©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regions of the JAK2 Tyrosine Kinase Required for Coupling to the Growth Hormone Receptor (*)

Stuart J. Frank (1) (3)(§), Woelsung Yi (1) (3), Yanming Zhao (2), Jeffrey F. Goldsmith (1) (3), Gretchen Gilliland (1), Jing Jiang (1), Ikuya Sakai (2), Andrew S. Kraft (2)

From the (1)Department of Medicine, Divisions of Endocrinology and Metabolism and (2)Hematology and Oncology, the (3)Department of Cell Biology, University of Alabama at Birmingham and theVeterans Administration Medical Center, Birmingham, Alabama 35294

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Growth hormone (GH) treatment of cells promotes activation of JAK2, a GH receptor (GHR)-associated tyrosine kinase. We now explore JAK2 regions required for GHR-induced signaling. Wild-type (WT) JAK2 and JAK2 molecules with deletions of the amino terminus (JAK2), carboxyl terminus (JAK2), or kinase-like domain (JAK2) were each transiently coexpressed in COS-7 cells with the rabbit GHR. The following responses were assayed: GH-induced transactivation of a luciferase reporter governed by a c-fos enhancer element; GH-induced shift in the molecular mass of a cotransfected epitope-tagged extracellular signal-regulated kinase molecule; and GH-induced antiphosphotyrosine immunoprecipitability of the transfected JAK2 form. In each assay, WTJAK2 and JAK2 allowed GH-induced signaling, whereas JAK2 and JAK2 did not. Anti-GHR serum coimmunoprecipitated WTJAK2, JAK2, and JAK2, but not JAK2. Finally, a chimera in which the JAK2 kinase domain replaced the GHR cytoplasmic domain signaled GH-induced transactivation. We conclude: 1) kinase-like domain deletion eliminates neither physical nor functional interaction between JAK2 and the GHR; 2) kinase domain deletion eliminates functional but not physical coupling of JAK2 to the GHR; 3) interaction with the GHR appears dependent on the NH-terminal one-fifth of JAK2; and 4) a GH-responsive signaling unit can include as little as the GHR external and transmembrane domains and the JAK2 kinase domain.


INTRODUCTION

Growth hormone (GH)()action is initiated by specific interaction of the polypeptide hormone with a cell surface glycoprotein, the GH receptor (GHR). A very early and apparently obligate step in GHR signal transduction is the activation of the GHR-associated cytoplasmic tyrosine kinase, JAK2(1) . In this respect, the GHR is similar to other members of the hematopoietin receptor family (such as receptors for prolactin, erythropoietin, interleukin-3, granulocyte macrophage-colony-stimulating factor, interleukin-6, leukemia inhibitory factor, oncostatin M, cillary neurotrophic factor, and interferon )(2, 3, 4, 5, 6, 7, 8, 9, 10, 11) . These receptors also use JAK2 to facilitate ligand-induced signaling. Still other members of this receptor family couple to the related JAK-family kinases TYK2, JAK1, and JAK3(12, 13, 14, 15, 16) .

Recent evidence indicates that association of JAK2 with the GHR is dependent on a perimembranous proline-rich region of the receptor cytoplasmic domain(17, 18, 19) . Further, removal of this region precludes GH-induced JAK2 and GHR tyrosine phosphorylation, mitogen activated protein kinase activation, and gene transcription(17, 18, 19, 20) . These data underscore the necessity for the proper physical association between the GHR and JAK2 to potentiate their GH-induced functional coupling.

In this study, we explore by mutagenesis the regions of JAK2 which are required for physical and functional interaction with the GHR. JAK2 is a protein of 1129 amino acids(6) . Like the other JAK family members (12-15) it is structurally characterized by the presence of: (a) a consensus kinase domain in the residue 849-1122 region; (b) a potential consensus motif for tyrosine autophosphorylation (-EYYKVKE-) at residues 1006-1012; (c) a kinase-like (or ``pseudokinase'') domain of unclear significance extending from residues 543 to 806; and (d) an amino-terminal half of the molecule with regions of similarity among JAK family members, including a potential tyrosine phosphorylation site (-IDGYYRL-) at residues 369-375. No clear SH2 or SH3 domains are found.

We used a COS-7 cell-based transient cotransfection system to test mutated JAK2 molecules in assays of hGH-induced transcriptional activation, extracellular signal-regulated kinase (ERK) SDS-PAGE mobility shift, JAK2 tyrosine phosphorylation, and GHR-JAK2 coimmunoprecipitation. Our findings indicate that internal deletion of the kinase-like domain does not prevent physical or functional coupling of JAK2 with the rabbit (r) GHR. A deletion of the JAK2 COOH terminus that disrupts the kinase domain prevents these aspects of hGH signaling, but does not inhibit JAK2 association with the rGHR. Amino-terminal truncation of one-fifth of the JAK2 molecule abrogates both physical and functional coupling to the rGHR.

Further, expression and testing of rGHR/JAK2 chimeras indicates that direct linkage of the COOH-terminal half of JAK2 (including the kinase-like and kinase domain) or the COOH-terminal one-third of JAK2 (including only the kinase domain) to the portion of the GHR including the external and transmembrane domains yields a receptor that can confer a GH-dependent transcriptional activation. Inclusion of the kinase-like domain only in such a chimera results in a receptor unable to transduce this signal. These results complement the JAK2 deletional analysis and support the conclusion that a role of the amino-terminal region of JAK2 is to facilitate the native interaction with the GHR.


EXPERIMENTAL PROCEDURES

Materials

Recombinant hGH was kindly provided by Eli Lilly Co. Routine reagents were purchased from Sigma unless otherwise noted. Restriction endonucleases were obtained from New England Biolabs (Beverly, MA). Oligonucleotides were synthesized and purified in the facility of Dr. J. Engler.

Cells and Cell Culture

COS-7 cells were maintained as described previously(19) . CV-1 cells were maintained in Dulbecco's modified Eagle's Medium (DMEM) (Biofluids, Rockville, MD) supplemented with 10% newborn calf serum (Biofluids) and 50 µg/ml gentamicin sulfate, 100 units/ml penicillin, and 100 µg/ml streptomycin (all Biofluids).

Plasmid Construction

The murine JAK2 cDNA was provided by Dr. J. Ihle (St. Jude Children's Research Hospital, Memphis, TN) and was ligated into the pRc/CMV expression plasmid (InVitrogen) as described previously(19) . Generation of the mutant JAK2 cDNAs in pRc/CMV is described in detail elsewhere.()In brief, the cDNA for JAK2 was created in pBluescript by replacing the region coding for residues 1-294 (using an endogenous EcoRI site at residue 294) with a PCR product encoding residues 240-294 with a restriction site and an ATG start codon at the 5` end. Similarly, the cDNA for JAK2 was created by replacing the region encoding residues 932 (an NdeI site) to 1129 with a PCR product encoding residues 932-999 with a termination codon on the 3` end. The cDNA for JAK2 was created by removal of the residue 523-746 region at two in-frame BglII sites in the cDNA and religation. All the cDNAs were subcloned into pRc/CMV, as previously described(19) .

The p2FTL fos-luciferase reporter plasmid (a gift of Dr. J. Kudlow, UAB, and Dr. W. Chen, Massachusetts Institute of Technology.) has been described(21) . In brief, from 5` to 3` it contains two copies of the c-fos 5`-regulated enhancer element (-357 to -276), a fragment of the herpes simplex virus thymidine kinase gene promoter (-200 to +70), and the gene for firefly luciferase. Each copy of the c-fos enhancer element contains the sis-inducible element, the serum response element, and an AP-1-binding site(22) .

pRc/CMV ERK2 was constructed by ligating a PCR fragment of the entire rat ERK2 cDNA (kindly provided by Dr. G. Yancoupoulos, Regeneron, Inc.) coding region by a 5` NcoI site and a 3` ClaI site into the pBluescript SK tag vector (a gift of Dr J. Woodgett, Ontario Cancer Institute). The ERK2 coding region with the 5` tag sequence (-YPYDVPDYASLGGP- of the influenza hemagglutinin) was removed from the pBluescript SK and subcloned via NotI and ApaI sites into the pRcCMV eukaryotic expression plasmid. The ERK2 and the 5` tag coding region sequences were confirmed by dideoxy DNA sequencing.

The creation of the pRc/CMV WTrGHR and pRc/CMV rGHR plasmids has been described(19) . To construct the rGHR/JAK2 and rGHR/JAK2 chimeras, we used the pBluescript mJAK2 (referred to above) as a template for PCR of the regions encoding residues 525-778 (called PK because it encodes the kinase-like or pseudokinase domain) and residues 753-1129 (referred to as K because it encodes the kinase domain). In each case restriction sites for BamHI (5`) and HindIII (3`) were included on the primers for PCR. After restriction digestion, the PK and K BamHI-HindIII fragments were ligated into the previously described (19) pBluescript rGHR plasmid at the BglII and HindIII sites. This yielded in each case cDNAs in pBluescript encoding the rGHR devoid of residues 278 to 292 (which harbor the Box1 element) directly fused in-frame to regions of JAK2 containing either the kinase-like domain or the kinase domain.

Creation of the chimera incorporating both the kinase-like and kinase domains rGHR/JAK2 was accomplished by removal via BglII and HindIII of the region encoding residues 746-1129 from pBluescript mJAK2 and its ligation into the BglII-HindIII digested pBluescript rGHR/JAK2. The rGHR/JAK2 cDNA thus encodes the severely truncated and Box1-deleted rGHR fused to JAK2 residues 525-1129 (the COOH-terminal 53% of the JAK2 molecule). Correct PCR and cloning were verified by dideoxy DNA sequencing of relevant regions, and each cDNA was subcloned into pRc/CMV, as described(19) , for eukaryotic expression. Notably, each of the three rGHR/JAK2 chimera cDNAs encodes the JAK2 residue 758-776 region and would thus be predicted to have reactivity with the anti-JAK2 peptide serum (see below) raised against that sequence.

Protein Expression

COS-7 cells were transfected at 60-80% confluence in 10 ml of DMEM medium in 100 20-mm tissue culture dishes (Becton-Dickinson) by the calcium phosphate precipitation method, as described previously(19, 23) . For individual experiments, the number of dishes transfected per condition is indicated in the figure legend. For experiments comparing the mutant and WT mJAK2, each dish was transfected with 30 µg of wild type or mutant rGHR-containing pRC/CMV along either 9 µg of the WT mJAK2, 36 µg of JAK2, 30 µg of JAK2, or 9 µg of JAK2 cDNA in pRc/CMV. In all cases, the total amount of DNA transfected was equalized by addition of pRc/CMV. The rGHR/JAK2 chimeras in pRc/CMV were transfected at 30 µg/dish. When used for cotransfection, p2FTL was added at 7 µg/dish and pRc/CMV ERK2 at 10 µg/dish.

For overexpression of mutant and WT mJAK2, 8 10 CV-1 cells/100 20-mm dish were infected with vvT7 pol vaccinia virus (24, 25) (kindly provided by Dr. M. Mulligan, UAB) at multiplicity of infections = 5 in 2 ml of DMEM without serum at 37 °C for 1 h. After infection, the cells were incubated in serum-containing medium for 2 h and then transfected with the above-mentioned JAK2 cDNAs in pRc/CMV using Lipofectin (Life Technologies, Inc.) at a ratio of 3-7 µg of plasmid plus 20-40 µl of Lipofectin reagent/dish for 4 h. Twenty-four h after transfection, the cells were harvested and solubilized as below.

c-fos-Luciferase Transactivation Assay

COS-7 cells (8 10/dish) were transfected as described above along with p2FTL. At 18 h after transfection, the cells were split into 24-well plates (approximately 80,000 cells/well). Serum starvation (substitution of 0.5% bovine serum albumin for fetal calf serum in the medium) was begun at 24 h after transfection and hGH (at 500 ng/ml or the dose indicated) or vehicle was added for the durations indicated, such that all stimulations (done in triplicate) were terminated simultaneously. Stimulations were ended by aspiration of the medium and addition of luciferase lysis buffer (1% Triton X-100, 10% glycerol, 2 mM dithiothreitol, 2 mMtrans-1,2-diaminocyclohexane-N,N,N`,N`-tetraacetic acid, 25 mM Tris-HCl, pH 7.8). Luciferase activity was assayed by mixture of equal volumes of the resulting cell extract and luciferase assay buffer (470 mM luciferin and 270 mM CoA) followed by counting for 10 s in a luminometer (Analytical Luminescence Laboratory). Results are plotted as the hGH-induced increase (mean ± 1 S.D.) relative to unstimulated (vehicle only) controls. The control values at each point are considered 100%.

Cell Stimulation, Protein Extraction, and GST Fusion Binding Assays

Serum starvation of COS-7 cells, usually at 24 h after transfection, was accomplished by substitution of 0.5% bovine serum albumin for fetal calf serum in the DMEM cuture medium for another 16-20 h. The stimulation protocol has been described(19) . Unless otherwise noted, hGH was used at a final concentration of 500 ng/ml for 10 min at 37 °C. Cells were washed once with ice-cold phosphate-buffered saline containing 0.4 mM sodium orthovanadate (PBS-vanadate) and then harvested by scraping in PBS-vanadate. After brief centrifugation, pelleted cells were then solubilized for 15 min at 4 °C in either RIPA lysis buffer (1% Nonidet P-40, 0.1% (w/v) SDS, 0.1% (w/v) sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 2 mM EDTA, 1 mM sodium orthovanadate, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin (Boehringer Mannheim)) or WLB buffer (1% Triton X-100, 137 mM NaCl, 20 mM Tris-HCl (pH 8.0), 10% (v/v) glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin) as indicated. After centrifugation at 15,000 g for 15 min at 4 °C, the detergent extracts were either immunoprecipitated or electrophoresed as below. CV-1 cells were harvested similarly and solubilized in either RIPA or fusion lysis buffer (1% Triton X-100, 150 mM NaCl, 10% glycerol, 50 mM Tris-HCl (pH 8.0), 100 mM NaF, 2 mM EDTA, 1 mM PMSF, 1 mM sodium orthovanadate, 10 µg/ml aprotinin). After centrifugation, these extracts were processed as below.

For assays of binding of mutant and WT mJAK2 to GST fusions, fusion protein induction and affinity purification on glutathione-Sepharose beads (Pharmacia) were as described previously(19) . Bacterial extract containing roughly 20 µg of the GST/hGHR fusion (which includes the entire hGHR cytoplasmic domain) bound to glutathione beads was incubated with fusion lysis buffer extract from CV-1 cells expressing either WT mJAK2 or JAK2 such that expression of the two proteins was roughly normalized. After incubation for 90 min at 4 °C, the beads were then washed five times with fusion lysis buffer and eluted in Laemmli sample buffer. 10% of the eluate was resolved by SDS-PAGE on a 9.5% gel and immunoblotted with anti-GST. The remainder was resolved on an 8% gel and immunoblotted with anti-JAK2 peptide antiserum. Equal fractions of the extracts that were subjected to bead binding were also electrophoresed and anti-JAK2 peptide antiserum immunoblotted to indicate the presence of similar amounts of WT mJAK2 and JAK2 in the extracts.

Immunopreciptiation, Electrophoresis, and Immunoblotting

The anti-JAK2 peptide antiserum (Upstate Biotechnology, Inc., Lake Placid, NY), PY20 monoclonal antiphosphotyrosine (APT) antibody (Transduction Laboratories, Lexington, KY), and 12CA5 monoclonal anti-influenza hemagglutinin (HA) peptide antibody (Berkeley Antibodies Co., Richmond, CA) were all purchased commercially. Anti-JAK2 peptide antiserum is directed at residues 758-776 of murine JAK2(6) . APT immunoprecipitation of COS-7 cell RIPA detergent extracts was performed with PY20 at 12 µg of antibody/immunoprecipitation as described previously(19, 26) . Aliquots of CV-1 cell RIPA extracts roughly normalized for similar expression levels of overexpressed mutant and WT mJAK2 proteins were precipitated similarly with PY20 to detect APT precipitability of these molecules, which were then immunoblotted with the anti-JAK2 peptide antiserum as below.

The anti-JAK2 used for immunoprecipitation experiments has been described. In brief, this antiserum was raised in rabbits against a gel-purified GST fusion protein incorporating residues 746-1129 of the murine JAK2. It was used at 18 µl/precipitation of RIPA extracts (0.5-0.7 ml total volume) of either COS-7 or CV-1 cells. Protein A-Sepharose (Pharmacia) was used to adsorb immune complexes and, after extensive washing with RIPA buffer, Laemmli sample buffer eluates were resolved by SDS-PAGE and immunoblotted with anti-JAK2 peptide antiserum.

The anti-hGHR serum used for immunoprecipitation has been previously described(26) . For coimmunoprecipitation experiments, hGH-stimulated (300 ng/ml for 5 min at room temperature) COS-7 cells expressing the rGHR and either mutant or WT mJAK2 were solubilized at approximately 1 10/ml in WLB buffer in the presence of 1 mM 3,3`-dithiobis(sulfosuccinimidyl propionate) (Pierce), a thiol-cleavable homobifunctional cross-linker, for 15 min on ice. After quenching of cross-linking with 10 mM ammonium acetate for 5 min and centrifugation at 15,000 g for 15 min, 80% of each detergent extract was immunoprecipitated with anti-hGHR or nonimmune rabbit serum (40 µl/sample) in the presence of 0.1% SDS and 0.1% sodium deoxycholate. For normalization purposes, 20% of each extract was immunoprecipitated with anti-JAK2, as above. Eluates in Laemmli sample buffer were resolved by SDS-PAGE under reducing conditions and immunoblotted with anti-JAK2 peptide antiserum.

Resolution of proteins under reduced conditions by SDS-PAGE, Western transfer of proteins, and blocking of nitrocellulose membranes (Schleicher and Schuell) with 2% bovine serum albumin were performed as described previously(19, 26) . For immunoblotting, anti-JAK2 peptide antiserum was used at 1:1000, and detection was accomplished using biotinylated protein A (Amersham Corp.) and a streptavidin alkaline phosphatase conjugate (Bethesda Reseach Laboratories) along with the BCIP/NBT detection system (Promega, Madison, WI), according to manufacturers' instructions. Anti-HA 12CA5 was used at 1:750 for immunoblotting and was detected with a goat anti-mouse immunoglobulin G (Fab`) alkaline phosphatase conjugate (Pierce), as described previously(19, 26) .

I-hGH Cross-linking Studies in rGHR/JAK2 Chimera-transfected COS-7 Cells

Competitive I-hGH (NEN DuPont, specific activity 85-130 µCi/µg) cross-linking experiments were performed on COS-7 cells transfected with 30 µg of the cDNAs for either WTrGHR or each of the three rGHR/JAK2 chimeras in the pRc/CMV expression plasmid according to procedures previously described(19) . In brief, transfected cells were incubated with I-hGH (4 ng/ml or approximately 0.18 nM) either in the presence or absence of excess unlabeled hGH (2 µg/ml) at 4 °C for 2 h. This was followed by incubation with 0.5 mM fresh bis-(sulfosuccinimidyl)suberate (BS; Pierce), a noncleaveable water-soluble homobifunctional cross-linker, for 15 min at room temperature. Cross-linking was quenched for 10 min by addition of 10 mM ammonium acetate, and the cells were washed, harvested, and solubilized directly in boiling Laemmli sample buffer. Proteins radioiodinated by virtue of being specifically bound and cross-linked by I-hGH were resolved by SDS-PAGE on 9.5% gels and detected by autoradiography.


RESULTS

Expression of Mutant JAK2 Molecules

To examine regions of JAK2 that are required for GH-induced signaling, we used PCR to generate cDNAs encoding the three deletion mutant murine (m) JAK2 molecules diagrammed with WT mJAK2 in Fig. 1A. The details of the creation of these mutants are described elsewhere. JAK2 lacks residues 2-239 and is thus an amino-terminal deletion. The carboxyl-terminal deletion mutant, JAK2, has a termination codon in place of residue 1000, resulting in deletion of residues 1000-1129, disruption of the tyrosine kinase domain, and removal of the consensus tyrosine autophosphorylation motif (-EYYKVKE-) at residues 1006-1012. JAK2 has in-frame internal deletion of residues 523-746 with removal of a large portion of the kinase-like (or pseudokinase) domain but maintenance of the kinase and consensus tyrosine autophosphorylation motifs.


Figure 1: A, diagram of WT mJAK2 and three JAK2 mutants created to evaluate interaction with the GHR. The positions of the kinase domain (residues 849-1122), the kinase-like (or pseudokinase) domain (residues 543-806), and potential tyrosine phosphorylation sites at the carboxyl-terminal (-EYYKVKE- at residues 1006-1012) and at the amino-terminal one-third (-IDGYYRL- at residues 369-375) are indicated. JAK2 lacks residues 2-239. JAK2 has a termination codon replacing residue 1000 and is thus lacking residues 1000-1129. JAK2 has an in-frame internal deletion of residues 523-746. B, expression of JAK2 mutants in COS-7 cells. The pRc/CMV expression plasmid containing the cDNA for either WT mJAK2, JAK2, JAK2, JAK2, or the vector alone was transfected into COS-7 cells, as under ``Experimental Procedures'' (one 100 15-mm dish/lane). After harvesting and solubilization in RIPA lysis buffer, extracts were immunoprecipitated with the anti-JAK2 serum. Immunoprecipitated proteins were resolved by SDS-PAGE on an 8% acrylamide gel, transferred to nitrocellulose, and immunoblotted with anti-JAK2 peptide antiserum. The positions of migration of each of the expressed JAK2 forms and of molecular mass standards are noted. Also detected more faintly in each precipitate is the endogenous WT monkey JAK2, which comigrates with the transfected WT mJAK2 at an molecular mass of roughly 120 kDa.



Expression plasmids including each cDNA were used to direct synthesis of the WT and mutant JAK2 molecules in COS-7 cells (Fig. 1B). Each protein was detergent soluble and recognized by two different JAK2 antisera. JAK2 and JAK2 species were detected at approximately 100 kDa while JAK2 migrated at roughly 110 kDa, each consistent with its predicted molecular mass. Notably, transfected WT mJAK2 comigrated with the endogenous monkey JAK2 (seen in all samples) at approximately 120 kDa. Considering that only a small fraction of the total cells examined are typically expressing the transfected gene in such a transient assay, we conclude that the level of the transfected JAK2 molecules greatly outweighs that of the endogenous molecule in those cells that are transfected.

Signaling Properties of the Mutant JAK2 Molecules

Our previous work (19) indicated that hGH-induced APT immunoprecipitability of JAK2 in COS-7 cells could be detected if the rGHR was transiently cotransfected with WT mJAK2, but not if the rGHR alone was expressed. This finding, along with the robust relative expression of the mutant JAK2 molecules seen in Fig. 1B, led us to consider use of this system to evaluate signaling and biochemical properties of these JAK2 mutants. The hGH signaling events addressed have been previously shown to be dependent on GHR-JAK2 association.

c-fos-Luciferase Transactivation

Others (27, 28, 29, 30, 31) have demonstrated in various cells types the hGH-dependent acute and transient increase in transcription of the c-fos gene. This effect has been mapped to an upstream region (-361 to -264 base pairs) of the c-fos promoter(32) , a region that includes the serum response element, the sis-inducible element, and an AP-1 binding site(22) . To gauge the ability of our transiently coexpressed rGHR and JAK2 molecules to mediate this gene activation, we used the p2FTL luciferase reporter plasmid. p2FTL includes two tandem copies of the murine c-fos enhancer fragment, the herpes simplex virus thymidine kinase promoter, and the luciferase gene and has been used in studies of epidermal growth factor receptor signaling(21) .

As seen in Fig. 2, A and B, transient transfection of the rGHR, WT mJAK2, and p2FTL into COS-7 cells resulted in an hGH-induced time- and concentration-dependent accumulation of luciferase activity with appreciable increases (greater than 2-fold) seen as early as 4 h into hGH treatment. Maximal stimulation (3-4.6-fold) was routinely achieved with a treatment of 18-24 h with 500 ng/ml hGH. Notably, no hGH-induced augmentation of luciferase activity was detected if the WT mJAK2 expression plasmid was omitted from the transfection (Fig. 2C). Therefore, we conclude that either the expression level/cell of the endogenous COS-7 JAK2 or its particular nature (monkey versus mouse) does not allow sufficient coupling to the transfected rGHR to promote significant hGH-induced activation of transcription. Consistent with our previous data demonstrating a lack of endogenous hGH-binding sites in COS-7 cells(19) , no hGH-induced luciferase activation was detected if the rGHR was omitted from the transfection mix (not shown).


Figure 2: hGH-induced transactivation of a c-fos enhancer-driven luciferase reporter in transiently transfected COS-7 cells. A, time course of transactivation mediated by WT mJAK2. Cells (0.8 million cells/100 15-mm dish) were transfected with pRc/CMV-WTrGHR, pRc/CMV-WT mJAK2, and p2FTL (as described under ``Experimental Procedures''). At 18 h after transfection, the cells were split into 24-well plates (approximately 80,000 cells/well). Under serum-starved conditions (as under ``Experimental Procedures''), hGH (500 ng/ml) or vehicle was added for the durations indicated, such that all stimulations (done in triplicate) were terminated simultaneously. Detergent extraction and luciferase activity assay were as described under ``Experimental Procedures.'' Results are plotted as the hGH-induced increase (mean ± 1 S.D.) relative to unstimulated (vehicle only) controls. The control values at each point are considered 100%. (There was less than 5% deviation among the mean control values at each time point.) B, hGH concentration dependence of transactivation mediated by WT mJAK2. The experiment was the same as that in A except that the dose of hGH used was varied and all incubations lasted 4.5 h at which point luciferase activity was measured. C, hGH-induced transactivation in COS-7 cells expressing JAK2 mutants. Cells transiently expressing the transfected WT rGHR and either the transfected WT mJAK2 (as above in A and B), JAK2, JAK2, JAK2, or no transfected JAK2 (vector) along with p2FTL were serum-starved and stimulated with hGH (500 ng/ml) for 22 h, as in A and B. The resulting hGH-induced luciferase activity (the ratio of hGH-treated to untreated samples ± 1 S.D. of triplicates) is shown. The luciferase activity (in relative light units) of the unstimulated samples for each of the five transfections shown were within 40% of each other (not shown), indicating roughly comparable transfection efficiencies. The experiment shown is representative of seven such experiments. Note that no significant increase in luciferase activity was seen when the transfected rGHR was expressed in the COS-7 cells with JAK2 or JAK2 or in the absence of transfected mJAK2 (vector sample).



We tested the ability of each of the JAK2 mutants to support this hGH-induced gene activation when coexpressed with the rGHR (Fig. 2C). Using transfection conditions that resulted in ample expression of each of the JAK2 molecules (not shown), neither JAK2 nor JAK2 expression mediated significant hGH-induced luciferase accumulation when compared to expression of WT mJAK2. Expression of JAK2, however, resulted in consistently detectable hGH-induced augmentation of luciferase activity, although levels were lower than for WT mJAK2 (1.8- versus 3.4-fold in the experiment shown).

Mobility Shift of Epitope-tagged ERK2

Another GH-induced response that has been identified is the phosphorylation and activation of ERKs, also known as mitogen-activated protein kinases(17, 33, 34, 35) . When phosphorylated, the ERKs characteristically exhibit retarded migration on SDS-PAGE gels(33) . This change in mobility provides a convenient way to monitor their phosphorylation state. We expressed an epitope-tagged (influenza hemagglutinin) rat ERK2 molecule in COS-7 cells along with the rGHR and mJAK2. As seen in Fig. 3(top panel), the appearance of an hGH-induced shift in ERK2 migration was detected by anti-HA immunoblotting when the WT mJAK2 and rGHR were coexpressed, but not when the mJAK2 was omitted from the transfection. Testing of the JAK2 mutants in this assay (Fig. 3, bottom panel) revealed a readily detectable ERK2 mobility shift in response to hGH treatment when the WT mJAK2 (lane 2) or the JAK2 mutant (lane 8) was expressed, but this shift was not seen when either JAK2 or JAK2 were expressed (lanes 4 and 6). These results do not prove that endogenous COS-7 cell ERK(s) is necessarily involved in eliciting the hGH-induced transcriptional activation in the transfected cells. However, the evidence does indicate that the JAK2 structural mutations have qualitatively similar effects on the ability of hGH to activate signaling in both the transcriptional activation and ERK2 mobility shift assays.


Figure 3: hGH-induced shift in molecular mass of epitope-tagged ERK2. Top panel, WT rGHR, along with either WT mJAK2 or vector only and ERK2 or vector only (as indicated), were coexpressed in COS-7 cells, as described under ``Experimental Procedures.'' After serum starvation, cells were treated with hGH (500 ng/ml) (+) or vehicle only (-) for 10 min at 37 °C. Cells were harvested and solubilized in WLB lysis buffer. Detergent extracts (corresponding to one dish of transfected cells/condition) were resolved by SDS-PAGE on a 10% gel, transferred to nitrocellulose, and immunoblotted with the 12CA5 anti-HA monoclonal antibody, as under ``Experimental Procedures.'' Note the hGH-induced appearance of an ERK2 band with retarded migration only in the sample in which the WT mJAK2 was expressed (lane 4). Bottom panel, the experiment was the same as that in the top panel, except that either WT mJAK2, JAK2, JAK2, or JAK2 (as indicated) was coexpressed with the rGHR and ERK2. Note the hGH-induced appearance of the shifted (retarded) ERK2 only in the samples in which the WT mJAK2 or JAK2 was expressed (lanes 2 and 8, respectively). The experiment shown is representative of four such experiments.



JAK2 APT Precipitability

Since the initiating step in multiple aspects of GH signaling appears to be JAK2 tyrosine kinase activation with resultant tyrosine phosphorylation of JAK2 and other cellular proteins, we examined the ability of the WT mJAK2 and the JAK2 mutants to undergo hGH-induced tyrosine phosphorylation. To enhance sensitivity of detection, we relied on our previously characterized ability to detect WT mJAK2 in APT immunoprecipitates of extracts of hGH-treated COS-7 cell transfectants(19) . In each case, cotransfection of cDNAs encoding the rGHR and WT mJAK2 or JAK2 mutant was followed by treatment for 10 min at 37 °C with or without hGH, detergent solubilization, and immunoprecipitation with the PY20 APT monoclonal antibody (75% of the extract) or anti-JAK2 (25% of the extract). Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with anti-JAK2 peptide serum.

The results of comparisons of each JAK2 mutant with WT mJAK2 in this assay are shown in Fig. 4, A-C. In each case, the hGH-induced appearance of APT-precipitable WT mJAK2 is evident as a positive control. Similarly, a robust signal is induced by hGH treatment of transfectants expressing JAK2, although basal signal is also apparent for this mutant (Fig. 4C). In distinction, despite significant levels of expression, neither JAK2 nor JAK2 became detectably APT precipitable in response to hGH treatment (Fig. 4, A and B). We note that while JAK2 has an interrupted kinase domain and lacks the potential consensus tyrosine autophosphorylation site, this region is intact in JAK2. Indeed, as seen in Fig. 4D, JAK2 is immunoprecipitable with the APT antibody when vastly overexpressed in CV-1 cells infected with the vaccinia virus-encoded T polymerase. JAK2, though amply expressed and anti-JAK2 precipitable, is, as predicted, not detectable with the APT antibody in the same assay. As also predicted, WT mJAK2 and JAK2 were APT precipitable when similarly overexpressed (not shown). The inability of JAK2 to become APT precipitable in response to hGH in rGHR-cotransfected COS-7 cells, therefore, appears not to be due to a defect in its potential to function as a tyrosine kinase and/or substrate.


Figure 4: APT precipitability of activated WT mJAK2 and JAK2 mutants. A, comparison of WT mJAK2 and JAK2 transiently expressed in COS-7 cells. The rGHR and either WT mJAK2 or JAK2 (as indicated) were transiently coexpressed in COS-7 cells, as under ``Experimental Procedures.'' After serum starvation and treatment with (+) or without (-) hGH (500 ng/ml) for 10 min at 37 °C, RIPA detergent extracts were immunoprecipitated with the PY20 monoclonal APT antibody (the equivalent of three transfected dishes/condition) (left panel) or the anti-JAK2 antiserum (one dish/condition) (right panel), resolved by SDS-PAGE, and immunoblotted with the anti-JAK2 peptide serum. Note the failure of JAK2 to become detectably APT precipitable in response to hGH treatment, despite ample expression. The experiment shown is representative of three such experiments. B, comparison of WT mJAK2 and JAK2 transiently expressed in COS-7 cells. The experiment was performed as in A, except that JAK2 was compared to WT mJAK2. Note that JAK2 also fails to become detectably APT precipitable in response to hGH treatment. The experiment shown is representative of three such experiments. C, comparison of WT mJAK2 and JAK2 transiently expressed in COS-7 cells. The experiment was the same as in A and B, except that JAK2 was compared to WT mJAK2. Note the easily detectable augmentation of JAK2 APT precipitability in response to hGH treatment. The experiment shown is representative of three such experiments. D, JAK2, but not JAK2, is APT precipitable when overexpressed. JAK2 and JAK2 were each transiently expressed in CV-1 cells that were also infected with the vvT7 pol vaccinia virus, as described under ``Experimental Procedures.'' RIPA extract of unstimulated cells was immunoprecipitated with either PY20 (roughly 0.8 and 0.4 dish equivalents/precipitation for JAK2 and JAK2, respectively) or anti-JAK2 (roughly 0.1 and 0.05 dish equivalents/precipitation for JAK2 and JAK2, respectively) and resolved and anti-JAK2 peptide antiserum immunoblotted, as in A-C. Note that despite evaluation of similar amounts of the two vastly overexpressed proteins, only JAK2 exhibits APT precipitability. The band seen above JAK2 in the APT precipitation and in the cell extract sample of Fig. 5D is an artifact of the immunoblotting detection system sometimes present when large amounts of CV-1 cell extract are utilized and is independent of the particular JAK2 form expressed (not shown).



Physical Association of Mutant JAK2 Molecules and the GHR

To further explore the unresponsiveness of the JAK2 and JAK2 mutants, we examined their ability to associate with the rGHR. Others have shown in murine preadipocytes and in rat GHR Chinese hamster ovary cell transfectants that JAK2 becomes coimmunoprecipitable with the GHR after GH treatment of the cells(1, 17) . Likewise, we have shown that our anti-GHR antibodies can specifically coimmunoprecipitate JAK2 from human IM-9 cells if a cleavable chemical cross-linker is used to stabilize the interaction (19). Though present in the unstimulated state, the amount of JAK2 that becomes anti-GHR coprecipitable from IM-9 cells is also augmented by hGH treatment(19) .

In the experiments shown in Fig. 5, A-C, COS-7 cells transfected with the cDNAs for the rGHR and mJAK2 (either WT or mutant) were treated with hGH, solubilized in a detergent lysis buffer, cross-linked with a thiol-cleavable cross-linker, and immunoprecipitated with anti-GHR (80% of the extract) or anti-JAK2 (20% of the extract). The eluates were resolved by SDS-PAGE under reducing conditions, thereby cleaving the cross-linker, and immunoblotted with anti-JAK2 peptide serum. The results reveal that WT mJAK2 (Fig. 5, A-C), JAK2 (Fig. 5B), and JAK2 (Fig. 5C) were each coimmunoprecipitated with similar efficiency with the anti-GHR antibody. To prove specificity, a duplicate coexpression of rGHR and JAK2 was processed identically, except that nonimmune serum was used instead of anti-GHR for immunoprecipitation (Fig. 5C). Despite ample directly precipitated JAK2 no JAK2 was detected in the nonimmune precipitate. In contrast to the WT mJAK2 and the other JAK2 mutants, JAK2 (Fig. 5A) was not coimmunoprecipitable with anti-GHR, indicating a markedly diminished interaction between this mutant and the rGHR. In the experiment shown in Fig. 5D, this finding was confirmed by the inability of JAK2 extracted from transfected vaccinia virus infected-CV-1 cells to interact in vitro with an immobilized GST fusion protein incorporating the full hGHR cytoplasmic domain. WT mJAK2, similarly overexpressed, does interact with this GST/hGHR fusion, consistent with our previous observations(19) . These findings indicate that the amino-terminal 239 residues of JAK2, absent in JAK2, are required for physical and, therefore, functional interaction with the GHR.


Figure 5: Association of the rGHR with WT mJAK2 and JAK2 mutants. A, comparison of WT mJAK2 and JAK2 transiently expressed in COS-7 cells. The rGHR and either WT mJAK2 or JAK2 (as indicated) were transiently coexpressed in COS-7 cells. After serum starvation and treatment with hGH (300 ng/ml) for 5 min at 25 °C, WLB detergent lysates were exposed to the cross-linking agent 1 mM 3,3`-dithiobis(sulfosuccinimidyl propionate (1 mM), as detailed under ``Experimental Procedures.'' After quenching with 10 mM ammonium acetate, extracts were immunoprecipitated with either anti-GHR (five dishes/precipitation) or anti-JAK2 (one dish/precipitation) in the presence of 0.1% SDS and 0.1% sodium deoxycholate. Eluted proteins were resolved under reducing conditions by SDS-PAGE and immunoblotted with the anti-JAK2 peptide serum. Note the lack of JAK2 coimmunoprecipitable with anti-GHR. The experiment shown is representative of three such experiments. B, comparison of WT mJAK2 and JAK2 transiently expressed in COS-7 cells. The experiment was performed as in A, except that JAK2 was compared to WT mJAK2. Note the coimmunoprecipitability of JAK2 with anti-GHR. The experiment shown is representative of two such experiments. C, comparison of WT mJAK2 and JAK2 transiently expressed in COS-7 cells. The experiment was performed as in A and B, except that JAK2 was compared to WT mJAK2. Note the coimmunoprecipitability of JAK2 with anti-GHR. In addition, when nonimmune serum was used instead of anti-GHR, no detectable JAK2 was observed illustrating the specificity of this coimmunoprecipitation procedure. The experiment shown is representative of two such experiments. D, WT mJAK2, but not JAK2, interacts with a GST fusion protein containing the hGHR cytoplasmic domain. WT mJAK2 and JAK2 were each transiently expressed in CV-1 cells that were also infected with the vvT7 pol vaccinia virus, as described under ``Experimental Procedures'' and in Fig. 4D. Fusion lysis buffer extracts were made of each population of transfected cells. This extract (0.9 dish equivalents for JAK2 and 0.1 dish equivalents for WT mJAK2) was subjected to a binding assay with the glutathione bead-bound fusion protein, GST/hGHR (which contains the entire hGHR cytoplasmic domain), as described under ``Experimental Procedures'' and Ref. 19. Eluates of these precipitates were separated by SDS-PAGE and immunoblotted with anti-JAK2 peptide serum (90% of the eluate) or anti-GST antibodies (10% of the eluate), as indicated. Unfractionated extract (one-tenth of the amount applied to the beads for each sample) was also separated by SDS-PAGE and immunoblotted with anti-JAK2 peptide serum for purposes of normalization. Note that despite evaluation of similar amounts of each JAK2 form and the presence of similar amounts of GST/hGHR in each sample, only WT mJAK2 interacted with the fusion protein. The experiment shown is representative of two such experiments.



Expression of GHR/JAK2 Chimeras

In light of the above findings that the JAK2 amino terminus contains region(s) required for this interaction, we tested whether chimeric proteins incorporating the GHR external and transmembrane domains and various JAK2 regions other than the amino terminus could signal in response to hGH. The chimeras created are diagrammed in Fig. 6A. Each includes rGHR residues 1-277 and 293-296 (i.e. rGHR with deletion of the residue 278-292 region that includes Box1, the proline-rich motif in the GHR that is essential for association with and activation of JAK2(17, 18, 19) ). The chimera rGHR/JAK2 has this rGHR region fused to residues 525-778 of mJAK2, a region inclusive of most of the kinase-like domain, but devoid of the kinase domain. rGHR/JAK2 includes mJAK2 residues 753-1129, the kinase domain, whereas rGHR/JAK2 contains mJAK2 residues 525-1129 (both the kinase-like and kinase domains).


Figure 6: Expression of GHR/JAK2 chimeras in COS-7 cells. A, diagram of the WT rGHR, WT mJAK2, and three rGHR/JAK2 chimeras created. The kinase-like (or pseudokinase) and kinase domains of mJAK2 and the extracellular, transmembrane (horizontal line pattern), intracellular and proline-rich Box1 regions of the rGHR are indicated. The chimeras, rGHR/JAK2, rGHR/JAK2, and rGHR/JAK2, contain the extracellular and transmembrane portions (but not the Box1 region) of the rGHR fused to the indicated regions of mJAK2, as detailed under ``Experimental Procedures'' and ``Results.'' B, I-hGH cross-linking to rGHR and the rGHR/JAK2 chimeras expressed on the surface of COS-7 cells. The rGHR and rGHR/JAK2 chimeras, as indicated, were expressed following transfection, as under ``Experimental Procedures.'' Cross-linking of radiolabeled hGH in the absence (-) or presence (+) of excess unlabeled hGH was accomplished using BS. Total cellular proteins were solubilized in boiling reduced Laemmli sample buffer, resolved by SDS-PAGE, and subjected to autoradiography. C, hGH-induced transactivation in COS-7 cells expressing rGHR/JAK2 chimeras. Cells transiently expressing either the transfected WT rGHR and WT mJAK2 or each of the three transfected rGHR/JAK2 chimeras (as indicated) along with p2FTL were serum starved and stimulated with hGH (500 ng/ml) for 22 h, as in Fig. 2. The resulting hGH-induced luciferase activity (the ratio of hGH-treated to untreated samples ± 1 S.D. of triplicates) is shown. The results of the two experiments shown are representative of eight similar experiments.



We tested the integrity and hGH binding ability of each chimera by performing radiolabeled hGH cross-linking studies (Fig. 6B). COS-7 cells transiently expressing each chimera or the WT rGHR were incubated with I-hGH in the presence or absence of excess unlabeled hGH and then exposed to BS, a noncleavable, membrane-impermeant chemical cross-linker. SDS-PAGE and autoradiography revealed that each chimera specifically bound I-hGH at the cell surface and migrated with its predicted molecular mass. Further, the indistinct nature of the proteins likely reflects their glycosylation, presumably derived from their rGHR component since JAK2 is not believed to be a glycoprotein. Immunoprecipitation and immunoblotting (not shown) revealed that the chimeras each reacted with the anti-JAK2 and/or anti-JAK2 peptide sera in the pattern predicted, considering the JAK2 regions included in the particular chimera. Together, these data verify the structural integrity of the chimeric molecules and their ability to bind hGH at the cell surface.

The rGHR/JAK2 chimeras were tested for their ability to mediate the hGH-induced transcriptional activation of the c-fos-luciferase reporter in COS-7 cells. Two representative experiments are shown in Fig. 6C. Similar to the results in Fig. 2, cotransfection of p2FTL, WT rGHR, and WT mJAK2 enabled a 4.6-fold induction of luciferase activity after 22 h of hGH treatment. Transfectants expressing rGHR/JAK2, the chimera that has only the kinase-like domain of JAK2, reproducibly displayed no increase in luciferase activity in response to hGH. However, inclusion in the chimeras of the JAK2 kinase domain alone (rGHR/JAK2) or in addition to the kinase-like domain (rGHR/JAK2) allowed significant hGH-induced transactivation (1.8-3.0-fold). The signal transduced through these chimeras was, however, consistently less than that seen with cotransfection of the WT rGHR and WT mJAK2. Notably, coexpression of the WT mJAK2 with the previously characterized truncated rGHR, which corresponds to the rGHR portion of the chimeras, yielded no hGH-induced increase in luciferase activity (not shown). This is consistent with its lack of the Box1 motif and its inability to promote hGH-induced APT precipitability of JAK2(19) . Thus, our data indicate that a minimal unit capable of transmitting at least one type of hGH signal consists of the GHR extracellular and transmembrane domains covalently fused to the kinase domain of JAK2.


DISCUSSION

Interactions between signaling molecules (activated receptors, enzymes, docking molecules) are often dependent on the presence in the interacting proteins of SH2 and/or SH3 domains and their tyrosine phosphorylated or proline-rich binding sites(36, 37, 38, 39) . Likely, the hematopoietin receptors will also be shown to utilize such motifs in their associations with some of the proteins involved in their signaling complexes. However, the interaction between these receptors and JAK family kinases appears to be independent of such known motifs or modifications. JAKs have no obvious SH2 or SH3 domains and, though studies of the GHR have indicated a ligand-induced enhancement of receptor association with JAK2(1) , other hematopoietin receptors appear to be constitutively associated with their JAK-family kinase(2, 3, 5, 7, 9) . Indeed, we have observed that the in vitro association of a GST fusion protein containing the hGHR cytoplasmic domain with IM-9 cell-derived JAK2 is not dependent on prior hGH-treatment of those cells or tyrosine phopshorylation of either JAK2 or the hGHR(19) .

The proline-rich perimembranous region of the GHR cytoplasmic domain has been implicated by us and others as important in interaction with JAK2(17, 18, 19) . Though rich in proline residues, this so-called Box1 region does not constitute a canonical SH3-binding site. Box1 has similar counterparts in various hematopoietin receptors, which relate to association with JAKs(5, 7, 8, 40, 41) . In this study, we specifically map JAK2 regions involved in physical and functional interaction of the GHR and JAK2. Using similar approaches, we have also recently examined the JAK2 interaction with the granulocyte macrophage-colony-stimulating factor receptor chain.

Our findings of reconstitution experiments in COS-7 cells support the contention that GH signaling requires proper association of the GHR with JAK2 and enzymatic activation of JAK2 in response to the GH-GHR interaction. Specific results that allow this interpretation include the following. 1) Transient coexpression of WT mJAK2 and the rGHR leads to anti-GHR coprecipitability of JAK2. 2) Detectable hGH-induced biochemical events resulting from such coexpression include JAK2 APT precipitability, retardation of migration in SDS-PAGE (likely corresponding to phosphorylation) of an epitope-tagged ERK2, and transactivation of a luciferase reporter with c-fos enhancer elements. 3) None of the findings in 1 and 2 above occur to a significant degree if the rGHR is transiently expressed without transfected JAK2, presumably relating to the inadequate expression level or interacting capacity of the endogenous COS-7 monkey JAK2.

Reconstitution experiments with amino- and carboxyl-terminal deletion mutants of JAK2 further support these notions. Carboxyl-terminal deletion (JAK2) does not adversely affect physical association of JAK2 with the rGHR, but the lack of hGH induction of the biochemical signals correlates with the disruption of the kinase domain that results from this truncation. The amino-terminal deletion mutant, JAK2, was also unable to couple hGH treatment to any of the activating events despite the integrity of its kinase domain and its demonstrated ability to become APT reactive when vastly overexpressed. In this case, the inability of JAK2 to physically interact with the rGHR most likely underlies its signaling defects.

Since JAK2 lacks the amino-terminal one-fifth of the JAK2 molecule, we infer that this region contains elements necessary for interaction with the GHR. Whether this region is solely sufficient to confer this ability to interact with the GHR is not yet known. Likewise, the particular area(s) within this amino-terminal region which promote this interaction is yet to be determined. This same JAK2 is also unable to associate with or promote tyrosine phosphorylation of the GM-CSF receptor chain in a transient expression system. This indicates a likely similarity in the mechanism of interaction of JAK2 with each receptor. Given the topological similarity of structure among JAK-family members, we might expect that the amino termini of the other JAKs may also be involved in interaction with their cognate receptors. Despite these likely similarities, functional specificity of the receptor-JAK interaction is also evident. We note that coexpression of JAK3 (provided by J. J. O'Shea, NCI), which is 47% identical to JAK2(14, 15) , with the rGHR was unable to mediate hGH-induced fos-luciferase transactivation (not shown).

Interestingly, deletion of the kinase-like domain (JAK2) did not abrogate the ability of the mutant JAK2 to interact with the rGHR or to mediate hGH-induced fos-luciferase transactivation, ERK2 mobilty shift, or APT precipitability of the kinase itself. Although this implies that the kinase-like domain is not essential for transduction of the hGH-induced activating signal, it is conceivable that it subserves a modulatory function that would be better discerned in other signaling assays. We note that JAK2 basal APT precipitability was reproducibly robust (Fig. 4C) and that our studies of the GM-CSF receptor -JAK2 mutant interactions showed a GM-CSF-independent GM-CSF receptor tyrosine phopshorylation in CV-1 cells transiently coexpressing that receptor and JAK2 at very high levels. Although we are not certain, these findings may indicate that the attenuated hGH-induced signal seen with JAK2 relative to WT mJAK2 in the fos-luciferase transactivation assay, for instance, could relate in part to a basal disinhibition of kinase function in the mutant.

The results obtained with the chimeric GHR/JAK2 molecules (Fig. 6) are consistent with the JAK2 deletional analysis. Reasoning that the amino terminus of JAK2 serves as an interacting domain, we eliminated it and directly fused either the kinase-like domain, the kinase domain, or both domains to a severely truncated rGHR. The rGHR component is almost identical to our previously described rGHR, having in addition only residues 276-277 and 293-296 (none of which are included in the Box1 region). We have shown that rGHR, though able to bind hGH normally, does not mediate hGH-induced APT precipitability of cotransfected JAK2(19) . Others have shown that the analogously truncated rat GHR is also incapable of mediating GH-induced mitogen-activated protein kinase activation, gene transcription, proliferation, JAK2 association, substrate tyrosine phosphorylation, and insulin production in various cell types(17, 18, 20, 33, 42) .

Each of our chimeras bound hGH at the cell surface. When assayed for fos-luciferase transactivation capability, the chimeras that incorporate the JAK2 kinase domain (rGHR/JAK2 and rGHR/JAK2) transduced the hGH activating signal. The rGHR/JAK2 chimera served as a negative control in that its lack of elicitation of luciferase activity in response to hGH might be predicted in the context of the findings with JAK2. Interestingly, inclusion of the kinase-like domain may influence the signaling in that there was in general an increased ability of rGHR/JAK2 to transduce hGH-induced transactivation when compared to rGHR/JAK2 (not shown). We note, however, that none of the chimeras signaled as well in this assay as did the WT rGHR coexpressed with the WT mJAK2. We assume this reflects both the absence of potentially critical regions of the GHR cytoplasmic domain and the less than perfect assembly of unnatural chimeric molecules. Further studies using the rGHR/JAK2 molecules may be helpful in discerning the relative impact of the loss of these GHR region(s) on various measures of signal transduction since the chimeras may serve as selective activators of hGH-induced JAK2-mediated pathways.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants R29DK46395 (to S. J. F.) and RO1DK44741 (to A. S. K.) and by National Cancer Institute Grant CA13148 to the UAB Comprehensive Cancer Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Early Career Development Award recipient from the American Federation for Clinical Research. To whom correspondence should be addressed: University of Alabama at Birmingham, Rm. 756, DREB, UAB Station, Birmingham, AL 35294. Tel.: 205-934-9856; Fax: 205-934-4389.

The abbreviations used are: GH, growth hormone; GHR, growth hormone receptor; ERK, extracellular signal-regulated kinase; PAGE, polyacrylamide gel electrophoresis; h, human, r, rabbit; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; WT, wild type; APT, antiphosphotyrosine; BS bis-(sulfosuccinimidyl)suberate.

Zhao, Y., Wagner, F., Frank, S. J., and Kraft, A. S. (1995) J. Biol. Chem.270, 13814-13818


ACKNOWLEDGEMENTS

We gratefully acknowledge Drs. J. Ihle, W. Wood, M. Mulligan, W. Chen, J. Kudlow, J. Woodgett, and G. Yancoupoulos for contribution of plasmids and Eli Lilly Co. for the hGH. We appreciate helpful conversations with Drs. J. Kudlow, A. Paterson, T. Shin, and J. J. O'Shea and critical review of the manuscript by J. Kudlow and E. Chin.

Addendum-While this manuscript was under review, a study of JAK2 regions involved in interaction with the GHR cytoplasmic domain was reported. Those results also indicate importance for the amino-terminal region of JAK2 in that interaction(43) .


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