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Molecular Endocrinology 21 (7): 1642-1655
Copyright © 2007 by The Endocrine Society

Role of the Growth Hormone (GH) Receptor Transmembrane Domain in Receptor Predimerization and GH-Induced Activation

Ning Yang, Xiangdong Wang, Jing Jiang and Stuart J. Frank

Department of Cell Biology (N.Y., S.J.F.) and Department of Medicine (X.W., J.J., S.J.F.), Division of Endocrinology, Diabetes, and Metabolism, University of Alabama at Birmingham, Birmingham, Alabama 35294-0012; and Endocrinology Section (S.J.F.), Medical Service, Veterans Affairs Medical Center, Birmingham, Alabama

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 AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GH receptor (GHR) mediates GH effects by activating the GHR-associated cytoplasmic tyrosine kinase, Janus kinase 2. Recent studies indicate that GHRs exist as dimers independently of GH binding. Some authors suggest that receptor predimerization is mediated by the transmembrane domain (TMD) and that GH binding initiates signaling by triggering changes in the orientation of the two GHRs within the dimer. In this study, we investigate the role of GHR TMD in GH-independent receptor dimerization and ligand-induced activation. We prepared a GHR mutant, GHRLDLR, in which the TMD is replaced with the TMD of the human low-density lipoprotein receptor (LDLR). The resultant chimera has a TMD two residues shorter than the native GHR TMD; thus, in addition to possessing a different TMD, the altered GHRLDLR TMD helical register may change positions of the GHR extracellular domain (ECD) and intracellular domain relative to the TMD when compared with the wild-type (WT) receptor. When each was coexpressed with an intracellular domain-truncated GHR mutant, GHR1–274-Myc, both WT GHR and GHRLDLR were specifically coprecipitated with GHR1–274-Myc, indicating that the GHR TMD was not required for GHR heterodimerization with GHR1–274-Myc. We further examined the contribution of the so-called "dimerization interface," a GHR ECD region that is critical for GH-induced signaling, to receptor predimerization. Coimmunoprecipitation experiments with either WT GHR, a dimerization interface mutant (GHR-H150D), or a control mutant (GHR-T147D) with GHR1–274-Myc showed dramatically reduced coprecipitation of GHR-H150D with GHR1–274-Myc when compared with WT GHR or GHR-T147K. This result suggests that, in contrast to some recent models, the dimerization interface contributes to GHR predimerization. We also compared WT GHR with GHRLDLR and GHRLDLR{Delta}4 (a chimera in which the LDLR TMD has an internal deletion of four residues) with regard to response to GH stimulation. Although the chimeras had similar GH dose responses and time courses for signaling as WT GHR, they were markedly less sensitive to inhibition of signaling by a conformation-sensitive GHR ECD monoclonal antibody. Further, the chimeras were much less sensitive to inducible metalloprotease cleavage than was WT GHR, implying that the ECD conformations of the chimera receptors differ from WT GHR. Collectively, our data indicate that the composition and/or length of the TMD affect some aspects of GHR function, but do not affect receptor predimerization or GH-induced GHR activation. Further, they suggest that the GHR ECD-TMD is more flexible than previously thought in terms of the ability to achieve the active conformation in response to GH.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GH IS THE MAJOR regulator of postnatal growth. GH exerts its somatogenic, metabolic, and differentiative effects on its target cells and tissues by binding to the GH receptor (GHR) (1). The GHR is a member of the class I cytokine receptor family. It has an extracellular domain (ECD), a single transmembrane domain (TMD), and an intracellular domain (ICD). GH binding results in activation of the GHR-associated nonreceptor tyrosine kinase, Janus kinase 2 (JAK2), which interacts with GHR at a proline-rich region (Box 1) in the receptor ICD (2, 3, 4, 5). Once activated by GH, JAK2 promotes tyrosine phosphorylation of itself, the GHR ICD, and other signaling molecules, resulting in signaling cascades and GH biological effects (1, 6, 7).

Early studies of the mechanism of GHR activation suggested a model of GH-induced sequential dimerization of receptor monomers, in which site 1 of GH binds a GHR monomer with high affinity and is followed by site 2 of the same GH molecule binding to a second GHR monomer, allowing dimerization of the two GHR monomers in the tripartite complex (8, 9, 10). This model arose in part from experiments with cells expressing a chimera between the GHR ECD and the granulocyte colony-stimulating factor receptor (10). The chimeric receptor was activated by treatment with divalent monoclonal antibodies to the GHR ECD, but not with their monovalent Fab fragments (10). The finding of a bell-shaped GH dose-response curve in cell culture systems also supported the sequential dimerization model (8, 10).

In contrast to this model, more recent work suggests that cytokine receptors exist as dimers independent of ligand stimulation (11, 12, 13, 14, 15). The erythropoietin receptor (EpoR), for example, was found to exist as a dimer in crystals that did not include Epo (16, 17) and at the cell surface as Epo-independent preformed dimers mediated by the EpoR TMD (18, 19). This observation correlates with reports that the TMD of EpoR possesses a heptad motif of leucine residues and that it can strongly self-assemble in a ToxR assay (19, 20). The GHR was also found to dimerize independently of GH binding using coimmunoprecipitation of differentially tagged receptors or fluorescence and bioluminescence resonance energy transfer techniques (12, 13). Furthermore, based on experiments in which the GHR ECD and/or ICD were truncated, the observed association between two GHR dimer subunits was attributed to the receptor’s TMD/juxtamembrane domain. Notably, however, the TMD of GHR does not possess a heptad motif of leucine residues and does not significantly self-interact in the ToxR assay (19, 20).

Unlike the ECD (9, 21), there are no direct structural data about the TMD or ICD of the GHR. The 2:1 ECD-GH complex has three binding interfaces: GH binding site 1 with ECD1, GH binding site 2 with ECD2, and the so-called "dimerization interface" between the second subdomains of ECD1 and ECD2. Notably, recent work indicates that the ECD1-ECD2 interaction is stronger than the GH-ECD2 interaction (22). Mutation in the dimerization interface generally has no effect on the GH binding affinity of GHR, which suggests that GH binding site 1 and the formation of the dimerization interface are not functionally coupled (22, 23). The dimerization interface is comprised of eight residues from ECD1 and seven residues from ECD2 and the interface contains six hydrogen bonds (9, 22). Residues involved in this interface contribute differentially to ECD-ECD contact and to GH-induced signal transduction (22, 23, 24). Interestingly, the unliganded GHR ECD was recently crystallized, and comparison of the unliganded vs. GH-engaged GHR ECD structures revealed that the orientations of all the side chains, including those in the dimerization interface, are almost identical in both (13). The findings of predimerization and the lack of major difference between the structures of the unliganded and GH-engaged ECDs has led to the proposition by Brown et al. (13) that GH initiates signaling by inducing twisting within the inactive preformed dimer so as to change the relative orientations of the cytoplasmic domains and bring JAK2 molecules bound to each GHR dimer partner within proximity for transphosphorylation and activation. This model suggests that both the ECD and ICD juxtamembrane domains of the GHR adopt a rigid conformation to allow the transmission of the torque force resulting from the asymmetrical binding of GH. Furthermore, Brown et al. (13) found that GHR can be activated in the absence of ligand by inserting a defined number of alanine residues within the TMD, presumably by causing a specific rotation of the dimerized receptor monomers relative to each other. Thus, it was concluded by those authors that the length of the TMD affects the orientation of the receptor and/or the register of JAK2 binding to the ICD.

In the current study, we employed a well-characterized reconstitution system, the {gamma}2A cell line and derivatives (24, 25, 26, 27, 28), to directly test whether the GHR TMD is required for GH-independent receptor dimerization and the role of TMD in ligand-induced activation. By using a chimera between the TMD of the human low-density lipoprotein receptor (LDLR) and the ECD and ICD of the GHR, we observed that the GHR TMD is not required for GHR predimerization, as assessed by coimmunoprecipitation. However, our data indicate that mutation of histidine-150, an ECD residue involved in dimerization interface hydrogen bonding and critical for GH-induced signaling (24), disrupted receptor predimerization. Furthermore, although the LDLR TMD is two amino acids shorter than the GHR TMD, the chimeric receptor transmitted acute GH-induced signals indistinguishably from the WT GHR in terms of GH dose-dependence and time-course responses. Despite normal signal initiation, further characterization revealed that the chimeric receptor was less sensitive than WT GHR to inhibition of signaling by our previously characterized conformation-specific ECD antibody, anti-GHRext-mAb, and was less sensitive to phorbol ester-stimulated metalloproteolysis than WT GHR. These data suggest that in our system the composition and/or length of the TMD affects some aspects of GHR function, but does not affect receptor predimerization or GH-induced triggering of signal transduction. Rather, the latter two features appear dependent on an intact ECD dimerization interface.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
GHR TMD Is Not Required for Receptor Predimerization
To directly test the role of TMD in ligand-independent dimerization (predimerization) of GHR, we sought to replace the GHR TMD with that of another unrelated transmembrane protein. For this purpose, we chose the TMD of the human low-density lipoprotein receptor, a well-studied cell surface receptor that is not known to undergo TMD-mediated dimerization (29). We prepared a cDNA encoding the chimeric receptor mutant, GHRLDLR, in which GHR TMD is replaced by the TMD of the human (h) LDLR (see Materials and Methods) (Fig. 1AGo). Because the LDLR TMD possesses only 22 residues, the resultant chimera should have a TMD two residues shorter than the native GHR TMD (24 residues), as predicted by the PredictProtein algorithm (30). Thus, in addition to possessing a different TMD, the altered TMD helical register in GHRLDLR may change positions of the ECD and ICD relative to the TMD when compared with the wild-type (WT) receptor. We also constructed a second chimeric receptor mutant, GHRLDLR{Delta}4, that has a four amino acid deletion within the LDLR TMD, which is thus predicted to be six residues shorter than that of the WT GHR (Fig. 1AGo).


Figure 1
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  		Fig. 1. Comparison of the TMDs and Expression of GHR-LDLR TMD Chimeras

A, Schematic presentation of the TMDs of the rabbit GHR, GHRLDLR and GHRLDLR{Delta}4. The numbers of predicted TMD residues are listed on the right. B, Expression of the chimeric GHRs. HEK-293 cells were transiently transfected with the cDNAs indicated. Detergent extracts of the serum-starved cells were resolved by SDS-PAGE and immunoblotted by anti-GHRcyt-AL47. Mature (m; fully glycosylated) and precursor (p; not fully glycosylated) forms of the GHR are labeled. aa, Amino acid; WB, Western blot.

 
We first tested whether these mutant receptors could be expressed by transiently transfecting human embryonic kidney (HEK)-293 cells with eukaryotic expression vectors encoding WT GHR, GHRLDLR, GHRLDLR{Delta}4, or empty vector (as a negative control) (Fig. 1BGo). Transfected cells were serum starved and harvested. Cellular proteins were detergent extracted, resolved by SDS-PAGE, and subjected to immunoblotting with anti-GHRcyt-AL47, a rabbit antiserum that recognizes the cytoplasmic domain of the receptor (31). This analysis revealed that both chimeric GHR mutants were expressed at levels comparable to the WT GHR, as the overall signal was similar for all of them. In this system, the GHR is detected as two predominant glycoprotein forms (indicated by brackets in Fig. 1BGo), the mature (cell surface) form and the precursor (pre-trans-Golgi) form, which are distinguishable based on their electrophoretic migration; previous work indicates that the mature (endoglycosidase H-resistant) form exhibits retarded migration relative to precursor (endoglycosidase H-sensitive) form (25, 27, 32, 33, 34). The mutants and WT GHR exhibited comparable abundance of mature receptor and, as judged by the ratio of mature-precursor receptor, similar maturation efficiency. The chimeric GHR mutants exhibited slightly altered migration compared with WT GHR on reducing SDS-PAGE (Fig. 1BGo). This may suggest that the chimeras bind sodium dodecyl sulfate slightly differently compared with the WT because they possess a different TMD.

We tested formation of GH-independent receptor dimers by assessing the ability of WT or chimeric GHR mutant to associate with a tagged truncated form of the receptor. GHR1–274-Myc has an intact ECD and TMD, but lacks all but four ICD residues (32). We first assessed association of WT GHR with GHR1–274-Myc. An expression vector encoding WT GHR was transiently cotransfected with a vector encoding GHR1–274-Myc in {gamma}2AJAK2, a human fibrosarcoma cell that lacks endogenous GHR (35) (Fig. 2AGo). Transfected cells were serum starved, and detergent extracts were prepared and subjected to immunoprecipitation with either anti-Myc or, as a negative control, anti-hemagglutinin (HA). Eluted proteins were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47, revealing specific coimmunoprecipitation of WT GHR with GHR1–274-Myc. Extracted proteins used in each precipitation were also blotted with anti-GHRcyt-AL47 and anti-Myc, verifying that similar amounts of extract were sampled. To compare the coimmunoprecipitation of WT GHR vs. GHRLDLR, the same experiment was performed with cells transfected with expression plasmids encoding each receptor along with GHR1–274-Myc (Fig. 2BGo). As seen in the representative blot, both WT GHR and GHRLDLR were coimmunoprecipitated with GHR1–274-Myc. The degree of coprecipitation of each receptor normalized for its expression in the cell extracts was estimated densitometrically. This analysis revealed no difference in coprecipitation of GHRLDLR with GHR1–274-Myc in this assay compared with WT GHR coprecipitation with GHR1–274-Myc, suggesting that the chimera receptor forms heterodimers with the truncated receptors as efficiently as the WT GHR does in the absence of the ligand. The data also indicate that the ICD is not required for predimerization, which is consistent with previous findings (12, 13). Furthermore, the native GHR TMD, which is not present in GHRLDLR, is also not required in this process.


Figure 2
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  		Fig. 2. GHR Predimerization Does Not Require GHR TMD

A, Either WT GHR or GHRLDLR was coexpressed with GHR1–274-Myc, which encodes the GHR ECD and TMD, but lacks all but four ICD residues, by transient transfection of {gamma}2A-JAK2 cells. Detergent extracts were immunoprecipitated with anti-Myc (9E10) and immunoblotted with anti-GHRcyt-AL47. An aliquot of each extract was also directly immunoblotted with anti-GHRcyt-AL47 or with anti-Myc, as indicated. B, Several experiments as in panel A were evaluated by densitometry to quantify the relative coimmunoprecipitation signal of the GHRLDLR, which was expressed as a percentage of coimmunoprecipitation signal relative to the WT GHR (mean ± SE, n = 5). The coimmunoprecipitation signals of these two receptors were not statistically different [nonsignificant (NS)] by Student’s t test. Both the mature and precursor forms of the receptors were used in the quantitation analysis. C and D, {gamma}2A-JAK2 cells were transiently transfected with either no DNA (mock transfection) or expression vector for hLDLR (C) or GHR or GHRLDLR (D), along with the vector encoding GHR1–274-Myc. Detergent extracts were immunoprecipitated with anti-Myc and immunoblotted with anti-hLDLR (C) or anti-GHR (D). An aliquot of the extracts were also directly immunoblotted with anti-hLDLR, anti-GHR, or anti-Myc, as indicated. E, Three experiments as in C and in D were evaluated by densitometry to quantify the relative coimmunoprecipitation signal compared with the 5% input signal and normalized for the abundance of GHR1–274-Myc in the extract. This value was considered as the normalized coimmunoprecipitation and is graphed for LDLR and GHRLDLR as mean ± SE. Both the mature and precursor forms of the receptors were used in the quantitation analysis. IP, Immunoprecipitation; m, mature; p, precursor; WB, Western blot.

 
We further tested the specificity of this interaction by assessing coimmunoprecipitation of full-length (WT) hLDLR with GHR1–274-Myc (Fig. 2Go, C–E). {gamma}2A-JAK2 cells were transiently transfected with an expression plasmid encoding GHR1–274-Myc along with plasmids encoding either LDLR (Fig. 2CGo) or WT GHR or GHRLDLR (Fig. 2DGo). As a control, untransfected cells were used (first lanes of both Figs. 2CGo and 2DGo). Detergent extracts were subjected to immunoprecipitation with either anti-HA (negative control) or anti-Myc, as indicated, and eluates were blotted for LDLR (Fig. 2CGo) or GHR (Fig. 2DGo). In each case, extracts were also blotted for the expressed proteins (LDLR or GHR and GHR1–274-Myc, as indicated) to normalize for their abundance. Like GHR, LDLR is expressed as precursor and mature forms (Fig. 2CGo, middle panel) (36, 37). Interestingly, we found that LDLR coimmunoprecipitated with GHR1–274-Myc (Fig. 2CGo, upper panel). To appreciate this in context, we compared the degree of LDLR coprecipitation with that of GHR and GHRLDLR. In each case—LDLR (Fig. 2CGo) and GHR and GHRLDLR (Fig. 2DGo)—the extracts and immunoprecipitates were resolved on the same gel, blotted under the same conditions, and developed under the same conditions. Further, the fractions of extract subjected to immunoprecipitation and resolved without precipitation for blotting were the same for each. Thus, the relative degree of coprecipitation (upper panels) vs. the abundance in the extract (middle panels) could be compared for each protein, in particular when further normalized for the expression level of the directly precipitated GHR1–274-Myc (lower panels). This analysis revealed that both GHR and GHRLDLR were substantially more coprecipitated with GHR1–274-Myc than was LDLR. For LDLR vs. GHRLDLR, we analyzed three such experiments densitometrically, including in the analysis both the mature and precursor forms of each that were coprecipitated. We found that the enhanced coprecipitation of GHRLDLR was reliable and significant. These data indicate that although GHR1–274-Myc can heterodimerize with LDLR, presumably via their TMDs, the heterodimerization of GHR1–274-Myc with both GHR and GHRLDLR is more pronounced, further supporting the conclusion that it is mediated via other element(s) in addition to the TMD, likely residing in the ECD.

Further, careful inspection of the blot in Fig. 2CGo reveals that the coprecipitation of the mature LDLR with GHR1–274-Myc was greatly reduced compared with that of the precursor LDLR, when compared with their levels in the extracts. By densitometry, the ratio of mature-precursor LDLR in the GHR1–274-Myc precipitate was 5.9 ± 0.6%, whereas the same ratio was 69.8 ± 0.3% in the cell extract (n = 3; P < 0.0002). No such dramatic difference was seen for either GHR or GHRLDLR in the GHR1–274-Myc precipitate vs. the extract. Thus, when comparing only the mature forms, LDLR was only minimally coprecipitated with GHR1–274-Myc relative to either GHR or GHRLDLR. We do not know the reason for this highly reliable finding, but speculate that ability of GHR-LDLR TMD interactions to transiently foster heterodimers in the environment of the endoplasmic reticulum and early secretory pathway (where the precursor receptors reside) is not maintained as the proteins traverse the Golgi and appear at the cell surface (locations enriched in the mature receptors). Rather, the interactions between GHR ECDs that maintain the heterodimers may predominate at that stage. We believe this distinction may be functionally relevant. Others have implicated the GHR TMD as having a role in predimerization of the GHR (12, 13, 38). These conclusions were drawn from data that coimmunoprecipitation and/or fluorescence/bioluminescence resonance energy transfer could be detected if the ECD and/or ICD of the receptor were mutated or removed. Our data tested this question in a different way, asking instead whether the GHR TMD is required for predimerization, rather than whether it could mediate it. We found that the GHR TMD was not required; however it may contribute to receptor predimerization, perhaps especially to some degree at early stages in the secretory pathway.

GHR1–274-Myc does not possess Box 1, the proline-rich motif that mediates JAK2 association. Because both GHR and GHRLDLR coprecipitated with GHR1–274-Myc,it is thus unlikely that GHR-JAK2 association is important in the predimerization of the GHR. Indeed, we observed similar results as in Fig. 2Go when we coexpressed GHR or GHRLDLR with GHR1–274-Myc in {gamma}2A cells, which do not express JAK2 (data not shown). Together, these data suggest that the ligand-independent dimerization of the receptor does not require the presence of JAK2.

The Dimerization Interface Contributes to the Predimerization of the GHR
Because the ICD was not required for predimerization of GHR, we focused on the receptor ECD to explore determinants of this process that might reside outside the TMD. The dimerization interface in the subdomain 2 was first revealed by analysis of the 2:1 ECD-GH crystal structure (9). The ECD1-ECD2 contact interface incorporates eight ECD1 residues and seven ECD2 residues, with D152 and S201 contributing to the interaction from both sides; however, each residue contributes uniquely to the interaction (9, 22). Mutation of most residues in the interface impairs or abrogates GH activity or GH-induced signaling (23, 24, 39). Early models of GHR activation featured ECD-ECD dimerization as a critical GH-triggered event. A more recent model has stressed ligand-independent GHR predimerization, but holds that the ECD-ECD interaction via the dimerization interface is GH induced and critical for signaling (13). Because this contact area comprises six highly organized hydrogen bonds that render great specificity (9, 22), we hypothesized that the dimerization interface might also be involved in the predimerization of receptors.

To approach this hypothesis, we studied two previously characterized GHR point mutants, each with alteration of a residue involved in formation of the hydrogen bonds of the dimerization interface (Fig. 3Go). GHR-H150D has a mutation changing histidine-150 to aspartate, and GHR-T147K has threonine-147 changed to lysine (23). GHRH150D exhibits severe impairment of GH-induced JAK2 activation (24) and transcriptional signaling (23); thus, this mutation is believed to dramatically alter (lessen) ECD-ECD interaction. Conversely, the GHR-T147K mutant is only mildly impaired in GH-induced signaling (23, 24) and is predicted to have less impact on maintaining the ECD-ECD interaction (22, 23). We transiently expressed either WT GHR or the dimerization interface point mutants in {gamma}2A-JAK2-GHR1–274-Myc cells, which stably expressed GHR1–274-Myc, and performed coimmunoprecipitation experiments with anti-Myc (Fig. 3AGo, upper panel) as described above. As a specificity control, transient transfection of WT GHR was duplicated and the extract was immunoprecipitated with monoclonal anti-HA (a negative control). Precipitates (anti-Myc and anti-HA) were eluted and proteins were resolved by SDS-PAGE side by side and immunoblotted with anti-GHRcyt-AL47. [Positions of the mature and precursor GHR are indicated, as is a nonspecific band (NS) present in precipitated samples.] As expected, WT mature and precursor GHR were precipitated with anti-Myc, but not with anti-HA, verifying the specificity of coimmunoprecipitation of WT GHR with stably expressed GHR1–274-Myc. GHR-T147K coimmunoprecipitated with GHR1–274-Myc to a similar degree as WT GHR (Fig. 3Go, A and B). In contrast to WT GHR and GHR-T147K, coprecipitation of GHR-H150D with GHR1–274-Myc was much less (~31% that of WT GHR) (Fig. 3Go, A and B). Anti-Myc blotting of the precipitates verified similar abundance of GHR1–274-Myc in each sample (Fig. 3AGo, middle panel). Blotting of the cell extracts with anti-GHR (Fig. 3Go, lower panel) also indicated similar receptor expression levels in all transfected cells. These results indicate that the two mutants differ substantially in their propensity to undergo ligand-independent association (dimerization) with GHR1–274-Myc. Their different degrees of association correlate with the previous observations of GH-induced signaling of these two mutants and suggest that the H150D mutation impairs predimerization of the GHR, perhaps accounting for the disrupted signaling with this mutant. By extension, these data implicate the dimerization interface in the ligand-independent dimerization of the GHRs.


Figure 3
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  		Fig. 3. Contribution of the Dimerization Interface to GHR Predimerization

A, {gamma}2A-JAK2 cells stably expressing GHR1–274-Myc were used to transiently express WT GHR, GHR-H150D, or GHR-T147K. Detergent extracts were immunoprecipitated with anti-Myc or anti-HA, as indicated, and immunoblotted with anti-GHRcyt-AL47. The blot was stripped and reprobed with anti-Myc. The extracts were also immunoblotted with anti-GHRcyt-AL47 to indicate the abundance of the receptors expressed. B, Four experiments as in panel A were evaluated by densitometry to quantify the relative coimmunoprecipitation signal of the ECD mutants, which was expressed as a percentage of coimmunoprecipitation signal relative to the WT GHR (mean ± SE). The coimmunoprecipitation of GHR-H150D was roughly 31% of the WT GHR (P < 0.001), whereas there was no statistical difference between the coimmunoprecipitation of GHRT147K and the WT-GHR. IP, Immunoprecipitation; m, mature; p, precursor; WB, Western blot.

 
We note that Gent et al. (38) have previously examined certain residues within the dimerization interface with regard to effects on predimerization. Specifically, those investigators also used a coimmunoprecipitation assay to evaluate GHRs with mutations N143K, D152H, Y200D, and S201K. In those experiments, stably transfected cells expressing each mutant GHR were transiently transfected with a truncated epitope-tagged receptor. Endogenous full-length mutant receptors were precipitated, and transiently transfected receptors that were coprecipitated were detected by blotting via the epitope tag. There was no difference between the mutants and the WT GHR in their ability to coprecipitate the epitope-tagged receptor, leading to the conclusion that the dimerization interface was not important in predimerization of the GHR (38). In contrast to the methods of Gent et al., we stably expressed a truncated epitope-tagged GHR and evaluated the degree to which precipitation of this receptor via the tag allowed coprecipitation of transiently transfected WT GHR vs. GHR-T147K or GHR-H150D. We found clear evidence for a defect in predimerization of GHR-H150D. Notably, this mutant was not evaluated in the study by Gent et al. (38). Further, the use of GHRT147K serves as an important control in our experiments in that T147 also participates in the dimerization interface, but is apparently not as critical as is H150, based on previous signaling data (23, 24). Because our experimental protocol should favor detection of the transiently transfected (and therefore highly expressed) full-length GHRs (WT or mutants) but specifically did not detect coprecipitation of GHR-H150D, we conclude that there is likely a substantial role for this GHR region in predimerization. However, it is evident that not all dimerization interface residues necessarily contribute to this phenomenon.

Further, it is conceivable that the dimerization interface may be less important at the initiation of predimerization in the endoplasmic reticulum, but that interaction at or governed by this interface is crucial in maintaining dimers as they traverse to the cell surface. This is evident from the observation that much less mature form of the LDLR appeared to coprecipitate with GHR1–274-Myc. In this model, the TMD could have a role in the predimerization initiation, but the dimerization interface could suffice if TMD interactions are not possible. Further mutagenesis will be required to determine whether such cooperation between the TMD and dimerization interface (with the latter being more stringently required) may reconcile our findings with those of other groups.

GH-Induced Signaling Is Intact in TMD Mutants
The length of the GHR TMD or the relative orientation of the receptor resulting from a certain length of TMD has been suggested to be important in the GHR activation (13). In light of this suggestion and our data that the GHR-LDLR TMD chimeras predimerize normally, we examined the ability of GHRLDLR and GHRLDLR{Delta}4 to transduce GH-induced signaling (Fig. 4Go). We first transfected {gamma}2A-JAK2 cells with GHRLDLR or GHRLDLR{Delta}4 and selected stably transfected clonal cell lines, screening for clones with similar levels of GHR by anti-GHR immunoblotting. {gamma}2A-JAK2-GHRLDLR and {gamma}2AJAK2-GHRLDLR{Delta}4 cells were also found to have GHR levels similar to that found in our previously characterized {gamma}2A-JAK2-GHR (C14) cells (25), which express WT GHR (Fig. 4AGo and data not shown).


Figure 4
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  		Fig. 4. GH Signaling in Cells Stably Expressing GHR-LDLR TMD Chimeras

A, GHR TMD mutants form GH-induced disufide-linkage similar to WT GHR. Serum starved {gamma}2A-JAK2-GHR, {gamma}2A-JAK2-GHRLDLR, and {gamma}2A-JAK2-GHRLDLR{Delta}4 cells were treated with GH (500 ng/ml) or vehicle (–) for 10 min. Detergent extracts were resolved by SDS-PAGE under nonreduced conditions and immunoblotted with anti-GHRcyt-AL47. Positions of the disulfide-linked (dsl) and non-disulfide-linked GHR are indicated. The experiments shown are representative of three such experiments. B and C, {gamma}2A-JAK2-GHR, {gamma}2A-JAK2-GHRLDLR, and {gamma}2A-JAK2-GHRLDLR{Delta} cells have similar dose-dependence (B) and time-course (C) responses to GH stimulation. Serum-starved cells were treated with GH for 10 min using the indicated concentrations (A) or with 500 ng/ml GH for the indicated durations (B). Immunoblots for anti-pTyr-JAK2 and anti-JAK2 were performed, as indicated. The experiments shown are representative of three such experiments.

 
We initially tested whether GH induced conformational changes in the chimeric receptors in comparison with WT GHR (Fig. 4AGo). We previously demonstrated that GH induces formation of a high Mr form of the GHR detectable by anti-GHR immunoblotting of cell extracts resolved under nonreducing conditions (24, 40, 41). Formation of this disulfide-linked GHR reflects attainment of the active signaling conformation of the receptor. Serum-starved {gamma}2A-JAK2-GHRLDLR, {gamma}2A-JAK2-GHRLDLR{Delta}4, and {gamma}2A-JAK2-GHR cells were treated with (+) or without (–) GH (500 ng/ml) for 10 min, after which they were harvested and detergent solubilized. Extracts were resolved by SDS-PAGE under nonreducing conditions and immunoblotted with anti-GHRcyt-AL47, revealing that GH induced the appearance of the high-Mr disulfide-linked form of the GHR in each cell type. Although it is conceivable that the degree of disulfide linkage may subtly vary, these data strongly suggest that the active signaling conformation can be achieved in response to GH even when the receptor TMD is replaced by a non-GHR TMD that is either two or six residues shorter than the native receptor TMD.

We next examined GH-induced JAK2 activation in the three cell lines. Serum-starved cells were treated with GH (various concentrations over the 0–500 ng/ml range) for 10 min (Fig. 4BGo) or with 500 ng/ml for various durations over the 0–60 min range (Fig. 4CGo) before detergent extraction, SDS-PAGE under reduced conditions, and sequential immunoblotting with anti-pTyr-JAK2 (an antibody that recognizes the tyrosine-phosphorylated activation loop and therefore correlates with kinase activity) and anti-JAK2AL33. We found that the chimera receptors showed very similar dose-dependent responses compared with WT GHR, with detectable tyrosine phosphorylation of JAK2 at 20 ng/ml GH treatment and maximum JAK2 activation between 125 and 500 ng/ml. Similarly, the time course of JAK2 activation was similar for the WT GHR and the chimera mutants. These data complement those in Fig. 4AGo and indicate that the presence of the LDLR TMD or a dramatically foreshortened version of that TMD did not affect the ability of GH to trigger JAK2 activation. Similarly, in these experiments the signal transducer and activator of transcription (STAT)5 activation profile was unaffected in the chimeras (data not shown).

Previous elegant studies by Brown et al. (13) have indicated that constitutive JAK2 activity was dramatically induced by insertion of four alanine residues within the TMD of the GHR or one alanine residue in the receptor juxtamembrane cytoplasmic domain of the GHR. Insertion of one to three or five to seven transmembrane residues or two to four juxtamembrane residues did not cause constitutive activation. They reasoned that these insertions did not change the relative vertical positions of these receptor regions relative to the JAK2-binding domain, but rather that a 400° rotation (100° per alanine residue) in the TMD helix (i.e. a nominal 40° clockwise rotation compared with no insertion) or a 100° clockwise rotation of the (presumed) juxtamembrane helix would twist the TMD/juxtamembrane regions such that JAK2 molecules bound at Box 1 would be brought into proximity for trans-autophosphorylation and activation. By the same reasoning, our replacement of the LDLR TMD for the GHR TMD (both assumed to be helical) would cause a two-residue loss of TMD length and the LDLR{Delta}4 TMD would cause a loss of six residues relative to the GHR TMD; they would thus result in 200° and 600° counterclockwise rotations, respectively, which correspond to 160° and 120° nominal clockwise rotations, respective to no deletion. Both of these clockwise rotations in the model of Brown et al. (13) would not result in constitutive activation and, indeed, it is notable that we observed no constitutive JAK2 activity for our chimera mutants.

Brown et al. (13) postulated that the natural activation by GH might be predicted to result in a clockwise rotation of the JAK2 binding region (via TMD/juxtamembrane torsion) of roughly the same degree as that achieved by constitutive activation (i.e. 40° to 100° clockwise) in their insertion mutants, but GH-induced activation of cells harboring those mutants was not reported. If we add such a GH-induced 40°-100° clockwise increase in rotation to the already 160° and 120° nominal clockwise rotations presumably present in our chimeras, this would clearly put such rotation beyond that predicted to be necessary; yet, GH signaling (dose response and time course) is not altered in our chimeras compared with WT GHR. Thus, our findings suggest that the final degree of rotation achieved is unlikely to be the only factor that governs the GH-triggered JAK2 activation.

Inhibition of GH-Induced Signaling by Anti-GHRext-mAb Is Markedly Reduced for GHR-TMD Chimeras
Using signal initiation as an indicator, the data in Fig. 4Go suggest that there is little change in response to GH in the GHR-TMD chimeras compared with the WT GHR. However, to explore this issue further, we tested the effects of an antagonistic antibody on signaling. We previously characterized anti-GHRext-mAb, a mouse monoclonal antibody raised against the rabbit GHR ECD that cross-reacts with human GHR (24, 41, 42). Anti-GHRext-mAb engages the receptor on the surface of intact cells, and preliminary mapping indicates that it reacts with subdomain 2, but not subdomain 1 or the juxtamembrane stem region of the ECD (24). The antibody is conformationally sensitive in that it immunoreacts with the GHR much less well when the receptor is engaged by GH, even though its epitope likely resides away from the GH-binding region of the receptor ECD (24, 41). Indeed, pretreatment of cells with anti-GHRext-mAb impairs radiolabeled GH binding only modestly, but markedly inhibits subsequent GH-induced activation of JAK2 and STAT5 signaling (24). We hypothesize that pretreatment with anti-GHRext-mAb is inhibitory by virtue of the antibody’s ability to bind the receptor and lock it in an inactive state, thereby inhibiting subsequent GH-induced activation.

In the experiments shown in Fig. 5AGo, serum-starved {gamma}2A-JAK2-GHR, {gamma}2AJAK2-GHRLDLR, and {gamma}2A-JAK2-GHRLDLR{Delta}4 cells were incubated with the indicated concentrations of anti-GHRext-mAb for 10 min at 37 C before treatment with (+) or without (–) GH (500 ng/ml) for another 10 min. Pretreatment with a different monoclonal antibody, anti-GHRcyt-mAb, served as a negative control; this antibody reacts with the GHR ICD and thus does not have access to its binding site when incubated with intact cells (24, 41). Detergent extracts were resolved by SDS-PAGE and blotted sequentially with anti-pTyr-JAK2 and anti-JAK2AL33. As expected, anti-GHRext-mAb inhibited GH-induced JAK2 activation in {gamma}2A-JAK2-GHR cells in a dose-dependent manner, consistent with our previous observations (24). This inhibition was specific in that anti-GHRcyt-mAb pretreatment had no effect. In contrast to the findings in i2A-JAK2-GHR cells, anti-GHRext-mAb pretreatment had relatively little effect on GH-induced JAK2 activation in {gamma}2A-JAK2-GHRLDLR and {gamma}2A-JAK2-GHRLDLR{Delta}4 cells. We estimated the degree of anti-GHRext-mAb inhibition in each cell type by densitometric quantitation of immunoblots from three independent experiments (Fig. 5BGo). This analysis makes evident the reduced inhibition by the antibody in the GHR-LDLR TMD chimera-expressing cells. At the highest concentration of anti-GHRext-mAb used (16 µg/ml), GH-induced JAK2 activation in {gamma}2A-JAK2-GHR cells was only 1% of the signal (99% reduction) observed in GH-treated cells that were not preincubated with antibody. However, the cells expressing chimera receptors and pretreated with anti-GHRext-mAb at the same concentration exhibited only approximately 50% inhibition of GH-induced JAK2 activation. This insensitivity to inhibition was not explained by inability of anti-GHRext-mAb to interact with the chimeric receptors (Fig. 5CGo). Each receptor was immunoprecipitated with anti-GHRext-mAb to a relatively similar degree as it was precipitated by anti-GHRcyt-mAb (which is a monoclonal antibody that recognizes the ICD of the receptor). This experiment indicated that the replacement of the GHR TMD with the LDLR and LDLR{Delta}4 TMDs did not destroy the epitope for anti-GHRext-mAb.


Figure 5
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  		Fig. 5. Anti-GHRext-mAb Inhibition of GH Signaling Is Dramatically Reduced in GHR TMD Mutants

A, Serum-starved {gamma}2A-JAK2-GHR, {gamma}2A-JAK2-GHRLDLR, and {gamma}2A-JAK2-GHRLDLR{Delta}4 cells were pretreated for 10 min with the indicated concentrations of anti-GHRext-mAb or anti-GHRcyt-mAb before treatment with GH (500 ng/ml) or vehicle for 10 min. Immunoblots for anti-pTyr-JAK2 and anti-JAK2 were performed, as indicated. Note signaling by WT GHR is substantially inhibited by anti-GHRext-mAb, whereas the inhibition is dramatically less in TMD mutants. B, Several experiments as in panel A were evaluated by densitometry to quantify the relative abundance of phospho-JAK2 appearing in response of the treatments. This was expressed as a percentage of GH-stimulated level in the absence of the anti-GHRext-mAb (mean ± SE, n = 3). C, Binding of anti-GHRext-mAb to GHR TMD mutants is intact. Detergent extracts of serum-starved {gamma}2A-JAK2-GHR, {gamma}2A-JAK2-GHRLDLR, and {gamma}2A-JAK2-GHRLDLR{Delta}4 cells were immunoprecipitated with anti-GHRext-mAb or anti-GHRcyt-mAb, as indicated, and blotted with anti-GHRcyt-AL47. The experiment shown is representative of three such experiments. D, Ligand antagonist G120K can inhibit signaling in TMD mutants. Serum-starved {gamma}2A-JAK2-GHR, {gamma}2A-JAK2-GHRLDLR, and {gamma}2A-JAK2-GHRLDLR{Delta}4 cells were treated with vehicle, GH (500 ng/ml), G120K (indicated concentrations), or both for 10 min. Detergent extracts were resolved by SDS-PAGE and immunoblotted with antipTyrJAK2. The experiment shown is representative of three such experiments. IP, Immunoprecipitation.

 
Collectively, the data in Fig. 5Go, A–C, indicated that the ability of an antibody directed at subdomain 2 to inhibit GH signaling is diminished by changes in the TMD, suggesting that the conformation of the ECD, at least in the region affected by the conformation-sensitive anti-GHRext-mAb, is altered. This is despite the fact that a proper GH-induced signaling conformation is attained in the absence of the antibody. To further understand effects of the TMD changes on the capacity of the mutant GHR to be antagonized, we tested the effects of the GH antagonist, G120K (Fig. 5DGo). G120K is a GH analog with a point mutation at binding site 2 (glycine 120 to lysine). This mutation results in elimination of site 2 interaction with the GHR, whereas site 1 binding is intact. G120K binds to the GHR via site 1 but fails to activate the receptor; thus, G120K functions as a GH antagonist by competing with GH for site 1 binding (10, 41, 43).

As expected, treatment of cells expressing WT or GHR-LDLR TMD chimeras with G120K alone was unable to cause JAK2 activation (Fig. 5DGo, lane 3 vs. 2). To test G120K’s ability to antagonize GH signaling, cells were treated with a mixture of G120K and GH at either 1:1 or 3:1 molar ratios. GH signaling in WT GHR-expressing cells was inhibited by G120K in a dose-dependent manner, as we have previously observed (41, 44). Notably, unlike anti-GHRext-mAb, G120K inhibited GH-induced JAK2 activation in cells that expressed each of the GHR-LDLR TMD chimeras equally well as in cells expressing WT GHR (Fig. 5DGo, lanes 4 and 5 vs. 2). This similarity of G120K inhibition indicates that G120K binds well to all three GHR molecules and further underscores that the mechanism(s) of inhibition differ between G120K and anti-GHRext-mAb. In addition, these data suggest that the effects of TMD replacement and/or shortening on GHR ECD conformation may be restricted to the region including subdomain 2 rather than to the regions involved in GH (and G120K) binding, which comprise mainly subdomain 1 and the interdomain hinge region (9). This issue is worthy of further study, because it may lead to better understanding of the natural mechanisms of GHR activation.

Inducible Proteolysis Is Impaired for GHR-TMD Chimeras
We previously reported that the GHR is subject to inducible metalloproteolysis in various cell lines in response to treatment with a protein kinase C activator [the phorbol ester, phorbol 12-methyl 13-acetate (PMA)], platelet-derived growth factor, or serum that results in loss of the full-length receptor, appearance of a cell-associated TMD-ICD-containing fragment (the remnant), and release of a soluble GHR ECD (26, 28, 31, 32, 42, 45, 46, 47, 48). The cleavage of the GHR is catalyzed by TNF{alpha}-converting enzyme and occurs in the receptor’s ECD stem region eight or nine residues (depending on species) outside of the TMD (32, 46, 48). Interestingly, deletion of three ECD residues surrounding the cleavage site (GHR{Delta}237–239) prevents inducible GHR cleavage, but replacement of these same three residues with alanines has no effect (32), strongly suggesting that TNF{alpha}-converting enzyme recognizes structural cues (e.g. distance from the TMD, length of the stem region, closeness to the globular subdomain 1 region, etc.) rather than sequence-specific cues.

These observations prompted us to test the effect of alterations of the TMD in the GHR-LDLR TMD chimeras on inducible proteolysis of the receptors (Fig. 6Go). Serum-starved {gamma}2A-JAK2-GHR, {gamma}2A-JAK2-GHRLDLR, and {gamma}2A-JAK2-GHRLDLR{Delta}4 cells were treated with vehicle or PMA (0.1 µg/ml) for 0–60 min, as indicated. Detergent extracts containing equivalent amounts of protein were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47, which detects both receptor and remnant (Fig. 6AGo). As evidenced by GHR loss and remnant accumulation, WT GHR in {gamma}2A-JAK2-GHR cells underwent rapid and robust PMA-induced proteolysis (upper panel), consistent with previous observations in these cells (28). In contrast, PMA-induced receptor loss was greatly attenuated in both {gamma}2A-JAK2-GHRLDLR and {gamma}2A-JAK2-GHRLDLR{Delta}4 cells (middle and lower panels). Densitometric quantitation of receptor loss in several independent experiments indicated the dramatic differences in inducible proteolysis of the GHR-LDLR TMD chimeras compared with WT GHR (Fig. 6BGo). After 60 min of PMA treatment, approximately half of the chimera receptors remained, whereas very little WT GHR remained after 30 or 60 min.


Figure 6
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  		Fig. 6. Inducible GHR Protelysis Is Impaired in GHR-LDLR TMD Chimeras

A, Serum-starved {gamma}2A-JAK2-GHR, {gamma}2A-JAK2-GHRLDLR, and {gamma}2A-JAK2-GHRLDLR{Delta}4 cells were treated with vehicle or PMA (0.1 µg/ml) for the indicated durations. Detergent extracts were resolved by SDS-PAGE and immunoblotted with anti-GHRcytAL-47. The positions of GHR (bracket) and GHR remnant (arrow) are indicated. B, Densitometric quantitation of three independent experiments as described in panel A. In each experiment, the abundance within each treatment group of mature GHR in cells treated with vehicle was considered 100% and compared with its abundance after PMA stimulation. Data are plotted as the mean ± SE. P < 0.05 at 15, 30, and 60 min comparing {gamma}2A-JAK2-GHRLDLR or {gamma}2A-JAK2-GHRLDLR{Delta}4 with {gamma}2A-JAK2-GHR.

 
These results suggest that although the GHR-LDLR TMD chimera receptors could undergo PMA-induced proteolysis, the efficiency of the cleavage was dramatically lower than that of the WT GHR. We note that in the ECD (which contains the cleavage site) the mutant receptors possess the identical amino acid sequence as the WT receptor. Thus, the difference in the proteolysis may be explained by difference in the ECD conformation, in this case likely involving the juxtamembrane stem region, that is induced by alterations in the TMD. We do not yet know whether this altered conformation in the region of GHR proteolysis is attributable to changes in the orientation of the stem with regard to the plane of the membrane, an effective shortening of the stem region (akin to that which might explain the lack of cleavage of the GHR{Delta}237–239 mutant mentioned above), or other reasons. This will be a useful issue to pursue in future studies. Together with the observations in Fig. 5Go, A and B, these data collectively suggest that, despite the unchanged ECD sequence, both the GHRLDLR and GHRLDLR{Delta}4 adopt a subtly different conformation compared with the WT GHR that is uncovered by using the inhibition of signaling by anti-GHRext-mAb and the susceptibility to inducible metalloprotease cleavage as probes of ECD conformation. It will be important in future studies to further define whether these conformational differences arise from substitution of the native TMD vs. shortening of the TMD.

Conclusions
In this work, we studied the effects of replacement of the GHR TMD with a different TMD from the LDLR on the propensity of the GHR to dimerize independently of ligand stimulation and on the signaling and proteolysis susceptibility characteristics of the GHR. This analysis revealed several important findings, which are summarized schematically in Fig. 7Go. We found that predimerization of the receptor with an otherwise normal, but truncated GHR proceeded normally with either GHR-LDLR TMD chimera, indicating that the GHR TMD is not necessary for stable dimerization to be detected. Interestingly, mutation of an ECD residue (H150) in the dimerization interface that is critical for GH signaling dramatically lessened receptor predimerization, indicating that the integrity of this ECD region may be crucial to allowing predimerization as well as signaling. Further, although both GHR-LDLR TMD chimeras exhibited normal GH dose responses and time courses for activation of signaling, both were less sensitive to inhibition of signaling by the conformation-sensitive monoclonal antibody, anti-GHRext-mAb, and were relatively insensitive to inducible metalloprotease cleavage, suggesting that changes in the TMD have subtle effects on ECD conformation. These data and the future experiments they suggest will allow the development of more complete models of the determinants of GHR dimerization, signaling, and proteolysis.


Figure 7
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  		Fig. 7. Schematic Comparison of the Findings Derived with GHR and GHRLDLR TMD Chimeras

GHR TMD and LDLR TMD are represented by black and white blocks, respectively. Dimerization interface that harbors H150 and T147 and the GH-binding interface are indicated. As explained in the text, the data suggest the structure of the ECD, especially the membrane proximal part, is altered in GHRLDLR compared with GHR, and such change does not affect the receptor signaling.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Recombinant human GH was kindly provided by Eli Lilly & Co. (Indianapolis, IN). Recombinant hGH-G120K was kindly provided by Sensus Corp. (Austin, TX). The phorbol ester, PMA, and routine reagents were purchased from Sigma Aldrich Corp. (St. Louis, MO) unless otherwise noted. Zeocin and hygromycin B were purchased from Invitrogen Life Technologies, Inc. (Carlsbad, CA). G418 was from Mediatech (Herndon, VA). Fetal bovine serum, gentamicin sulfate, penicillin, and streptomycin were purchased from BioFluids (Rockville, MD).

Antibodies
The rabbit polyclonal antiserum, anti-GHRcytAL-47, was raised against a bacterially expressed N-terminally histidine-tagged fusion protein incorporating human GHR residues 271–620 (the entire cytoplasmic domain) and has been previously described (31). Anti-JAK2AL33 (directed at residues 746-1129 of murine JAK2) polyclonal serum has been described elsewhere (49). Anti-GHRcyt-mAb is a mouse monoclonal antibody directed against a bacterially expressed glutathione-S-transferase fusion protein incorporating human GHR residues 271–620 (41). Anti-GHRext-mAb is a mouse monoclonal antibody directed against a bacterially expressed glutathione-S-transferase fusion protein incorporating rabbit GHR residues 1–246; its generation and purification have been described previously (24, 41, 42, 50). Monoclonal anti-STAT5 (W-17) and monoclonal anti-Myc (9E10) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antiphospho-STAT5 affinity-purified rabbit polyclonal antibody [recognizing the tyrosine-phosphorylated form (Tyr-694) of STAT5a and STAT5b] was purchased from Zymed Laboratories Inc. (South San Francisco, CA). Polyclonal antiphospho-JAK2 (recognizing phosphorylated Tyr-1007 and Tyr-1008) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Monoclonal anti-HA(12CA5) was purchased from Roche (Indianapolis, IN). Polyclonal hLDLR antibody was a gift from Dr. Alan Attie (University of Wisconsin, Madison, WI). Horseradish peroxidase-conjugated antirabbit (1:15,000) or antimouse (1:15,000) were from Pierce Chemical Co. (Rockford, IL).

Plasmid Construction
The rabbit GHR cDNA (51) was a kind gift of Dr. W. Wood, Genentech, Inc. (South San Francisco, CA). The hLDLR expression vector was a gift of Dr. Alan Attie. Construction of the pcDNA-rbGHR1–274-Myc-His has been described elsewhere (32). cDNA expression vectors encoding the GHR TMD mutants, GHRLDLR and GHRLDLR{Delta}4, were constructed using the ExSite (Stratagene, La Jolla, CA) PCR-based site-directed mutagenesis method and the pSX-rbGHR (50) as the template. Sequences for the mutagenic oligonucleotides are available upon request. The deletion mutation of GHRLDLR{Delta}4 removed in-frame four contiguous amino acids in the middle of the TMD of LDLR (see diagram in Fig. 1AGo). This mutant was isolated adventitiously in the process of construction of GHRLDLR. The entire protein coding sequence of each selected mutant cDNA was subjected to dideoxy DNA sequencing (University of Alabama at Birmingham Genetics Core Facility), which verified the presence of the desired mutations and the absence of unwanted mutations.

Cells, Cell Culture, and Transfection
Both transient and stable transfections were performed using Lipofectamine Plus (Invitrogen Life Technologies, Inc.) according to the manufacturer’s instructions. {gamma}2A is a JAK2-deficient human fibrosarcoma cell line (52) kindly provided by Dr. G. Stark (Cleveland Clinic Foundation, Cleveland, OH). A stable {gamma}2A cell line expressing rabbit GHR and mouse JAK2 ({gamma}2A-JAK2-GHR, previously referred to as C14) was achieved by stable transfection of {gamma}2A-GHR with murine JAK2 as previously described (25) and was maintained in DMEM (1 g/liter glucose) (Mediatech) supplemented with 10% fetal bovine serum, 50 µg/ml gentamicin sulfate, 100 U/ml penicillin, 100 µg/ml streptomycin, 200 µg/ml G418, 100 µg/ml hygromycin B, and 100 µg/ml zeocin. A stable {gamma}2A cell line expressing murine JAK2 ({gamma}2A-JAK2) was achieved by introducing pcDNA3.1+-JAK2-zeocin into cells as described elsewhere (35) and was maintained in the medium for {gamma}2A-JAK2-GHR, except for the hygromycin B. Stable transfection of GHRLDLR, GHRLDLR{Delta}4, or GHR1–274-Myc-His was achieved by cointroducing pSX-GHRLDLR, pSXGHRLDLR{Delta}4, or pcDNA-rbGHR1–274-Myc-His and pSX-hygromycin-HA, into {gamma}2AJAK2 cells. Each cell line was selected in DMEM growth medium supplemented with G418, zeocin, and 200 µg/ml hygromycin. Mutant GHR expression was screened by blotting with anti-GHRcytAL-47 (for {gamma}2A-JAK2-GHRLDLR and {gamma}2A-JAK2-GHRLDLR{Delta}4) or anti-Myc (for {gamma}2A-JAK2-GHR1–274-Myc-His).

Cell Stimulation, Protein Extraction, Immunoprecipitation, Electrophoresis, and Immunoblotting
Serum starvation of all cell lines was accomplished by substitution of 0.25% (wt/vol) BSA (fraction V; Roche) for serum in their respective culture media for 16 h before experiments. All the stimulations were carried out at 37 C 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] unless noted otherwise. Stimulations were terminated by washing cells twice with ice-cold PBS in the presence of 0.4 mM sodium orthovanadate (PBS-vanadate). Cells were solubilized in lysis buffer [150 mM NaCl, 10% (vol/vol) glycerol, 50 mM Tris-HCl (pH 7.4), 50 mM sodium fluoride, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium orthovanadate, 10 mM 1,10-phenanthroline, 1 µg/ml leupeptin, and 10 µg/ml aprotinin] with 1% (wt/vol) Triton X-100 (0.5% Triton X-100 for immunoprecipitation) on ice for 20 min. The detergent extracts were collected by spinning the lysate at 20,000 x g for 15 min at 4 C. In immunoprecipitation experiments, the extracts were incubated with the indicated monoclonal antibody overnight at 4 C on rolling rack and with protein G-sepharose for 2 h. The protein G-sepharose was then collected by centrifugation, washed with the lysis buffer twice and then with PBS twice. Addition of 2x Laemmli SDS-PAGE sample buffer to the sepharose was followed by boiling samples at 95 C for 5 min.

Extracted proteins or eluates from immunoprecipitation were then resolved by SDS-PAGE and immunoblotted as previously described (53). Immunoblotting detection reagents (SuperSignal West Pico chemiluminescent substrate) were from Pierce Chemical Co. Stripping and reprobing of blots was accomplished according to the manufacturer’s suggestions.

Densitometric Analysis
Densitometric quantitation of immunoblots was performed using a high-resolution scanner and the ImageJ 1.30 program [developed by W. S. Rasband, Research Services Branch, National Institute of Mental Health, National Institutes of Health (NIH), Bethesda, MD]. Pooled data from several experiments are displayed as mean ± SE. The significance (P value) of differences of pooled results was estimated by t tests.


   ACKNOWLEDGMENTS
 
We thank Drs. Y. Huang, K. He, K. Loesch, L. Deng, J. Cowan, X. Li, R. Black, T. Clemens, J. Collawn, J. L. Messina, R. Serra, and V. Mishra for helpful conversations.


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

Parts of this work were presented at the 88th Annual Meeting of The Endocrine Society in Boston, June 2006.

Disclosure Summary: N.Y., X.W., J.J., and S.J.F. have nothing to declare.

First Published Online April 24, 2007

Abbreviations: ECD, Extracellular domain; EpoR, erythropoietin receptor; GHR, GH receptor; HA, hemagglutinin; ICD, intracellular domain; JAK2, Janus kinase 2; LDLR, low-density lipoprotein receptor; PMA, phorbol 12-methyl 13-acetate; STAT, signal transducer and activator of transcription; TMD, transmembrane domain; WT, wild type.

Received for publication November 1, 2006. Accepted for publication April 18, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
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