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Molecular Endocrinology, doi:10.1210/me.2007-0138
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Molecular Endocrinology 21 (7): 1537-1551
Copyright © 2007 by The Endocrine Society

Determinants of Growth Hormone Receptor Down-Regulation

Luqin Deng, Kai He, Xiangdong Wang, Ning Yang, Chellappagounder Thangavel, Jing Jiang, Serge Y. Fuchs and Stuart J. Frank

Department of Cell Biology (L.D., N.Y., S.J.F.), and Department of Medicine (K.H., X.W., J.J., S.J.F.), Division of Endocrinology, Diabetes, and Metabolism, University of Alabama at Birmingham, Birmingham, Alabama 35294-0012; Department of Animal Biology (C.T., S.Y.F.), University of Pennsylvania, Philadelphia, Pennsylvania 19104-6046; and Endocrinology Section (S.J.F.), Medical Service, Veterans Affairs Medical Center, Birmingham, Alabama 35233

Address all correspondence and requests for reprints to: Stuart J. Frank, University of Alabama at Birmingham, 1530 Third 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 receptor (GHR) is a cytokine receptor family member that responds to GH by activation of the receptor-associated tyrosine kinase, JAK2 (Janus family of tyrosine kinase 2). We previously showed that JAK2, in addition to being a signal transducer, dramatically increases the half-life of mature GHR, partly by preventing constitutive GHR down-regulation. Herein we explored GHR and JAK2 determinants for both constitutive and GH-induced GHR down-regulation, exploiting the previously characterized GHR- and JAK2-deficient {gamma}2A reconstitution system. We found that JAK2’s ability to protect mature GHR from rapid degradation measured in the presence of the protein synthesis inhibitor, cycloheximide, depended on the presence of GHR’s Box 1 element and the intact JAK2 FERM (band 4.1/Ezrin/Radixin/Moesin); domain, but not the kinase-like or kinase domains of JAK2. Thus, GHR-JAK2 association, but not JAK2 kinase activity, is required for JAK2 to inhibit constitutive GHR down-regulation and enhance GHR half-life. In cells that expressed JAK2, but not cells lacking JAK2, GH markedly enhanced GHR degradation. Like JAK2-induced protection from constitutive down-regulation, GH-induced GHR down-regulation required the GHR Box 1 element and an intact JAK2 FERM domain. However, a JAK2 mutant lacking the kinase-like and kinase domains did not mediate GH-induced GHR down-regulation. Likewise, a kinase-deficient JAK2 was insufficient for this purpose, indicating that kinase activity is required. Both lactacystin (a proteasome inhibitor) and chloroquine (a lysosome inhibitor) blocked GH-induced GHR loss. Interestingly, GH-induced GHR ubiquitination, like down-regulation, was prevented in cells expressing a kinase-deficient JAK2 protein. Further, a GHR mutant, of which all the cytoplasmic tyrosine residues were changed to phenylalanines, was resistant to GH-induced GHR ubiquitination and down-regulation. Collectively, our data suggest that determinants required for JAK2 to protect mature GHR from constitutive degradation differ from those that drive GH-induced GHR down-regulation. The latter requires GH-induced JAK2 activation and GHR tyrosine phosphorylation and is correlated to GHR ubiquitination in our reconstitution system.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GH IS AN ANTERIOR pituitary-derived peptide hormone that in humans and other vertebrates is important in promotion of growth and regulation of metabolism and energy balance, and likely has a role in longevity (1, 2). GH signaling is initiated by interaction with the cell surface receptor [GH receptor (GHR)] on target tissues and results in activation of several intracellular signaling pathways and expression of a diverse set of genes (3, 4). The GHR is a type 1 glycoprotein that is a member of the cytokine receptor superfamily (5). Like other cytokine receptor family members, GHR couples to a Janus family kinase; specifically, GHR utilizes JAK2 (Janus family of tyrosine kinase 2) (6). JAK2 is a tyrosine kinase that has no transmembrane domain but physically and functionally associates with the dimerized transmembrane GHR both in the secretory pathway and at the cell surface by virtue of a proline-rich perimembranous cytoplasmic domain element of the receptor called Box 1 and the N-terminal FERM (band 4.1/Ezrin/Radixin/Moesin) domain of JAK2 (7, 8, 9, 10, 11, 12, 13, 14).

An essential determinant of tissue and cellular sensitivity to GH relates to regulation of cell surface GHR abundance and availability, as well as the ability of the GH response to be terminated or dampened. These issues have received much attention in recent years, as mechanisms regulating the biogenesis, trafficking, and down-regulation of cell surface receptors in general have become better appreciated. GHR is synthesized in the endoplasmic reticulum, where it rapidly dimerizes as a high-mannose (endoglycosidase H-sensitive) glycoprotein precursor and then traffics through the Golgi complex, acquiring a mature (endoglycosidase H-resistant) glycosylation pattern, and is transported to the cell surface (8, 14, 15, 16, 17). Surface GHR abundance in the absence of ligand binding is subject to modulation by two major mechanisms. First, the receptor is a target for inducible metalloprotease-mediated cleavage in the proximal extracellular domain, which releases the extracellular domain as a GH-binding protein, lessens receptor abundance, and reduces sensitivity to subsequent GH stimulation (18, 19, 20, 21, 22, 23). Second, the surface GHR can also undergo ligand-independent (constitutive) down-regulation via a process that is inhibited by disruption of proteasomal and lysosmal function (24, 25). Recently, we have become interested in the role(s) of JAK2 in regulating GHR surface abundance in the absence of ligand stimulation. We found that JAK2 enhances the level of surface GHR by increasing the efficiency of biogenesis and by enhancing the mature receptor’s stability (14, 25). This latter effect is quite significant and presumably relates to effects on the rate of constitutive (ligand-independent) GHR endocytosis and lysosomal downregulation.

GH-induced GHR down-regulation proceeds via clathrin-coated pit-mediated endocytosis and lysosomal degradation. Intriguingly, elegant studies of Strous and colleagues and others (26, 27, 28, 29, 30) indicate that an intact ubiquitin-proteasome system is required for ligand-induced receptor down-regulation and that a cytoplasmic domain sequence downstream of the Box 1 element called the ubiquitin-dependent endocytosis motif is required for efficient GHR endocytosis. Ligand-induced GHR down-regulation has been described; however, the degree to which GH enhances constitutive GHR degradation is relatively unclear, with some authors suggesting only a modest change induced by GH (31, 32). Likewise, the role of JAK2 in GH-induced GHR down-regulation is also uncertain. Using chemical kinase inhibitors such as the tyrphostin AG490 and staurosporine, previous studies suggested that blockade of GH-induced JAK2 activation prevents receptor degradation (33, 34); however, these inhibitors lack specificity, making firm conclusions regarding effects of JAK2 per se difficult to draw. In contrast, Strous and colleagues (35) found that, in Chinese hamster ts20 cell stable transfectants, a receptor with a mutation of the Box 1 element underwent GH-induced down-regulation similar to a wild-type GHR and concluded that GHR degradation is independent of signal transduction via JAK2.

In this study, we use our well-characterized human fibrosarcoma cell GHR and JAK2 reconstitution system to investigate determinants of constitutive and GH-induced GHR down-regulation. We find that constitutive GHR down-regulation is strongly inhibited by the association of GHR and JAK2 and that JAK2 kinase activity is not essential for this effect. GH treatment markedly enhances GHR degradation, an effect that relies upon GHR-JAK2 association, but also requires JAK2 kinase activity and GHR tyrosine phosphorylation. We further find that, contrary to previous findings in other systems, GH-dependent ubiquitination of GHR requires activation of JAK2 in that ubiquitination of GHR is impaired in cells expressing a kinase-dead JAK2. Likewise, a mutant GHR, in which all tyrosine residues in the intracellular domain are replaced by phenylalanine, although able to bind GH and allow JAK2 activation, does not undergo either GH-induced ubiquitination or degradation. These findings suggest critical roles for both JAK2 activation and GHR tyrosine phosphorylation in GHR down-regulation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
JAK2 Association, But Not the JAK2 Kinase Domain, Is Required for Stabilization of Mature GHR
{gamma}2A is a human fibrosarcoma cell that lacks both GHR and JAK2 (36, 37). We previously demonstrated that stable reconstitution with GHR and JAK2 ({gamma}2A-GHR-JAK2 cells), when compared with GHR alone ({gamma}2A-GHR cells), allowed GH-induced signaling and enhanced surface GHR abundance (14, 25). This enhanced abundance was substantially accounted for by the ability of JAK2 to selectively stabilize the mature form of the receptor. We sought to determine the regions of GHR and JAK2 required for this stabilizing effect of JAK2.

To assess GHR degradation rates, we used cycloheximide (CHX) to inhibit new protein synthesis and followed the fate of previously synthesized GHR by immunoblotting using an anti-GHR serum that recognizes the receptor cytoplasmic domain (38). In the experiment shown in Fig. 1Go, four stable transfectant cell lines were compared. These included the previously described {gamma}2A-GHR and {gamma}2A-GHR-JAK2 cells as well as newly prepared stable clones that express a GHR with an in-frame internal deletion of the Box 1 element (diagrammed in Fig. 1AGo), either in the absence ({gamma}2A-GHR{Delta}Box1) or presence ({gamma}2A-GHR{Delta}Box1-JAK2) of JAK2. We previously reported that this GHR mutant lacking Box 1 is structurally intact but cannot interact with JAK2 or signal in response to GH (9). Serum-starved cells were treated with CHX for 0–5 h, as indicated (Fig. 1BGo), and detergent-extracted proteins were resolved by SDS-PAGE and anti-GHR immunoblotted. With increasing CHX treatment duration, precursor abundance decreased rapidly to a similar degree in all four cell lines. This result indicates that the precursor is quite unstable, consistent with previous data (25), and that precursor degradation rate was unaffected by the presence of JAK2 or its ability to associate with GHR. These data are shown graphically in Fig. 1CGo (left panel), in which the results of multiple such experiments were evaluated densitometrically and the receptor remaining after CHX treatment is expressed as a percentage of the level in untreated cells.


Figure 1
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  		Fig. 1. The Box 1 Element of GHR Is Required for JAK2’s Stabilizing Effect on Mature GHR

A, Diagram of wild-type GHR compared with GHR{Delta}Box1. EC, extracellular; TM, transmembrane; IC, intracellular. B, CHX-induced GHR loss. Serum-starved {gamma}2A-GHR, {gamma}2A-GHR-JAK2, {gamma}2A-GHR{Delta}Box1, and {gamma}2A-GHR{Delta}Box1-JAK2 cells were treated with CHX (20 µg/ml) for 0–5 h, as indicated. Detergent extracts were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47 and anti-EGFR. The positions of the mature (bracket) and precursor (arrowhead) GHR forms are indicated. The data shown are representative of three such experiments. C, Densitometric quantitation of anti-GHR blots, including those in panel B. The GHR abundance at time zero was considered 100%, respectively, for each experiment (mean ± SE; n = 3 independent experiments). M, Mature; P, precursor; WB, Western blot.

 
In contrast to the precursor, the kinetics of mature GHR loss after CHX treatment differed substantially among the cells (Fig. 1Go, B and C, right panels). As we previously demonstrated (25), the stability of wild-type mature GHR was greatly enhanced by JAK2 [compare {gamma}2A-GHR (t1/2 ~ 1.5 h) vs. {gamma}2A-GHR-JAK2 (t1/2 > 4 h)]. However, in cells that expressed the Box 1-deleted GHR, the stability of the receptor was similar to that in {gamma}2A-GHR and was unaffected by expression of JAK2 (compare {gamma}2A-GHR{Delta}Box1 and {gamma}2A-GHR{Delta}Box1-JAK2 with the other cells and with each other). Thus, the GHR Box 1 element is required for JAK2’s stabilizing effect on mature GHR, suggesting that this stabilization is mediated via JAK2-GHR interaction.

We sought to confirm this model in a reciprocal fashion by testing the capacity of JAK2 mutants to stabilize the mature GHR (Fig. 2Go). In addition to the GHR Box 1 element, GHR-JAK2 association requires an intact FERM domain, which resides in the N-terminal 450 residues of JAK2 (14, 39, 40). Removal of the N-terminal 47 residues of JAK2 (JAK2{Delta}1–47), which includes the first FERM subdomain, renders JAK2 unable to physically or functionally interact with GHR (14). Another previously characterized JAK2 mutant, JAK21–511-HA, includes the first 511 residues and thus has the FERM domain, but lacks the pseudokinase and kinase domains (14) (see diagram in Fig. 2AGo). CHX treatment of cells stably expressing wild-type GHR and JAK2{Delta}1–47 ({gamma}2A-GHR-JAK2{Delta}1–47 cells) yielded rapid loss of mature GHR (Fig. 2BGo, left panel, and Fig. 2CGo, graph), indicating that this FERM domain mutant JAK2 was unable to prevent degradation of the mature GHR. In contrast, the GHR in {gamma}2A-GHR-JAK21–511-HA cells was substantially stabilized in the same assay (Fig. 2BGo, right panel, and Fig. 2CGo). Collectively, these data indicate that association of JAK2 with GHR, mediated by the receptor Box 1 element and the JAK2 FERM domain, is required for enhanced stability of the mature GHR. Conversely, the pseudokinase and kinase domains of JAK2 are not required for this effect.


Figure 2
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  		Fig. 2. An Intact N Terminus, But Not the Pseudokinase and Kinase Domains, of JAK2 Is Required to Stabilize Mature GHR

A, Diagram of JAK2{Delta}1–47 compared with JAK21–511-HA. B, {gamma}2A-GHR-JAK2{Delta}1–47 and {gamma}2A-GHR-JAK21–511-HA cells were treated with CHX (20 µg/ml) for 0–5 h, as in Fig. 1Go, and detergent extracts were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47, anti-JAK2AL33, or anti-HA, as indicated. The data shown are representative of three such experiments. C, Densitometric quantitation of anti-GHR blots, including those in panel B. The mature GHR abundance at time zero was considered 100%, respectively, for each experiment (mean ± SE; n = 3 independent experiments). For comparison purposes, densitometric data for {gamma}2A-GHR-JAK2 and {gamma}2A-GHR{Delta}Box1-JAK2 cells shown in Fig. 1CGo are reproduced. M, Mature; P, precursor; WB, Western blot.

 
GH Markedly Enhances GHR Down-Regulation in a JAK2-Dependent Fashion
The experiments in Figs. 1Go and 2Go addressed the effect of JAK2 on constitutive GHR down-regulation. There are conflicting reports concerning the degree to which GH affects the rate of GHR degradation and whether GHR-JAK2 association influences this. We used our stably transfected cells to address these issues. First, serum-starved {gamma}2A-GHR-JAK2 cells were treated with CHX alone vs. CHX plus GH for 0–5 h. Anti-GHR blotting of cell extracts revealed marked GH-induced enhancement of GHR loss. Densitometric evaluation of three independent experiments like that shown in Fig. 3Go revealed that GH treatment in this context reduced the GHR half-life from more than 4 h to approximately 1 h (Fig. 3Go).


Figure 3
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  		Fig. 3. GH Markedly Accelerates Degradation of Mature GHR

Serum-starved {gamma}2A-GHR-JAK2 cells were treated with CHX (20 µg/ml) alone or cotreated with CHX (20 µg/ml) plus GH (500 ng/ml) for 0–5 h, as indicated. Detergent extracts were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47 and anti-JAK2AL33. The data shown are representative of three similar independent experiments. M, Mature; P, precursor; WB, Western blot.

 
To examine the requirement for JAK2 for GH-dependent receptor loss, JAK2-deficient ({gamma}2A-GHR) and JAK2-replete ({gamma}2A-GHR-JAK2) cells were compared (Fig. 4AGo). In cells that lacked JAK2, GH did not decrease the levels of mature or precursor GHR (Fig. 4AGo, left panel). In contrast, GH treatment caused marked loss of mature GHR in {gamma}2A-GHR-JAK2 cells (Fig. 4AGo, right panel), suggesting that JAK2 expression is required for this effect of GH. [In other experiments (data not shown), GH-induced GHR degradation in this setting was dose-dependent.] Blotting for signal transducer and activator of transcription (STAT)5 and epidermal growth factor receptor (EGFR) in the same extracts served as a loading control. Notably, GH also down-regulated mature GHR in Chinese hamster ovary (CHO) cells stably expressing GHR (Fig. 4BGo, left panel) and in 3T3-F442A cells, murine preadipocytes that endogenously express GHR (Fig. 4BGo, right panel).


Figure 4
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  		Fig. 4. JAK2 Is Required for GH to Enhance GHR Down-Regulation

A, Serum-starved {gamma}2A-GHR and {gamma}2A-GHR-JAK2 cells were treated with GH (500 ng/ml) for 0–5 h, as indicated. Detergent extracts were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47, anti-JAK2AL33, anti-STAT5, and anti-EGFR. The data shown are representative of three such experiments. B, Serum-starved CHO-GHR and 3T3-F442A cells were treated with GH (500 ng/ml) for 0–3 h, as indicated. Detergent extracts were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47. The data shown are representative of three such experiments. M, Mature; P, precursor; WB, Western blot.

 
GH-Dependent GHR Down-Regulation Requires GHR-JAK2 Association and JAK2 Kinase Activity
The data in Fig. 4AGo indicate that JAK2 expression is required for GH-induced GHR down-regulation but do not indicate what role JAK2 plays in this process. To approach this question, we first assessed whether GHR-JAK2 association was required by comparing {gamma}2A-GHR-JAK2, {gamma}2A-GHR{Delta}Box1-JAK2, and {gamma}2A-GHR-JAK2{Delta}1–47 cells (the first having wild-type GHR and JAK2, the second having the Box 1-deleted GHR and wild-type JAK2, and the third having wild-type GHR and the FERM domain-disrupted JAK2). Serum-starved cells treated with GH were evaluated by anti-GHR blotting (Fig. 5AGo). In contrast to the marked GH-dependent mature GHR down-regulation seen in {gamma}2A-GHR-JAK2, GH did not cause mature or precursor GHR loss in cells bearing either the Box 1- or FERM-deleted GHR or JAK2, respectively (Fig. 5BGo). These data strongly suggest that association with JAK2 is required for this GH-dependent down-regulation of GHR.


Figure 5
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  		Fig. 5. GH-Induced GHR Loss Requires GHR-JAK2 Association

A, Serum-starved {gamma}2A-GHR{Delta}Box1-JAK2, {gamma}2A-GHR-JAK2{Delta}1–47, and {gamma}2A-GHR-JAK2 cells were treated with GH (500 ng/ml) for 0–5 h, as indicated. Detergent extracts were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47 and anti-JAK2AL33. The data shown are representative of three such experiments. B, Densitometric quantitation of the anti-GHR blots, including those in panel A. The mature GHR abundance at time zero was considered 100%, respectively, for each experiment (mean ± SE; n = 3 independent experiments). M, Mature; P, precursor; WB, Western blot.

 
We next evaluated whether GHR-JAK2 association is sufficient to allow GH-induced receptor degradation. Cells coexpressing wild-type GHR and either wild-type [hemagglutin (HA)-tagged] JAK2 ({gamma}2A-GHR-JAK2-HA) or the JAK2 mutant encoding only the first 511 residues ({gamma}2A-GHR-JAK21–511-HA) were compared (Fig. 6Go). As expected, expression of JAK2-HA allowed marked GH-dependent receptor loss. However, in cells expressing JAK21–511-HA, GH did not cause GHR disappearance. Thus, although required, the FERM domain of JAK2 is not sufficient to allow GH-dependent receptor down-regulation in this system.


Figure 6
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  		Fig. 6. GHR-JAK2 Association Is Not Sufficient for GH-Induced GHR Degradation

A, Serum-starved {gamma}2A-GHR-JAK21–511-HA and {gamma}2A-GHR-JAK2-HA cells were treated with GH (500 ng/ml) for 0–5 h, as indicated. Detergent extracts were resolved by SDS-PAGE and blotted with anti-HA. The data shown are representative of three such experiments. B, Densitometric quantitation of the anti-GHR blots, including those in panel A. The mature GHR abundance at time zero was considered 100%, respectively, for each experiment (mean ± SE; n = 3 independent experiments). M, Mature; P, precursor; WB, Western blot.

 
We sought to investigate the role of JAK2 catalytic activity in our genetically tractable system (Fig. 7Go). JAK2KD has point mutations at residues 1020 (W to G) and 1024 (E to A) that abrogate its phosphotransferase activity (41) (Fig. 7AGo). GH treatment of {gamma}2A-GHR-JAK2KD failed to down-regulate GHR (Fig. 7Go, B and C), indicating that JAK2 protein kinase activity is required for this effect.


Figure 7
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  		Fig. 7. JAK2 Catalytic Activity Is Required for GH-Induced GHR Down-Regulation

A, Diagram of JAK2 compared with JAK2KD. B, Serum-starved {gamma}2A-GHR-JAK2 and {gamma}2A-GHR-JAK2KD cells were treated with GH (500 ng/ml) for 0–5 h, as indicated. Detergent extracts were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47, anti-JAK2AL33, and anti-STAT5. The data shown are representative of three such experiments. C, Densitometric quantitation of the anti-GHR blots, including those in panel B. The mature GHR abundance at time zero was considered 100%, respectively, for each experiment (mean ± SE; n = 3 independent experiments). M, Mature; P, precursor; WB, Western blot.

 
Effects of Proteasome and Lysosome Inhibitors on GH-Induced GHR Down-Regulation
Using the {gamma}2A reconstitution system, we previously showed that proteasome inhibitors and lysosome inhibitors both blocked constitutive (in the absence of ligand) GHR down-regulation measured in the presence of CHX (25). In the experiments shown in Fig. 8Go, we tested the effects of the proteasome inhibitor, lactacystin, and the lysosome inhibitor, chloroquine, on GH-induced GHR degradation in this same system. Serum-starved {gamma}2A-GHR-JAK2 cells were pretreated with lactacystin (Fig. 8AGo) or chloroquine (Fig. 8BGo) or their vehicles and then exposed to GH or vehicle for 3 h. Anti-GHR blotting of detergent extracts revealed that the profound GH-induced GHR loss was blocked by pretreatment with each inhibitor. Densitometric quantitation of several such experiments confirmed these results. Although interpretation of these data obtained using chemical inhibitors has known specificity caveats, the results are consistent with the notion that, like constitutive GHR down-regulation, inhibition of either proteasome activity or lysosome activity blocks efficient GH-induced GHR down-regulation.


Figure 8
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  		Fig. 8. Effects of Proteasome and Lysosome Inhibitors on GH-Induced GHR Down-Regulation

A and B, Serum-starved {gamma}2A-GHR-JAK2 cells were pretreated with either lactacystin (A) or chloroquine (B) or their vehicles before stimulation with GH for 3 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. Densitometric quantitation of the anti-GHR blots, including those in the upper panels, is shown in the lower panels of A and B. The mature GHR abundance at time zero was considered 100%, respectively, for each experiment (mean ± SE; n = 2 independent experiments in panel A and three independent experiments in panel B). M, Mature; P, precursor; WB, Western blot.

 
Tyrosine Phosphorylation of GHR Is Required for Its Ubiquitination and Down-Regulation by GH
GH-induced GHR ubiquitination has previously been demonstrated in other cell systems (31, 42). The role of JAK2 signaling in allowing this receptor modification, however, has yet to be directly tested. We used our reconstitution system to probe this issue (Fig. 9AGo). For these experiments, GHR was immunoprecipitated, resolved by SDS-PAGE, and blotted with an antiubiquitin antibody. Stimulation of {gamma}2A-GHR-JAK2 cells with GH indeed yielded a diffuse, slower migrating species detected by antiubiquitin antibody (Fig. 9AGo, left panel). This is consistent with the migration of the ubiquitinated GHR reported previously (26, 42). In the same experiment, we asked whether JAK2 kinase activity was required for GH-induced receptor ubiquitination. In contrast to the cells that harbor wild-type JAK2, GH did not increase GHR ubiquitination in {gamma}2A-GHR-JAK2KD cells (Fig. 9AGo, left panel), in which GHR was not down-regulated by GH. Stripping and reprobing revealed similar GHR abundance in the two cells (Fig. 9AGo, right panel). Along with the data in Fig. 7Go, these data strongly suggest that GH-induced JAK2 kinase activity is required for GH-induced receptor ubiquitination and down-regulation. These results differ substantially from those reported by Alves dos Santos et al. (35), from which it was concluded that GH-induced GHR ubiquitination (and down-regulation) occurred even if GH-induced JAK2 signaling was blocked; this point is further addressed in the Discussion.


Figure 9
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  		Fig. 9. GH-Induced GHR Ubiquitination Requires JAK2 Kinase Activity and GHR Tyrosine Phosphorylation

A and B, Serum-starved {gamma}2A-GHR-JAK2 vs. {gamma}2A-GHR-JAK2KD cells (A) and {gamma}2A-pGHR-JAK2 vs. {gamma}2A-MYFc8-JAK2 cells (B) were treated with GH or vehicle for 30 min. Detergent extracts were immunoprecipitated with anti-GHRcyt-AL47. Eluates were resolved by SDS-PAGE and sequentially immunoblotted with anti-ubiquitin antibody (left panels) and anti-GHRcyt-AL47 (right panels). The positions of the ubiquitinated GHR (left panels) and mature (bracket) and precursor (arrowhead) GHR forms (right panels) are indicated. The data shown are representative of two such experiments. IP, Immunoprecipitation; M, mature; P, precursor; Ub-GHR, ubiquitinated GHR; WB, Western blot.

 
The data in Figs. 5–7GoGoGo indicate that GHR-JAK2 association and intact JAK2 kinase activity are required for GH-induced GHR down-regulation. There are multiple cellular proteins that become tyrosine phosphorylated in response to GH in a JAK2-dependent fashion; these include the GHR, JAK2 itself, STAT5, and others (3, 4). Elegant studies using a GHR in which all cytoplasmic domain tyrosine residues were mutated to phenylalanine revealed that GHR tyrosine phosphorylation is not required for GH-induced JAK2 activation, but is necessary for STAT5 tyrosine phosphorylation and action (43, 44). We tested whether this mutant GHR could undergo GH-induced down-regulation.

For these experiments, we used the MYFc8 porcine GHR (pGHR) in which all eight cytoplasmic tyrosine residues were changed to phenylalanine (44) (a gift of Drs. E. List and J. J. Kopchick) (Fig. 10AGo). For control, we used the wild-type pGHR. Each was stably expressed in {gamma}2A-JAK2 cells to yield {gamma}2A-MYFc8-JAK2 and {gamma}2A-pGHR-JAK2 cells. Consistent with the previous reports in other cell systems, GH stimulation acutely activated JAK2 and caused its tyrosine phosphorylation, but did not cause tyrosine phosphorylation of STAT5 (Fig. 10BGo). As expected, GH treatment caused loss of the wild-type pGHR (Fig. 10CGo, right panel), just as occurred in cells expressing rabbit and mouse GHR (Figs. 3–8GoGoGoGoGoGo). Notably, however, GH did not substantially down-regulate the mutant GHR in {gamma}2A-MYFc8-JAK2 cells. This result strongly suggests that either receptor tyrosine phosphorylation itself or the association of an important trafficking molecule with the tyrosine-phosphorylated GHR enables the receptor to undergo degradation in response to GH.


Figure 10
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  		Fig. 10. Tyrosine Phosphorylation of GHR Is Required for Its GH-Induced Degradation

A, Diagram of wild-type pGHR compared with MYFc8. B, GH induces tyrosine phosphorylation of JAK2, but not STAT5, in {gamma}2A-MYFc8-JAK2 cells. Serum-starved {gamma}2A-pGHR-JAK2 and {gamma}2A-MYFc8-JAK2 cells were treated with GH or vehicle for 15 min. Detergent extracts were resolved by SDS-PAGE and sequentially blotted with anti-pJAK2 and JAK2 (left panel) or anti-pSTAT5 and STAT5 (right panel). The data shown are representative of three such experiments. C, Serum-starved {gamma}2A-pGHR-JAK2 and {gamma}2A-MYFc8-JAK2 cells were treated with GH (500 ng/ml) for 0–5 h, as indicated. Detergent extracts were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47, anti-JAK2AL33, and anti-STAT5. The positions of the mature (bracket) and precursor (arrowhead) GHR forms are indicated. The data shown are representative of three such experiments. EC, Extracellular; IC, intracellular; M, mature; P, precursor; TM, transmembrane; WB, Western blot.

 
Results in Fig. 9AGo demonstrated that GH-induced GHR ubiquitination required the ability of JAK2 to be active as a kinase. We further asked whether GHR tyrosine phosphorylation is required for receptor ubiquitination (Fig. 9BGo). GH caused ubiquitination of wild-type pGHR, but not the MYFc8 mutant receptor. Thus, both GH-induced receptor ubiquitination and down-regulation required not only activation of JAK2, but also tyrosine phosphorylation of the receptor itself.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In addition to the levels of JAK2, STAT5, and other intracellular GH signaling elements, cell surface GHR abundance is an important factor that modulates biological responses to GH at target tissues (3, 4, 20, 22, 45, 46, 47, 48). JAK2 is the critical proximal signal transducer for GH action; in cells that lack JAK2 or in which JAK2 cannot associate with the GHR, GH is unable to trigger the major signaling pathways downstream of the receptor (6, 9, 10, 11, 12, 13). We recently uncovered additional effects of JAK2 on GHR. By virtue of its interaction with GHR, JAK2 impacts GHR biogenesis, the stability of the mature cell surface receptor, and susceptibility of the receptor to metalloproteolysis (Refs. 14 , 23 , and 25 ; and Loesch, K., L. Deng, X. Wang, K. He, J. Jiang, and S. J. Frank, submitted).

In the current study, we further explored the role of JAK2 in regulating stability of the mature GHR and examined GHR and JAK2 determinants for GH-induced receptor down-regulation. We found that, like its effects on biogenesis and metalloprotease sensitivity, the capacity of JAK2 to prevent constitutive GHR down-regulation requires the ability of the two molecules to associate. This conclusion is based on the fact that such association is disrupted by mutation in either JAK2 (JAK2{Delta}1–47) or GHR (GHR{Delta}Box1). Further, the protective effect of JAK2 does not require either the pseudokinase or kinase domains (the JAK21–511 mutant). Thus, the ability of JAK2 to bind the receptor is apparently sufficient to fulfill this role.

We also studied whether JAK2 affected GH-induced GHR down-regulation. We first established that GH in our reconstitution system greatly accelerated the rate of GHR degradation. Likewise, we observed a robust ligand-induced GHR loss in a separate reconstitution system (CHO-GHR in Fig. 4BGo) and in cells that endogenously express GHR and JAK2 (3T3-F442A in Fig. 4BGo and H4IIE hepatoma cells in Ref. 49).

GH-induced GHR down-regulation in our reconstitution system was quite dependent on JAK2; GH promoted no loss of GHR in cells that lack JAK2. In contrast, GH-induced GHR degradation was substantial in cells reconstituted with JAK2 (Fig. 4AGo). This is an important result, because it addresses an issue about which there is some disagreement in the literature and which has not previously been addressed in the definitive fashion allowed by use of the JAK2 reconstitution system employed in our studies. Alves dos Santos et al. (35) found that cells that express a GHR in which all four Box 1 proline residues are mutated displayed no difference in GH-induced GHR degradation in pulse chase experiments, although the GH effect was rather modest even for wild-type receptor in the cell system used in those studies (32). To the contrary, our data in Fig. 5AGo with a previously characterized (9, 23) Box 1 deletion mutant GHR indicated that this receptor was not down-regulated at all in response to GH, despite the presence of JAK2. Thus, we conclude that GH-induced GHR degradation depends on the presence of JAK2 and its ability to associate with GHR.

Our mutational studies of JAK2 confirmed that JAK2 association was required ({gamma}2A-GHR-JAK2{Delta}1–47; Fig. 5AGo), but was not sufficient ({gamma}2A-GHR-JAK21–511-HA; Fig. 6Go) for GH-induced GHR down-regulation. Interestingly, this is distinctly different from our findings about constitutive receptor down-regulation, for which GHR-JAK2 association was required and sufficient to protect the receptor (see above and Fig. 2Go). Further, GH-induced GHR down-regulation was not supported by a catalytically inactive JAK2 mutant ({gamma}2A-GHR-JAK2KD; Fig. 7Go). This result strongly implicates GH-induced JAK2 kinase activity as necessary for GH-induced GHR down-regulation.

Strous and colleagues (29) first demonstrated GH-induced GHR ubiquitination and proposed that an intact ubiquitin-conjugating system was required for GH-induced endocytosis and degradation of the receptor, but later concluded that receptor ubiquitination per se was not required for GH-induced GHR internalization (30). This conclusion was drawn from experiments performed with a truncated (rather than full-length) GHR in which all remaining lysine residues were replaced with arginine (and thus were not available for ubiquitin conjugation) and which was still internalized in response to GH (30). Notably, GH-induced GHR degradation was not monitored in that study; thus, conclusions concerning whether GHR ubiquitination was required for down-regulation to occur could not be drawn (30). Further work in the same system (referred to above) indicated that GH-induced GHR ubiquitination proceeded even for a GHR with a mutated Box 1 element, suggesting to the authors that it was independent of GH-induced JAK2 signaling (35).

In the current study, we also detected GH-induced GHR ubiquitination in cells that express wild-type GHR and JAK2 (Fig. 9Go). However, our finding that this modification was not observed in cells that harbored a kinase-deficient JAK2 is in sharp contrast to those mentioned above. Furthermore, our data indicate that the lack of GH-induced GHR ubiquitination in {gamma}2A-GHR-JAK2KD cells correlates to the inability of GH to cause receptor down-regulation in those cells. This relationship is correlative, but not necessarily causal, however, and further experiments would be needed to establish that ubiquitination is required for down-regulation of the receptor. We are uncertain as to why our conclusions that GH-dependent JAK2 kinase activation is necessary for GH-induced GHR ubiquitination and down-regulation differ from those drawn previously from studies of GH-induced ubiquitination and degradation of a Box 1-mutated GHR in the temperature-sensitive Chinese hamster fibroblast cellular system reported by Alves dos Santos et al. (35).

Our experiments using a GHR with all cytoplasmic tyrosine residues mutated to phenylalanine further tested the basis for the requirement for intact JAK2 kinase function for GH-induced GHR ubiquitination and degradation. Interestingly, we found that GH-induced JAK2 kinase activation, although required, was insufficient for these effects on GHR. Apparently, GHR itself needs to undergo tyrosine phosphorylation to become efficiently ubiquitinated (Fig. 9BGo) and down-regulated (Fig. 10Go). Although other explanations are possible, these data suggest to us that tyrosine phosphorylation of the receptor cytoplasmic domain allows direct binding via a phosphorylated tyrosine(s) to an E3 ubiquitin ligase or proteins involved in ubiquitination and/or down-regulation indirectly (e.g. other kinases or phosphatases). One also cannot rule out that the tyrosine-phosphorylated receptor is a better target for another modification (e.g. phosphorylation at serine or threonine residues) by other enzymes that then enhance the receptor’s susceptibility to ubiquitination and degradation.

Tyrosine phosphorylation is critical to down-regulation of other surface receptors. EGFR, for example, undergoes EGF-induced autophosphorylation, which allows association with ubiquitin ligase activity and EGFR ubiquitination; this allows interaction with proteins in the endocytic/lysosomal pathway and ultimate degradation in lysosomes (50). Indeed, the tyrosine-phosphorylated GHR binds well to several Src homology 2 (SH2)-containing proteins involved in signaling and trafficking (17, 51, 52, 53, 54). We previously found that the SH2-containing protein tyrosine phosphatase (SHP-2), binds specifically to a tyrosine-phosphorylated glutathione-S-transferase fusion protein incorporating the distal 40% of the human GHR cytoplasmic domain, which harbors four tyrosine residues (55). Stofega et al. (52) demonstrated that mutation of one of these tyrosine residues in the rat GHR (corresponding to Y-577 in the human and rabbit GHR) markedly reduced SHP-2 association and prolonged GH-induced signaling. This mutant GHR was also down-regulated somewhat less rapidly than the wild-type GHR, but the effect was subtle compared with the dramatic lack of GH-induced degradation of the MYFc8 receptor we observed in Fig. 10CGo. Thus, the degree to which the prolongation of signaling seen with the Y-577 mutant GHR was explained by a lack of SHP-2-mediated dephosphorylation of the receptor vs. diminished GH-induced receptor degradation is not clear.

Another SH2-containing protein that binds to the tyrosine-phosphorylated GHR is the suppressor of cytokine signaling family protein CIS (cytokine inducible SH2 domain-containing protein) (53, 54). Recent work suggests that CIS, by virtue of its interaction with the GHR, promotes receptor internalization in response to GH and thereby contributes to desensitization of GH signaling (56). The contribution of CIS to GH-induced GHR degradation per se (as opposed to internalization) has yet to be directly studied. Interestingly, CIS, like other suppressor of cytokine signaling proteins, is likely linked to Cullin5-based E3 ubiquitin ligase complex, which may serve to recruit proteins to the proteasome for degradation (57).

Recently, another E3 ubiquitin ligase, ß-TrCP (ß-transducin repeats-containing protein), has been implicated in the ligand-induced ubiquitination and proteolysis of three cytokine receptor family members including the interferon-{alpha} receptor 1 (IFNAR1) and the prolactin receptor and erythropoietin receptor (58, 59, 60, 61, 62). Those studies suggest that phosphorylation of specific serine residues within the cytoplasmic domain of these receptors are critical for ligand-induced association with ß-TrCP and subsequent receptor ubiquitination and down-regulation (59, 61). Intriguingly, catalytic activity of TYK2 is required for IFNAR1 ubiquitination because it was essential for phosphorylating IFNAR1 within the ß-TrCP-binding motif (63). Furthermore, it has been reported that JAK2 catalytic activity is essential for the ubiquitination of erythropoietin receptor (64). Although a role for ß-TrCP in GH-induced GHR down-regulation has not been definitively found, it is notable that GHR contains a similar cytoplasmic domain sequence (DSG and surrounding amino acids) as that found in the prolactin receptor and IFNAR1, and erythropoietin receptor to mediate phosphoserine-dependent recruitment of ß-TrCP (61). Our results with the MYFc8 mutant make us consider whether ß-TrCP might affect GHR down-regulation and, if so, whether receptor tyrosine phosphorylation, by impacting serine phosphorylation, could allow GHR-ß-TrCP interaction. Future studies will be required to discriminate which, if any, of these mechanisms are at play.

The potential impact of receptor ubiquitination and specific ubiquitin ligases in regulation of GH-induced GHR down-regulation and the effects of proteasome inhibitors on this process also raise the issue of the proteasome’s involvement in these processes. Our work suggests that the proteasome is likely directly involved in degrading the GHR or its fragments under certain circumstances. We previously showed, for instance, that the soluble cytoplasmic domain of the receptor that results from intramembraneous cleavage by {gamma}-secretase is degraded in a proteasome-dependent fashion (65). Recent work also suggests that in the absence of JAK2, newly synthesized GHR is degraded in an endoplasmic reticulum-associated degradation-like fashion and that this is blocked by proteasome inhibitors (Loesch et al., submitted). Although it is possible that the proteasome itself is involved in GH-induced GHR down-regulation, we cannot rule out an indirect effect of proteasome inhibitors on this process because proteasome inhibition might suppress the lysosomal pathway via depletion of the ubiquitin pool (66). Future studies will address this important mechanistic issue and explore the E3 ubiquitin ligases involved in GHR ubiquitination.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Recombinant hGH was kindly provided by Eli Lilly Co. (Indianapolis, IN). CHX, 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). G418 and hygromycin (Cellgro, Herndon, VA) were purchased from Mediatech, Inc. (Holly Hill, FL). Fetal bovine serum, gentamicin sulfate, penicillin, and streptomycin were purchased from Biofluids (Rockville, MD).

Antibodies
The rabbit polyclonal antiphosphorylated JAK2 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal anti-STAT5 and anti-EGFR were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antiubiquitin monoclonal antibody was purchased from Stressgen Biotech Corp. (Victoria, British Columbia, Canada). Antiphosphorylated STAT5 polyclonal antibody was purchased from Zymed Laboratories, Inc. (South San Francisco, CA). The 3F10 anti-HA rat monoclonal antibodies was purchased from Roche (Indianapolis, IN). Anti-JAK2AL33 (directed at residues 746-1129 of murine JAK2) polyclonal serum has been described (67). 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 (38).

Plasmids
The rabbit GHR cDNA (68) was a gift from Dr. W. Wood (Genentech, Inc., South San Francisco, CA). All JAK2 cDNAs used encode murine JAK2. Ligation of the rabbit GHR cDNA into the pSX expression plasmid has been described previously (55), as have been the generation of pSX-GHR{Delta}278–292 (GHR{Delta}Box1) (9), pEF-BOS-JAK2{Delta}VIII (JAK2KD) (a gift from Dr. P. P. Sayeski, University of Florida, Gainesville, FL) (41, 69), pCI-Neo-JAK2-HA (a gift of Dr. O. Silvennoinen, University of Tampere, Finland) (70), pMet-IG-pGHR and pMet-IG-MYFc8 (a gift of Dr. J. J. Kopchick, Ohio State University, Columbus, OH) (44), and the pRC/CMV-JAK2{Delta}1–47 and pcDNA3.1+-JAK21–511-HA expression vectors (14).

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) (36). A stable {gamma}2A cell line expressing rabbit GHR ({gamma}2A-GHR) and its cell culture conditions have been described elsewhere (38). A stable {gamma}2A cell line expressing rabbit GHR and mouse JAK2 ({gamma}2A-GHR-JAK2, previously referred to as Clone 14 or C14 cells) was achieved by stable transfection of {gamma}2A-GHR with murine JAK2, as described previously (14). Stable transfection and maintenance of cell lines {gamma}2A-JAK2, {gamma}2A-GHR{Delta}Box1, and {gamma}2A-GHR{Delta}Box1-JAK2 were described previously (23).

Stable {gamma}2A-GHR-JAK2-HA, {gamma}2A-GHR-JAK21–511-HA, {gamma}2A-GHR-JAK2{Delta}1–47, and {gamma}2A-GHR-JAK2KD cell lines were achieved by introducing pCI/Neo-JAK2-HA, pcDNA 3.1+-JAK21–511-HA (which encodes zeocin resistance), pRC/CMV-JAK2{Delta}1–47, and pEF-BOS-JAK2{Delta}VIII plasmids into {gamma}2A-GHR cells individually using Lipofectamine Plus (Invitrogen) according to the manufacturer’s instructions. For transfection of pCI/Neo-JAK2-HA, pRC/CMV-JAK2{Delta}1–47, and pEF-BOS-JAK2{Delta}VIII, cells were cotransfected with the pcDNA 3.1– -Zeocin plasmid, which encodes zeocin resistance. Cells were selected in DMEM {gamma}2A-GHR growth medium supplemented with 400 µg/ml zeocin and screened 3–5 wk after dilution cloning and selection for full-length or mutant JAK2 expression by blotting with anti-JAK2AL-33 or 3F10 antibody. {gamma}2A-pGHR-JAK2 and {gamma}2A-MYFc8-JAK2 were generated by stable transfection of {gamma}2A-JAK2 cells with pMet-IG-pGHR or pMet-IG-MYFc8, respectively, in the same fashion as the generation of {gamma}2A-GHR{Delta}Box1-JAK2 (23). These stable cell lines were maintained in the same media as used for {gamma}2A-GHR-JAK2.

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. Adherent cells were stimulated with human GH (500 ng/ml) in DMEM (low glucose) with 0.5% (wt/vol) BSA at 37 C. 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, 10 µg/ml aprotinin), as indicated. For experiments in which ubiquitination was assayed, N-ethylmaleimide (5 mM) was added to the lysis and immunoprecipitation buffers to prevent postlysis debiquitination of proteins. 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 anti-GHRcyt-AL47, 3 µl of antiserum was used per precipitation. Protein-A Sepharose (Amersham Biosciences, 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 Biosciences) with 2% BSA were performed as previously described (14, 37, 38, 71). Immunoblotting with anti-GHRcyt-AL47 (1:4000), anti-JAK2AL33 (1:4000), anti-EGFR (1:1000), anti-STAT5 (1:1000), 3F10 (100 ng/ml), antiubiquitin (1:1000), anti-P-JAK2 (1:1000), or anti-P-STAT5 (1:1000) and horseradish peroxidase-conjugated antirabbit secondary antibodies (1:10,000), antirat secondary antibodies (1:2000), or antimouse secondary antibodies (1:5000) and enhanced chemiluminescence detection reagents (SuperSignal West Pico chemiluminescent substrate; all from Pierce Chemical Co., Rockford, IL) and stripping and reprobing of blots were accomplished according to the manufacturer’s suggestions.

Degradation of GHR after Blockade of Protein Synthesis (CHX Chase)
Cells were grown to 90% confluence in six-well plates, serum-starved overnight, and then incubated with CHX (20 µg/ml) for 0–5 h. The treatments were ended by washing with ice-cold PBS, and cells were harvested and detergent extracts prepared as described above. Cell lysates were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47, anti-JAK2AL33, or anti-EGFR.

Effects of Proteasome and Lysosome Inhibitors on GH-Induced GHR Degradation
Cells were grown to 90% confluence in six-well plates and serum starved overnight. After incubation with lactacystin (15 µM) or chloroquine (100 µM) for 2 h, GH (500 ng/ml) or vehicle was added for 3 h. Cell lysates were resolved by SDS-PAGE and blotted with anti-GHRcyt-AL47.

Densitometric and Statistical Analysis
Immunoblots were scanned using a high-resolution scanner (Hewlett-Packard Co., Cupertino, CA). Densitometric quantification of images exposed in the linear range was performed using an image analysis program, Image J (developed by W. S. Rasband, Research Services Branch, National Institute of Mental Health, Bethesda, MD). Pooled data of densitometry from several experiments are displayed as mean ± SE.


   ACKNOWLEDGMENTS
 
We appreciate helpful conversations with Drs. K. Loesch, J. Cowan, Y. Huang, and X. Li and the generous provision of reagents by those named in the text.


   FOOTNOTES
 
This work was supported by National Institutes of Health (NIH) Grant DK58259 and in part by NIH Grant DK46395, and a Veterans Affairs Merit Review Award (to S.J.F.) and by NIH Grant CA115281 (to S.Y.F.).

Results from this work were presented in part at the 87th and 88th Annual Meetings of The Endocrine Society in San Diego in June 2005 and in Boston in June 2006, respectively.

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

First Published Online May 8, 2007

Abbreviations: CHO, Chinese hamster ovary; CHX, cycloheximide; CIS, cytokine-inducible SH2 domain-containing protein; EGFR, epidermal growth factor receptor; FERM, band 4.1/Ezrin/Radixin/Moesin; HA, hemagglutin; IFNAR1, interferon-{alpha} receptor 1; JAK2, Janus family of tyrosine kinase 2; SH2, Src homology 2; SHP-2, SH2-containing protein tyrosine phosphatase; ß-TrCP, ß-transducin repeats-containing protein.

Received for publication March 13, 2007. Accepted for publication April 30, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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