (Fig. 1AGo, lane 5). Notably, deglycosylation with F/N caused all detected GHR to migrate with the endoH-sensitive form (Fig. 1AGo, lane 6 vs. 5), suggesting that the endoH-resistant receptors were deglycosylated by an enzyme that does not discriminate immature from mature forms. These data indicated that a substantial fraction of transfected rbGHR achieves endoH resistance even in the absence of JAK2.

We next examined the impact of transient coexpression of JAK2 on GHR glycosylation status and cell surface expression. {gamma}2A cells transfected with rbGHR were compared with those cotransfected with rbGHR and murine JAK2 with regard to endoH sensitivity (Fig. 1BGo, lanes 1 and 2 vs. lanes 3 and 4). JAK2 coexpression did not substantially change the fraction of rbGHRs that achieved endoH resistance. To verify JAK2 coexpression, aliquots of the same transfected cells analyzed in Fig. 1BGo were stimulated with either GH or its vehicle, and JAK2 abundance and tyrosine phosphorylation were assessed. Anti-JAK2AL33 (42) precipitation and blotting verified that JAK2 was present only in the cotransfected cells (Fig. 1CGo, lower panel, lanes 3 and 4 vs. lanes 1 and 2), and reprobing with anti-pY indicated that GH caused JAK2 tyrosine phosphorylation (Fig. 1CGo, upper panel, lanes 4 vs. 3). Thus, in this transient cotransfection system, a level of JAK2 expression sufficient to confer GH-dependent signaling did not substantially affect GHR glycosylation status.

GHR surface expression was qualitatively assessed by [125I]human (h) GH cell surface cross-linking (Fig. 1DGo). {gamma}2A cells transiently expressing rbGHR (lanes 1 and 2) or rbGHR with JAK2 (lanes 3 and 4) were serum-starved and incubated with radiolabeled GH in the presence (+) or absence (-) of excess unlabeled GH at 4 C, followed by covalent cross-linking, SDS-PAGE, and autoradiography. Affinity-labeled surface GHR was detected in cells transfected both with and without JAK2. Immunoblotting of aliquots of the same cells revealed JAK2 only in the cotransfectants and that transfected rbGHR levels were similar in both pools of cells (Fig. 1EGo, upper and lower panels, respectively). In other experiments (not shown), surface GH binding capacity was similar in rbGHR vs. rbGHR-JAK2 transient transfectants by [125I]hGH binding assay.

In light of the reports that JAKs may affect the surface expression of cytokine receptors (27, 33, 34), we also analyzed {gamma}2A cells stably expressing GHR and JAK2. We began with {gamma}2A-rbGHR, the result of stable transfection of rbGHR into {gamma}2A (38). {gamma}2A-rbGHR was stably transfected with a JAK2 expression plasmid, and zeocin-resistant clones were analyzed by immunoblotting with anti-GHRcyt-AL47 and anti-JAK2AL33. The analysis of six representative clones with varying levels of JAK2 expression compared with {gamma}2A-rbGHR is shown in Fig. 2AGo. Relative JAK2 abundance in extracts containing equal protein amounts from each clone was assessed by anti-JAK2-AL33 precipitation and blotting (Fig. 2AGo, lower panel). As expected, JAK2 was not detected in {gamma}2A-rbGHR cells, but a range of JAK2 (arrow) abundance was observed in the other clones.



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Fig. 2. Stable Expression of rbGHR and JAK2 in {gamma}2A Cells

A, rbGHR pattern in clones of {gamma}2A-rbGHR cells stably transfected with JAK2. {gamma}2A-rbGHR cells were transfected with a JAK2 cDNA expression plasmid, and stable clones were selected with zeocin. The parental cells ({gamma}2A-rbGHR) and six independent JAK2-expressing clones were serum-starved, and detergent extracts containing equal amounts of total protein were either immunoprecipitated with anti-JAK2AL33, resolved by SDS-PAGE, and immunoblotted with anti-JAK2AL33 (lower panel) or resolved without immunoprecipitation and immunoblotted with anti-GHRcyt-AL47 (upper panel). The positions of the mature (bracket) and precursor (arrowhead) rbGHR forms are indicated in the upper panel. The position of transfected JAK2 is indicated by an arrow in the lower panel. Note the relative increase in mature vs. precursor forms with increased JAK2 levels among the clones. The data shown are representative of three such experiments. B, Effect of stable JAK2 expression on rbGHR glycosylation status. Serum-starved {gamma}2A-rbGHR and {gamma}2A-rbGHR-JAK2 clone 14 cells were subjected to anti-GHRcyt-AL37 immunoprecipitation, endoH deglycosylation, and anti-GHRcyt-AL47 immunoblotting as described in Fig. 1BGo. Note the marked relative decrease in endoH-sensitive vs. endoH-resistant rbGHR in clone 14 compared with {gamma}2A-rbGHR cells. The data shown are representative of two such experiments. C, Comparison of JAK2 abundance and relative rbGHR maturation among JAK2 transfectant clones. Immunoblots in A were subjected to densitometric analysis, as described in Materials and Methods, to derive estimates of the relative ratio of mature/immature rbGHR abundance (ordinate) vs. the relative JAK2 abundance (abscissa) among the JAK2 stable transfectants and the parental {gamma}2A-rbGHR cells. r2 = 0.82 for the best-fit straight line for this dataset.

 
The rbGHR in each clone was detected by anti-GHRcyt-AL47 immunoblotting of equal aliquots of cell extract (Fig. 2AGo, upper panel). Interestingly, although the GHR in all clones was that originally present in {gamma}2A-rbGHR (the target of the transfection), the pattern of receptor migration varied such that clones stably expressing relatively increased JAK2 levels also displayed relatively more of the broad 115- to 140-kDa GHR form (bracket) compared with the roughly 100 kDa form (arrowhead). We confirmed that these forms corresponded to the mature (endoH-resistant) and immature (endoH-sensitive) forms of the rbGHR, respectively, by comparing the electrophoretic behaviors of the immunoprecipitated receptors from the JAK2- deficient {gamma}2A-rbGHR (Fig. 2BGo, lanes 1 and 2) and the high level JAK2-expressing clone 14 (lanes 3 and 4) after treatment of the precipitates with endoH (Fig. 2BGo). Both cells contained endoH-resistant GHRs (bracket), but clone 14 contained relatively much less of the endoH-sensitive form (arrows) than did {gamma}2A-rbGHR. In other experiments (not shown), this same pattern was seen with endoH digestion for the other JAK2 transfectant clones. The ratio of mature/immature rbGHR forms was estimated by densitometry and plotted against the densitometrically determined relative abundance of JAK2 for the clones and {gamma}2A-rbGHR (Fig. 2CGo). This plot indicated robust correlation between the JAK2 expression level and the degree of mature rbGHR detected in the steady state by immunoblotting. We conclude that, although unnecessary for the GHR to achieve a mature glycosylation pattern and surface expression, JAK2 may govern the degree to which the receptor transitions from endoH sensitivity to endoH resistance and/or may preferentially protect the mature GHR from degradation.

An Intact FERM Domain Is Required for Physical and Functional Interaction of JAK2 with GHR
The data in Fig. 1Go with transient JAK2 reconstitution of {gamma}2A cells suggested that this system could be used to further map the functional interaction of JAK2 N- terminus with the GHR. For these studies we transiently expressed wild-type (WT) and JAK2 mutants into {gamma}2A-rbGHR cells. The JAK2 mutants tested are diagrammed in Fig. 3Go, as are JAK2-1–999 and JAK2-{Delta}1–239, which have been previously studied (20, 43). We first tested the ability to productively reconstitute {gamma}2A-rbGHR with WT murine JAK2 (Fig. 4Go). {gamma}2A-rbGHR cells were transiently transfected with WT JAK2, serum-starved, and treated with (+) or without (-) GH for 10 min. Anti-JAK2AL33 precipitation and immunoblotting with anti-pY (Fig. 4AGo, upper panel) and anti-JAK2AL33 (Fig. 4AGo, lower panel) demonstrated that the transfected JAK2 underwent tyrosine phosphorylation in response to GH (lanes 2 vs. 1). JAK2 can be specifically coimmunoprecipitated with our antisera to the GHR cytoplasmic domain (5, 44, 45). Anti-JAK2AL33 immunoblotting of anti-GHRcyt-AL37 precipitates from the same extracts showed the coimmunoprecipitation of JAK2 with the receptor in the WT JAK2-transfected cells (Fig. 4BGo, lanes 1 and 2), and that the GHR-JAK2 association was enhanced after GH exposure, consistent with previous results (5, 45). Precipitation with nonimmune serum did not allow the detection of either GHR or JAK2 (not shown). To further assess the functionality of JAK2 reconstitution, we cotransfected a plasmid encoding a luciferase (luc) reporter driven by tandem copies of the Spi2.1 {gamma}-interferon-activated sequence (GAS)-like element (GLE), the trans-activation of which we have previously shown reflects GH- induced GHR- and JAK2-mediated STAT5 activation (46, 47). Cotransfected cells responded in a GH concentration-dependent fashion with inducible luc activity when WT JAK2 was expressed (Fig. 4CGo; as below in Fig. 5CGo, vector transfection alone did not allow trans-activation). This indicates the utility of this assay in our reconstitution system.



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Fig. 3. JAK2 Mutants Studied and Summary of Results

Diagram of JAK2 mutants previously studied (20 ) (JAK2-1–999 and JAK2-{Delta}1–239) and those newly prepared for the current study. Each mutant is named so as to indicate the remaining JAK2 residues, the residues deleted ({Delta}), or the residues substituted (as for JAK2FAAAA), as detailed in Materials and Methods. The FERM, kinase-like, and kinase domains are indicated. The ability of the indicated mutants to allow GH-induced JAK2 tyrosine phosphorylation, STAT5 tyrosine phosphorylation, and/or Spi2.1 trans-activation in Figs. 4–8GoGoGoGoGo is summarized (+ or -).

 


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Fig. 4. Comparison of JAK2-{Delta}1–120 with WT JAK2

A and B, GH-induced tyrosine phosphorylation and GHR-JAK2 association. A, {gamma}2A-rbGHR cells transiently transfected with either WT JAK2 (lanes 1 and 2) or JAK2-{Delta}1–120 (lanes 3 and 4) 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 (upper panel) and anti-JAK2AL33 (lower panel). Bold and dotted arrows indicate the positions of WT JAK2 and JAK2-{Delta}1–120, respectively. Note the absence of GH-induced tyrosine phosphorylation of JAK2-{Delta}1–120. B, Detergent extracts of serum-starved cells, transfected and GH stimulated as described in A, were subjected to immunoprecipitation with anti-GHRcyt-AL37. Precipitated proteins were separated by SDS-PAGE and immunoblotted sequentially with anti-GHRcyt-AL37 (lower panel) and anti-JAK2AL33 (upper panel). Note the absence of basal or GH-induced coimmunoprecipitation of JAK2-{Delta}1–120 with rbGHR. The data shown in A and B are representative of three such experiments. C, GH-induced Spi2.1 trans-activation. Serum-starved cells, transfected as described in A and B, were treated with varying concentrations of GH for 18 h before measurement of luciferase activity as described in Materials and Methods. Data for this concentration dependence experiment are plotted as the fold increase in activity at each concentration relative to untreated samples (mean ± SE of triplicate determinations).

 


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Fig. 5. Comparison of JAK2 N-Terminal Deletants with WT JAK2

A, GH-induced tyrosine phosphorylation. {gamma}2A-rbGHR cells transiently transfected with WT JAK2 (lanes 1 and 2), JAK2-{Delta}1–20 (lanes 3 and 4), JAK2-{Delta}1–47 (lanes 5 and 6), JAK2-{Delta}1–102 (lanes 7 and 8), or JAK2-{Delta}1–36 (lanes 9 and 10) 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 (upper panel) and anti-JAK2AL33 (lower panel). A bracket indicates the range of positions of the transfected JAK2 proteins. The data shown are representative of four such experiments. B, GH-induced Spi2.1 trans-activation. Mutants were analyzed for reporter gene trans-activation as described in Fig. 4CGo. Data are plotted as the GH-induced fold increase in activity (mean ± SE; n = 3 independent experiments), using that mediated by WT JAK2 within each experiment as 100%, as described in Materials and Methods.

 
We began mapping the N terminus of JAK2 by generating JAK2-{Delta}1–120, in which the first 120 residues are removed (see diagram in Fig. 3Go) by PCR mutagenesis. This mutant retains the epitope(s) for recognition by anti-JAK2AL33. After expression in {gamma}2A-rbGHR cells, JAK2-{Delta}1–120 was detected by immunoprecipitation and immunoblotting at approximately 110 kDa (Fig. 4AGo, lower panel, lanes 3 and 4), consistent with its expected position in SDS-PAGE roughly 12–15 kDa less than WT JAK2. Despite being expressed, no GH-inducible tyrosine phosphorylation of JAK2-{Delta}1–120 was detected (Fig. 4AGo, upper panel, lanes 3 and 4 vs. lanes 1 and 2), nor was this mutant coprecipitated with the GHR in cells treated either with or without GH (Fig. 4BGo, upper panel, lanes 3 and 4 vs. lanes 1 and 2). In contrast to WT JAK2, JAK2-{Delta}1–120 also did not allow GH- induced trans-activation of Spi-GLE-Luc (Fig. 4CGo). These findings indicated that deletion of its N- terminal 120 residues, like our previous deletion of the first 239 residues, renders JAK2 unable to physically or functionally interact with the GHR.

We next tested a series of progressive N-terminal truncation mutants, including JAK2-{Delta}1–102, JAK2-{Delta}1–47, JAK2-{Delta}1–36, and JAK2-{Delta}1–20 (diagrammed in Fig. 3Go). When transiently expressed in {gamma}2A-rbGHR cells, each mutant was immunologically detected with anticipated molecular mass relative to WT JAK2 (Fig. 5AGo, lower panel), indicating that each retained the structural integrity required to be recognized by anti-JAK2AL33. Like JAK2-{Delta}1–120, JAK2-{Delta}1–102 and JAK2-{Delta}1–47 were both incapable of undergoing GH-induced tyrosine phosphorylation (Fig. 5AGo, upper panel, lanes 5 and 6 and lanes 7 and 8 vs. lanes 1 and 2) or coprecipitation with the GHR (data not shown). Likewise, neither mutant mediated GH-induced Spi-GLE-Luc trans-activation (Fig. 5BGo). In contrast, both JAK2-{Delta}1–20 and JAK2-{Delta}1–36 underwent GH-induced tyrosine phosphorylation in a pattern similar to WT JAK2 (Figs. 5AGo, lanes 3 and 4 and lanes 9 and 10 vs. lanes 1 and 2). Further, both mutants normally mediated GH-induced Spi2.1-GLE-Luc trans-activation (Fig. 5BGo). The first FERM subdomain of JAK2 is predicted to begin at residue 37 (23). Thus, these findings strongly suggested that the most N-terminal part of JAK2’s FERM domain is required for productive interaction with the GHR.

To determine whether this region, in addition to being required, might be sufficient to mediate this effect, we tested JAK2 {Delta}1–20 {Delta}48–524, a JAK2 mutant in which residues 21–47 were fused N terminal to residues 525-1129 (roughly the C-terminal half of the molecule, which includes both the pseudokinase and kinase domains; diagrammed in Fig. 3Go). We have previously shown that this C-terminal region of JAK2, when fused to the GHR extracellular and trans- membrane domains, can confer GH-inducible activation of signaling (20). JAK2 {Delta}1–20 {Delta}48–524 was expressed in {gamma}2A-rbGHR cells and, as expected, was basally tyrosine phosphorylated (Fig. 6AGo, lane 3, upper and lower panels). Unlike WT JAK2 (lanes 1 and 2), however, JAK2 {Delta}1–20 {Delta}48–524 was unable to mediate appreciable GH-induced activation and signaling (Fig. 6AGo, lanes 4 vs. 3, and Fig. 6BGo), indicating that the N-terminal region containing only the first FERM subdomain was necessary, but not sufficient, to confer GH-inducible JAK2 activation. These findings suggested that, as for certain other JAK2-cytokine receptor interactions (25, 26, 27, 28), the GHR-JAK2 interaction is probably dependent on the intactness of the entire FERM domain.



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Fig. 6. Comparison of JAK2-{Delta}1–20 {Delta}48–524 with WT JAK2

A, GH-induced tyrosine phosphorylation. {gamma}2A-rbGHR cells transiently transfected with either WT JAK2 (lanes 1 and 2) or JAK2-{Delta}1–20 {Delta}48–524 (lanes 3 and 4) 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 (upper panel) and anti-JAK2AL33 (lower panel). Bold and dotted arrows indicate the positions of WT JAK2 and JAK2-{Delta}1–20 {Delta}48–524, respectively. The data shown are representative of two such experiments. B, GH-induced Spi2.1 trans-activation. JAK2-{Delta}1–20 {Delta}48–524 was analyzed for reporter gene trans-activation, as described in Figs. 4CGo and 5BGo. Data are plotted as the GH-induced fold increase in activity (mean ± SE; n = 3 independent experiments), using that mediated by WT JAK2 within each experiment as 100%.

 
We also addressed whether the Y231RFRR region of JAK2 is important in GH-induced transcriptional signaling. This sequence is critical for mediating physical association of JAK2 with the AT1R and for angiotensin II-induced STAT1 nuclear translocation and gene transcription (31). Expression of JAK2-FAAAA allowed GH-induced JAK2 activation similar to that afforded by WT JAK2 (Fig. 7AGo, lanes 3 and 4 vs. lanes 1 and 2). To determine whether this mutant JAK2 could also support GH-induced STAT5 signaling, we cotransfected a STAT5B expression plasmid along with vector only (Fig. 7BGo, lanes 1 and 2), WT JAK2 (lanes 3 and 4), or JAK2-FAAAA (lanes 5 and 6) and evaluated GH-induced STAT5 tyrosine phosphorylation by immunoblotting with a state-specific antibody (anti-pSTAT5). Both WT JAK2 and JAK2-FAAAA allowed GH-induced STAT5 tyrosine phosphorylation. Correspondingly, JAK2-FAAAA expression resulted in a pattern of GH-induced Spi-GLE-Luc trans-activation similar to that of WT JAK2 (Fig. 7CGo). In addition to this concentration dependence experiment, separate experiments performed with 500 ng/ml GH (n = 4; not shown) indicated no alteration in Spi-GLE-Luc trans-activation with JAK2-FAAAA vs. WT JAK2. Thus, while AT-II-induced STAT1-mediated gene trans-activation was lessened for JAK2-FAAAA (31), GH-induced STAT5-mediated gene trans-activation was not affected. These findings suggested that the structural bases for productive GHR-JAK2 association and AT1R-JAK2 association may substantially differ. The results obtained in Figs. 4–7GoGoGoGo are summarized graphically with the mutants in Fig. 3Go.



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Fig. 7. Comparison of JAK2-FAAAA with WT JAK2

A and B, GH-induced tyrosine phosphorylation of JAK2 and STAT5. A, {gamma}2A-rbGHR cells transiently transfected with either WT JAK2 (lanes 1 and 2) or JAK2-FAAAA (lanes 3 and 4) were serum-starved and treated with (+) or without (-) GH for 15 min before detergent lysis and anti-JAK2AL33 immunoprecipitation. Precipitated proteins were resolved by SDS-PAGE and immunoblotted sequentially with anti-pY (upper panel) and anti-JAK2AL33 (lower panel). The data shown are representative of two such experiments. B, {gamma}2A-rbGHR cells transiently transfected a STAT5b expression vector (lanes 1–6) along with WT JAK2 (lanes 3 and 4), JAK2-FAAAA (lanes 5 and 6), or empty vector (lanes 1 and 2) were serum-starved and treated with (+) or without (-) GH for 10 min before detergent lysis. Proteins were resolved by SDS-PAGE and immunoblotted with anti-pSTAT5 antibody. The data shown are representative of two such experiments. C, GH-induced Spi2.1 trans-activation. JAK2-FAAAA was analyzed for reporter gene trans-activation as described in Fig. 4Go. Samples were exposed to varying concentrations of GH for 18 h before measurement of luciferase activity as described in Materials and Methods. Data are plotted as the fold increase in activity at each concentration relative to untreated samples (mean ± SE of triplicate determinations).

 
Evidence for Functional Complementation between JAK2 Molecules within the GHR/JAK2 Signaling Complex
One model of JAK activation by cytokines suggests that a JAK associated with an unliganded receptor is brought into proximity with another JAK associated with another unliganded receptor in response to binding of the cytokine or hormone. Closely approximated, the JAKs would then be positioned to influence one another by causing activation loop tyrosine phosphorylation in trans and thereby enhanced tyrosine kinase activation, autophosphorylation, and tyrosine phosphorylation of the receptors and other molecules. Our JAK2 mutants allowed us to probe elements of this proposed mechanism for GH-induced activation of the GHR-JAK2 system in {gamma}2A-rbGHR cells.

JAK2-{Delta}1–47, which lacks the first FERM subdomain, but has an intact kinase domain, could not couple to the GHR to allow GH-induced activation of tyrosine autophosphorylation or trans-activation of the Spi-GLE-luc reporter. In contrast, JAK2-1–999, which can associate with the GHR, cannot confer GH signaling because it lacks an intact kinase domain (Ref.20 and this study). We tested whether these two JAK2 mutants, nonfunctional for different reasons, could complement one another in assays of GH signaling. {gamma}2A-rbGHR cells were transiently transfected with empty vector (negative control), WT-JAK2 (positive control), JAK2-{Delta}1–47, JAK2-1–999, or the combination of JAK2-{Delta}1–47 plus JAK2-1–999 (Fig. 8AGo). In each case, a STAT5b expression vector was cotransfected. Serum-starved cells were exposed to either vehicle or GH for 10 min, after which detergent extracts were immunoblotted with anti-pSTAT5 (Fig. 8AGo, upper panel) and anti-STAT5 (Fig. 8AGo, lower panel). As expected, GH induced STAT5 tyrosine phosphorylation in {gamma}2A-rbGHR cells when WT-JAK2 was expressed (Fig. 8AGo, upper panel, lanes 2 vs. 1), but not when either JAK2-{Delta}1–47 or JAK2-1–999 was individually expressed (lanes 3 and 4 and lanes 5 and 6, respectively), despite ample STAT5 expression in all samples (Fig. 8AGo, lower panel, lanes 1–8). Notably, coexpression of JAK2-{Delta}1–47 with JAK2-1–999, in contrast to their individual expression, partially restored GH- induced tyrosine phosphorylation of STAT5 (Fig. 8AGo, upper panel, lanes 8 vs. 7). We also tested the ability of these JAK2 mutants to complement one another for trans-activation of the Spi-GLE-Luc reporter (Fig. 8BGo). Again, neither JAK2-{Delta}1–47 nor JAK2-1–999 alone allowed GH-induced gene activation. As seen for STAT5 tyrosine phosphorylation, coexpression of JAK2-{Delta}1–47 with JAK2-1–999 substantially reconstituted GH-induced Spi-GLE-Luc trans-activation (35% of WT-JAK2), suggesting that the tyrosine phosphorylation of STAT5 culminated in downstream signaling allowed by functional collaboration of the two mutant JAKs.



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Fig. 8. Functional Complementation between JAK2 Mutants

A–C, GH-induced STAT5 tyrosine phosphorylation and Spi2.1 trans-activation. A, {gamma}2A-rbGHR cells transiently transfected with STAT5b (lanes 1–8) along with WT JAK2 (lanes 1 and 2), JAK2-{Delta}1–47 (lanes 3 and 4), JAK2-1–999 (lanes 5 and 6), or JAK2-{Delta}1–47 plus JAK2-1–999 (lanes 7 and 8) were serum-starved and treated with (+) or without (-) GH for 10 min before detergent lysis. Proteins were resolved by SDS-PAGE and immunoblotted sequentially with anti-pSTAT5 antibody (upper panel) and anti-STAT5 (lower panel). The data shown are representative of three such experiments. B and C, JAK2-{Delta}1–47 plus JAK2-1–999 (B) and JAK2-{Delta}1–239 plus JAK2-1–511 (C) were analyzed for reporter gene trans-activation as described in Figs. 5Go and 6Go. Data are plotted as the GH-induced fold increase in activity (mean ± SE; n = 3 independent experiments in B and C), using that mediated by WT JAK2 within each experiment as 100%.

 
Although JAK2-1–999 lacks the kinase domain activation loop and is thus not a phosphotransferase (20, 48), this mutant still possesses the amino terminus of the kinase domain and the entire pseudokinase domain. JAK2-{Delta}1–47, whereas it lacks the extreme N terminus of the FERM domain and is otherwise intact, containing the full pseudokinase and kinase domains. To more carefully assess JAK2’s structural requirements for GH-induced signaling, we tested the ability of JAK2-1–511, a mutant lacking both the pseudokinase and kinase domains, to complement the GHR association-defective JAK2-{Delta}1–239 (Fig. 8CGo). As expected, neither JAK2-{Delta}1–239 nor JAK2-1–511 individually mediated GH-induced Spi-GLE-Luc trans-activation. Coexpression of the two mutants, however, allowed GH-induced trans-activation. In other experiments (not shown), similar findings were obtained for GH-induced STAT5 tyrosine phosphorylation. The results of Fig. 8Go, A–C (summarized in Fig. 3Go), suggested that the functional complementation of the mutant JAKs does not require that both JAKs possess a pseudokinase domain and occurs even if one JAK lacks a substantial fraction of the FERM domain.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Productive engagement of GHR signaling pathways is believed to be heavily reliant, if not absolutely dependent, upon activation of JAK2 (1). The current study substantially extends our knowledge of the GHR-JAK2 interaction and presents new information on three aspects of JAK2 function: 1) the influence of JAK2 on GHR surface expression, 2) the role of the FERM domain in fostering GH signaling by allowing association with the GHR, and 3) the mechanism of JAK2 activation.

The influence of JAKs on surface expression of cytokine receptors is an important issue from both cell biology and signaling perspectives. Our data reveal several interesting features about JAK2 and GHR cell surface display. We found that JAK2 is not required for GHR surface expression, as transient or stable expression of rbGHR in {gamma}2A cells, even in the absence of JAK2, resulted in the presence of endoH-resistant GHRs that bound GH at the cell surface. This is consistent with our previous observations that inducible GHR proteolysis and GH binding protein shedding (which probably derive from the surface GHR pool) and GH-induced GHR disulfide linkage (also requiring surface receptor expression) are detected in {gamma}2A- rbGHR cells (38) and that a rbGHR lacking the cytoplasmic domain (rbGHR1-275), and thus incapable of JAK2 interaction, was expressed at the cell surface in COS-7 cells (16). Further, transient expression of JAK2 allowed GH-induced signaling without appreciably altering surface GHR expression or the receptor’s endoH resistance, suggesting that the cell surface GHR level in the absence of JAK2 is sufficient to allow productive GHR triggering by GH. However, we note caveats in our interpretations. {gamma}2A cells, although deficient in JAK2 expression, probably express JAK1 and TYK2, two other JAK family members. We have not detected GH-induced JAK1 tyrosine phosphorylation in {gamma}2A-rbGHR cells (data not shown), but are aware of previous studies showing GH-induced JAK1 activation in some settings (49). Thus, the JAK2-independent rbGHR surface expression we observe in {gamma}2A cells could conceivably be mediated by another JAK family member. Further, our methods could be inadequate to detect subtle changes in GHR glycosylation status or surface expression that might occur with transient expression of JAK2. Despite these caveats, our data are most consistent with the lack of an absolute requirement for JAK2 for surface GHR expression.

Interestingly, our study also provides clear evidence for augmentation of surface GHR expression by JAK2 in the stably transfected cells with a positive relationship among the different {gamma}2A-rbGHR-JAK2 clones between the JAK2 expression level and the degree of rbGHR endoH resistance. Because the cell ({gamma}2A- rbGHR) targeted for JAK2 transfection already stably expressed the receptor, and multiple JAK2-transfected clones were analyzed, it is unlikely that the differences in rbGHR maturation reflect mere clonal variation. Rather, rbGHR maturation was apparently fostered by JAK2 above a JAK2-independent threshold. This is reminiscent of recent findings for JAK2 and EpoR, JAK1 and oncostatin M receptor, and TYK2 and IFNAR1 in which JAKs increased cytokine receptor surface expression (27, 33, 34). However, our findings of a substantial JAK2-independent cell surface GHR cohort indicate that more than one control mechanism probably governs GHR surface expression. We cannot yet discriminate whether JAK2’s facilitation of GHR surface expression involves chaperoning of receptors during Golgi trafficking [as reported for JAK2/EpoR and JAK1/OSMR (27, 33)] or selective stabilization of the cell surface receptor pool [as reported for TYK2/IFNAR1 (34)]. Further studies of the fate of newly synthesized GHRs in the presence or absence of JAK2 will be revealing in this vein.

We found in our mapping studies that the entire FERM domain, but not the extreme N-terminal residues that precede it, was required for productive GHR association. Furthermore, the first FERM subdomain (residues 37–47) was not alone sufficient to mediate this interaction. This is relevant in that the analogous region of JAK1 also harbors elements that are probably critical for allowing interaction of that JAK family member with cytokine receptors (26). These findings thus point to the likelihood of either a multifocal interacting surface on JAKs or a requirement for intactness of the entirety of the FERM domain for a small region within it to interact with the cytokine receptor. Indeed, our attempts to express in {gamma}2A cells JAK2 molecules with in-frame internal deletions of regions within the FERM domain have yielded very little mutant protein, suggesting the relative instability of such mutants (not shown). While structural analysis of the FERM domain as it exists within JAK2 and in association with the GHR or other cytokine receptors has not been reported, we note that computer modeling of the entire JAK2 protein (50) suggests a compact structure for the FERM domain, consistent with our findings of instability of internal deletants. Furthermore, this modeling suggests that particular JAK2 FERM domain residues (M181, F236, and F240) might engage in hydrophobic interactions with the GHR Box 1 region, but that residues 37–47 would not be predicted to do so (50). Thus, we favor the likelihood that the first FERM subdomain, rather than directly interacting with the GHR, is needed for overall structural support of the FERM domain to allow receptor-JAK2 interaction.

We are intrigued by comparing our current findings obtained with the JAK2-FAAAA mutant with previous data concerning JAK2-AT1R interaction. This mutant, in which five residues, Y231RFRR, in the midst of the FERM domain are changed to F231AAAA, is activated in response to angiotensin II, despite its lack of stable interaction with the AT1R (31). However, the nuclear translocation of STAT1 and STAT1-mediated gene transcription normally accompanying angiotensin-II-induced JAK2 activation do not occur with JAK2-FAAAA (31). In examining physical and functional interaction of JAK2-FAAAA with the GHR in {gamma}2A-rbGHR cells, we found that GH caused tyrosine phosphorylation of both WT and mutant JAKs and also dose-dependently activated STAT5 signaling to a similar degree for each. We observed no difference in wild-type JAK2- vs. JAK2-FAAAA-mediated GH-induced trans-activation of a c-fos enhancer-driven reporter gene (not shown), suggesting that GH-activated STAT1-mediated signaling was similarly not affected by the FAAAA mutation. Our findings further highlight differences in the mechanisms by which the GHR (a cytokine receptor) and the AT1R (a serpentine receptor) physically associate with and use JAK2 in signaling. These differences can be added to previous observations that the AT1R-JAK2 interaction appears dependent on the catalytic competence of JAK2 (30), in distinction to our findings for GHR-JAK2 (20). Further studies directed at definition of the structural underpinnings of these differences, determination of whether the FERM domain per se is similarly important in serpentine receptor-JAK interaction, and investigation of whether other (accessory) proteins might be differentially involved in mediating JAK2’s interactions with cytokine vs. serpentine receptors appear quite worthwhile.

We believe that our findings of functional complementation of mutant JAK2 molecules are important to better understanding mechanisms of GH-induced activation of JAK2. It is now clear that the unliganded GHR, like several other cytokine receptors, exists to some extent as a dimer that undergoes conformational changes in response to GH to allow triggering of JAK2 (10, 11, 12, 13, 51, 52, 53, 54, 55, 56). However, there is little known structural detail about how the GHR-JAK2 interaction results in JAK2 activation. Although it has been shown for other cytokine-receptor interactions that juxtamembrane cytoplasmic elements N-terminal to Box 1 are critical for effective coupling (57, 58), the importance of analogous GHR elements is as yet unknown. In addition to these GHR-GHR and GHR-JAK2 interactions that influence the degree and tempo of JAK2 activation, our studies suggest that interaction between the JAK2 molecules that are associated with conformationally competent receptors contributes to GH-induced JAK2 activation.

Interactions between JAK molecules are known to occur and to modulate their kinase activities. For example, deletion of the JAK2 pseudokinase domain leads to enhanced basal JAK2 activity and a relative lack of inducible JAK2 activation (20, 39). Mutagenesis and computer modeling studies suggest that these effects relate to intramolecular interactions between the pseudokinase and kinase domains (59, 60, 61, 62). Another potentially critical regulatory interaction was identified in JAK3 between the FERM and kinase domains by Zhou et al. (28), who demonstrated that these two domains interacted and that their coexpression resulted in increased catalytic activity of the kinase domain. The influence of cytokine receptors on this interaction and the potential for the FERM kinase domain association contributing to the normal ligand-induced triggering of JAK activity were not explored.

Our analysis extends these findings in several important ways. We show that coexpression of two different combinations of JAK2 mutants (JAK2-{Delta}1–47 plus JAK2-1–999 and JAK2-{Delta}1–239 plus JAK2-1–511) along with the rbGHR into JAK2-deficient cells results in GH-inducible signaling, whereas the expression of any of the four mutants individually with the rbGHR does not allow GH responsiveness. The nature of our system (JAK2-deficient cells and several JAK2 mutants) encourages us to conclude that the complementation of an association-incompetent (FERM domain defective), but catalytically intact, JAK2 mutant by a catalytically incompetent (but FERM domain- intact) JAK2 mutant to allow GH-induced signaling probably occurs via interactions in trans between the JAK2 mutants. We do not yet know the extent of the required regions within each mutant to allow complementation, although the activity of the JAK2-{Delta}1–239 plus JAK2-1–511 combination suggests that neither pseudokinase domains nor intact FERM domains need be present in both of the partners.

Although the combinations do not function as well as the intact WT JAK2, the ligand inducibility of signaling suggests that our findings are relevant for understanding the normal GH-induced GHR-JAK2 activation mechanism. One function of the FERM domain-intact JAK2 could be to recruit the kinase-competent JAK2 into the signaling complex via JAK2-JAK2 interaction. It seems possible, therefore, that a mechanism of normal GH-induced JAK2 activation might be to allow the GHR-associated JAK2 to further recruit other non-GHR-associated JAK2 molecules to join the GHR-JAK2 complex, enhancing the likelihood of kinase domain interactions in trans between JAK2 molecules and thereby enhancing signaling. Such a scenario would suggest that GH may enhance the JAK2:GHR stoichiometry as at least part of its activation mechanism. The current absence of structural data concerning the GHR-JAK2 interaction (or that of any other cytokine receptor-JAK) does not allow us to definitively discount or embrace this interpretation. However, we do note that we and others have detected GH-induced augmentation of the abundance of JAK2 that coimmunoprecipitates with the GHR in several cell types (4, 5, 45). Although other possibilities exist to explain these findings, it is tempting to hypothesize that this increase in association could reflect a change in the JAK2:GHR stoichiometry consequent to GH-induced conformational changes in the GHR that render JAK2-JAK2 interaction more favorable. Future studies will address these issues.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Recombinant hGH was provided by Eli Lilly & Co. (Indianapolis, IN). Routine reagents were purchased from Sigma- Aldrich Corp. (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). Restriction endonucleases were obtained from New England Biolabs (Beverly, MA).

Plasmid Construction
The murine JAK2 cDNA (48) was provided by Dr. J. Ihle (St. Jude Children’s Research Hospital, Memphis, TN), and the ligation into the pRc/CMV expression plasmid (Invitrogen) and construction of the JAK2-1–999 and JAK2-{Delta}1–239 expression vectors have been described previously (20). Ligation of JAK2 cDNA into the pcDNA 3.1+ eukaryotic expression vector (which carries the zeocin resistance gene) was accomplished using NotI and ApaI restriction endonucleases. JAK2-FAAAA has been described (31). JAK2 deletion constructs (see Fig. 3Go) were generated by PCR using Pfu DNA polymerase and cloned into pRc/CMV-JAK2 WT at the NotI/BstEII restriction sites. In brief, the cDNAs for JAK2 mutants were created in pRc/CMV expression plasmid by replacing the region encoding residues 1–418 (using an endogenous BstEII site at residue 418) with PCR products encoding residues 21–418, 37–418, 48–418, 103–418, and 121–418, with a NotI site at the 5' end. The cDNA expression vector encoding JAK2-1–511 was generated in pcDNA 3.1+ by replacing the region in WT JAK2 encoding residues 418-1129 with a PCR product encoding residues 418–511 with an in-frame HA tag preceding a stop codon and an ApaI site at the 3' end. The cDNA expression vector encoding JAK2-{Delta}-1–20-{Delta}-48–524 was constructed using the ExSite (Stratagene, La Jolla, CA) PCR-based site-directed mutagenesis method and the pRc/CMV-JAK2-{Delta}-1–20 as the template. Sequences for the mutagenic oligonucleotides are available upon request.

The rbGHR cDNA (2) was a gift from Dr. W. Wood (Genentech, Inc. (South San Francisco, CA). Ligation of rbGHR cDNA into the pSX expression plasmid has been described previously (44). The plasmids encoding the Spi-GLE-Luc (46) (provided by Dr. William Lowe, Northwestern University, Chicago, IL) and cDNA for murine STAT5b (pME18S STAT5b) (63) (provided by A. Mui, DNAX Research Institute, Palo Alto, CA) have been previously described.

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 previously (42). The rabbit polyclonal antisera, anti-GHRcyt-AL47, raised against a bacterially expressed N-terminally histamine-tagged fusion protein incorporating human GHR residues 271–620 (the entire cytoplasmic domain) (38), and the rabbit polyclonal antisera anti-GHRcyt-AL37, which was directed against a bacterially expressed glutathione-S-transferase fusion with hGHR residues 271–620 (42), have been previously described. Rabbit antiphosphotyrosine-STAT5 polyclonal antibody (raised against a phosphopeptide surrounding phosphorylated Tyr694 of murine STAT5A, which is conserved in both STAT5A and STAT5B) was obtained from Zymed Laboratories, Inc. (San Francisco, CA). Mouse anti-STAT5 monoclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Cells, Cell Culture, and Transfection
{gamma}2A cells (a JAK2-deficient human fibrosarcoma cell line provided by Dr. G. Stark, Cleveland Clinic Foundation, Cleveland, OH) (36) 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, and 200 µg/ml G418. A stable {gamma}2A cell line expressing rbGHR ({gamma}2A-rbGHR) has been described previously (38) and was maintained in medium supplemented with 100 µg/ml hygromycin B.

Stable transfection of {gamma}2A-rbGHR with murine JAK2 was achieved by introducing pcDNA3.1+-JAK2 using Lipofectamine Plus (Invitrogen) according to the manufacturer’s protocol. Transfected cells were grown in complete Dulbecco’s modified Eagle’s growth medium for 48 h. After dilution, clones were selected in medium supplemented with 400 µg/ml zeocin and screened for JAK2 expression by anti-JAK2AL33 immunoprecipitation and immunoblotting. Transient transfection was achieved by introducing plasmids encoding rbGHR or JAK2 mutants using Lipofectamine Plus (Invitrogen) according to the manufacturer’s instructions.

Cell Stimulation, Protein Extraction, Immunoprecipitation, Electrophoresis, and Immunoblotting
Serum starvation of transfectant cells was accomplished by substitution of 0.5% (wt/vol) BSA (fraction V; Roche, Indianapolis, IN) for serum in their respective culture media for 16–20 h before the experiments. Unless otherwise noted, stimulations were performed at 37 C. Details of the hGH (500 ng/ml unless otherwise noted) treatment protocols have been described. 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 phenylmethylsulfonylfluoride, 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-AL37 sera, 3 µl of each were used per precipitation. Protein A-Sepharose (Amersham Pharmacia Biotech, Arlington Heights, IL) 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 Pharmacia Biotech) with 2% BSA were performed as previously described (5, 38, 42). Immunoblotting with antibodies 4G10 (1:2000), anti-GHRcyt-AL47 (1:1000), anti-STAT5 (1:1000), anti-pSTAT5 (1:500), and anti-JAK2AL33 (1:1000), with horseradish peroxidase-conjugated antimouse or antirabbit secondary antibodies (1:5000) and ECL detection reagents (all from Amersham Pharmacia Biotech) and stripping and reprobing of blots were accomplished according to the manufacturer’s suggestions.

Enzymatic GHR Deglycosylation
Cells were serum-starved overnight and then solubilized as indicated above. Supernatants from these lysates were subjected to immunoprecipitation with anti-GHRcyt-AL37. Precipitates were eluted with 0.5% SDS and 1% ß-mercaptoethanol at 100 C for 10 min. Eluted proteins were digested with endoH (New England Biolabs; 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 for 16 h in accordance with the manufacturer’s suggestions. Sodium dodecyl sulfate sample buffer eluates were resolved by SDS-PAGE and immunoblotted as indicated.

Trans-Activation Assay
{gamma}2A-GHR cells (one 70% confluent 100 x 20-mm dish per transfection) were transfected with Spi-GLE-Luc reporter plasmid and expression plasmids encoding WT JAK2, mutated JAK2, or combinations of mutated JAK2 using Lipofectamine Plus (Invitrogen) for 6 h. The cells were trypsinized and seeded into six-well plates at 4.5 x 105 cells/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 hGH (500 or 0.5–500 ng/ml, if indicated) 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 described previously (20, 44). Similarity of JAK2 expression levels was verified by immunoblotting (not shown). As indicated, data are displayed either as the mean (±SE of triplicate determinations) fold increase in GH vs. vehicle or as the GH-induced fold increase in activity (mean ± SE) of a number of independent experiments, using that mediated by WT JAK2 within each experiment as 100%.

Competitive [125I]hGH Cross-Linking
{gamma}2A cells were transfected, as described above, with the rbGHR expression plasmid with or without the WT JAK2 expression plasmid. For each transfection, a single 100 x 20-mm dish of transfected cells was split 16 h after transfection into two 60 x 15-mm dishes (1 x 106 cells/dish) and continued in culture for 8–12 h before undergoing serum starvation for 16 h as described above. After washing, cells were incubated at 4 C for 2 h in 1 ml PBS/1 mM dextrose to which was added [125I]hGH (New England Nuclear-DuPont, Wilmington, DE; specific activity, 85–130 µCi/pg) at a final concentration of 4 ng/ml (~0.18 nM) in the presence or absence of excess unlabeled hGH (2 µg/ml). Fresh bis-(sulfosuccinimidyl)suberate (Pierce Chemical Co., Rockford, IL), a homobifunctional noncleavable water-soluble cross-linker, was added (0.5 mM, final concentration) for 15 min at room temperature, and the cross-linking was then quenched for 10 min by the addition of 10 mM ammonium acetate. Cells were washed twice with ice-cold PBS and then harvested and solubilized in lysis buffer. The detergent extract was resolved by SDS-PAGE and subjected to autoradiography as has been described previously (16).

Densitometric Analysis
Densitometric quantitation of enhanced chemiluminescence immunoblots was performed using a high resolution scanner and the ImageJ 1.30 program (developed by W. S. Rasband, Research Services Branch, NIMH, Bethesda, MD).


    ACKNOWLEDGMENTS
 
We appreciate helpful conversations with Drs. E. Benveniste, G. Fuller, J. Kudlow, A. Theibert, J. Messina, Y. Huang, S.-O. Kim, K. Loesch, J. Cowan, and N. Yang, and the generous provision of reagents by those named in the text.


    FOOTNOTES
 
This work was supported by NIH Grants DK-46395 and DK-58259 (to S.J.F.). Parts of this work were presented at the 85th Annual Meeting of The Endocrine Society, Philadelphia, PA, 2003.

Abbreviations: AT1R, Angiotensin II receptor; {gamma}c, common {gamma}-chain; endoH, endoglycosidase H; EpoR, erythropoietin receptor; F/N, N-glycosidase F and neuraminidase; GAS, {gamma}-interferon-activated sequence; GHR, GH receptor; GLE, GAS-like element; h, human; IFNAR1, interferon-2 receptor-1; JAK, Janus kinase; luc, luciferase; rbGHR, rabbit growth hormone receptor; Spi2.1, serine protease inhibitor 2.1; STAT, signal transducer and activator of transcription; TYK, tyrosine kinase; WT wild type.

Received for publication June 27, 2003. Accepted for publication August 5, 2003.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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