©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Intracellular Tyrosine Residues of the Human Growth Hormone Receptor Are Not Required for the Signaling of Proliferation or Jak-STAT Activation (*)

(Received for publication, October 28, 1994; and in revised form, January 4, 1995)

Yi-Ding Wang Ka Wong William I. Wood (§)

From the Department of Molecular Biology, Genentech, Inc., South San Francisco, California 94080

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Ligand binding and dimerization of the growth hormone (GH) receptor leads to the rapid tyrosine phosphorylation of the intracellular kinase, Jak2, to the tyrosine phosphorylation and activation of STAT protein(s) and to the tyrosine phosphorylation of the receptor itself. Expression of the human GH receptor in the mouse promyeloid, interleukin-3-dependent cell line, FDC-P1, shows that this receptor can signal ligand-dependent proliferation in these cells as well as induce the tyrosine phosphorylation of Jak2 and the activation of transcription factors. We now examine the requirement for tyrosine phosphorylation of the GH receptor for these three events by expression of a receptor without tyrosine residues in the intracellular domain. Six of the seven intracellular tyrosine residues were removed by a carboxyl-terminal truncation, and the remaining tyrosine was changed to phenylalanine to yield the GH receptor D351Stop/Y314F. When expressed in FDC-P1 cells, this receptor retained its ability to induce the tyrosine phosphorylation of Jak2, to induce the activation of transcription factors, and to signal ligand-dependent cell proliferation. Thus, tyrosine phosphorylation of the GH receptor is not essential for the signaling of these three events at least in this system. This finding contrasts with that for the interferon- receptor system where data indicate that the specific tyrosine phosphorylation of the interferon- receptor leads to an association with the STAT protein, p91, that is the mechanism by which ligand couples the receptor to the signal transduction system.


INTRODUCTION

The GH (^1)receptor is a 130-kDa glycoprotein that transduces the signal for a variety of the biological actions of GH, including the regulation of long bone and soft tissue growth, fat metabolism, and insulin action(1) . The human GH receptor contains an extracellular hormone binding domain of 246 amino acids, a single transmembrane domain of 24 amino acids, and a 350-amino acid cytoplasmic domain(2) . It is a member of a family of proteins that includes the receptors for GH, prolactin, interleukins 2-7 and 11, G-CSF, GM-CSF, erythropoietin, thrombopoietin, ciliary neurotrophic factor, leukemia inhibitory factor, and oncostatin M(3, 4, 5) . Structural, mutational, and functional studies provide strong evidence that dimerization of receptors of this family by a single ligand molecule is the first step in receptor activation that ultimately leads to signal transduction(6, 7, 8) .

Studies of intracellular signaling events induced via the GH receptor (and by several other receptors of the GH/cytokine receptor family) show that tyrosine phosphorylation is an early consequence of ligand binding and receptor dimerization(9) . In various cell lines and in vivo, GH induces the rapid tyrosine phosphorylation of several proteins, including the GH receptor itself, the tyrosine kinase Jak2, the STAT protein p91 (or a related molecule), as well as other proteins(10, 11, 12, 13, 14, 15, 16, 17, 18, 19) . Recently, a direct signaling pathway utilizing tyrosine kinases of the Jak family and STAT proteins has been delineated for the IFN-alpha and IFN- receptors(20, 21, 22) . These experiments and data from the GH system support the hypothesis that Jak2 associates with the GH receptor following ligand binding and receptor dimerization(14) . This leads to activation of the Jak2 kinase which results in tyrosine phosphorylation of Jak2 itself, of the GH receptor, and of STAT proteins. The activated STAT proteins, possibly in a complex with other transcription factors, then migrate to the nucleus and bind DNA elements responsible for the modulation of gene transcription(10) . For the IFN- receptor, mutagenesis and phosphorylated peptide data show that Tyr of the intracellular domain of the alpha chain is essential for signaling(23, 24) . These data also show that the STAT protein, p91, specifically interacts with the phosphorylated receptor, indicating that this inducible association is the mechanism by which the ligand couples the receptor to the signal transduction system(24) .

Expression of the human GH receptor in the promyeloid cell line, FDC-P1, or in the pro-B cell line, Ba/F3, which both normally require IL-3 for maintenance and growth, leads to lines that proliferate in response to GH(25, 26) . We have used this system to demonstrate that GH receptor truncations containing as few as 54 amino acids of the cytoplasmic domain will signal a proliferative response upon ligand binding(27) . This proliferative response is preceded in these systems by the tyrosine phosphorylation of Jak2 and the activation of transcription factors(25) . Mutation of amino acids in the remaining 54-residue signaling region shows that there is a correlation of Jak2 phosphorylation, transcription factor activation, and cell proliferation, a finding that is consistent with the hypothesis that the Jak-STAT signaling pathway is required for the signaling of proliferation in this system(25) .

In the current work we examine the requirement for tyrosine phosphorylation of the GH receptor for the signaling of proliferation and for the activation of the Jak-STAT pathway. We find that the human GH receptor without tyrosine residues in the intracellular domain is capable of signaling the proliferation of FDC-P1 cells and that Jak2 tyrosine phosphorylation and transcription factor activation appear unaffected.


MATERIALS AND METHODS

GH Receptor Mutagenesis

DNA sequences encoding the full-length and D351Stop human GH receptor in pRKN2 expression vector were constructed previously(27) . The Y314F mutation shown in Fig. 1was made by in vitro mutagenesis as described(27) . The double mutant D351Stop/Y314F was constructed by swapping the EcoRI fragment from Tyr into D351Stop to give the plasmid pRKN2.hghr51.1.


Figure 1: Outline of the human GH receptor mutants. The location of the exons is shown. Numbers indicate the extent of the extracellular, transmembrane (TM), and cytoplasmic domain; residue 1 is the first amino acid of the mature protein(2) . The location of the cysteine residues (C) in the extracellular domain and of regions conserved in the GH/cytokine receptor family (WSXWS, Box 1, and Box 2) are indicated. The WSXWS motif is less conserved in the human GH receptor where the sequence is YGEFS. The amino acid sequence of the cytoplasmic domain of the D351Stop truncated GH receptor is shown as well as the location of the Y314F mutation.



Cell Lines

FDC-P1 cell line was kindly provided by Dr. Shigekazu Nagata. Untransfected cells were maintained in RPMI 1640 supplemented media (10% fetal calf serum, 10 mg/ml streptomycin sulfate, 50 mM 2-mercaptoethanol, and 2 mM glutamine) with addition of 10 units/ml IL-3. Cells were transfected with 30 µg of NsiI linearized expression plasmid by electroporation in 1 ml of serum-free RPMI 1640, at 325 V and 1600 microfarads(27) . Supplemented RPMI 1640 media containing 0.4 mg/ml G418 and 10 nM human GH were used to select a stable transfected cell population. Binding assays were performed as described (27) .

Immunoprecipitation and Protein Blots

Jak2 immunoprecipitations and phosphotyrosine immunoblots were performed as described(25) . GH receptor immunoprecipitations and blots were performed in the same manner, but with 10 million cells and with 10 µg of anti-GH receptor monoclonal antibody 263 (Agen, Inc.).

Cell Proliferation and Electrophoretic Mobility Shift Assays

Cells were starved overnight in GH-free supplemented medium (above) at 37 °C and assayed in triplicate wells with medium containing 10 nM human GH, 10 units/ml IL-3, or no addition as described(25) . Growth is expressed as a percentage of the IL-3 response after subtracting the value for no addition. Transcription factor complexation and electrophoretic mobility shift assays were performed with 10 million cells as described(25) . For SIE (high affinity m67) 5`-CTAGAGTCGACATTTCCCGTAAATCT and 5`-CTAGAGATTTACGGGAAATGTCGACT were used(28, 29) , and for GRR, 5`-TCTAGAGCATGTTTCAAGGATTTGAGATGTATTTCCCAGAAAAGAT and 5`-TCTAGATCTTTTCTGGGAAATACATCTCAAATCCTTGAAACATGCT were used(30) .


RESULTS

In order to determine whether tyrosine phosphorylation of the cytoplasmic domain of the GH receptor is essential for the signaling of a ligand-dependent growth response, we utilized plasmids encoding the three forms of the receptor shown in Fig. 1. Previous work has shown that the D351Stop truncated receptor (which lacks 77% of the cytoplasmic domain and six of the seven tyrosine residues) gives a growth response similar to that found for the full-length receptor when expressed in FDC-P1 or Ba/F3 cells(25, 27) . The one remaining tyrosine in the cytoplasmic domain, Tyr, was changed to phenylalanine in the D351Stop truncated GH receptor to eliminate all the tyrosines in the cytoplasmic domain. Characterization of the GH binding properties of the receptors when stably expressed in FDC-P1 cells gives affinities of K(d) = 0.61, 0.21, and 1.8 nM and receptor numbers of 5600, 500, and 3500 sites/cells for the full-length, D351Stop, and D351Stop/Y314F expressing cell lines, respectively (data not shown)(27) .

Tyrosine phosphorylation of the GH receptor in the expressing cell lines was assessed by immunoprecipitation of the receptor followed by phosphotyrosine immunoblot (Fig. 2). For the full-length receptor, a broad, GH-dependent band of the expected size (130 kDa) (2, 31) was found (FFR); this band was absent with untransfected cells (FDC-P1). These findings indicate that the GH receptor is tyrosine-phosphorylated in this system. No phosphorylated receptor band was found for the D351Stop/Y314F receptor, as expected (F51.5). No phosphorylated receptor band was found as well for the D351Stop receptor (F351), suggesting the possibility that much of the tyrosine phosphorylation found for the full-length receptor is in the distal six tyrosine residues.


Figure 2: Tyrosine phosphorylation of GH receptor mutants expressed in FDC-P1 cells. Whole cell extracts prepared from cells treated without(-) or with (+) 10 nM human GH were immunoprecipitated with an anti-GH receptor monoclonal antibody (263) and analyzed by phosphotyrosine immunoblot (8% polyacrylamide gel). FDC-P1, parental cells; FFR, cells expressing the full-length GH receptor; F351, cells expressing the D351Stop GH receptor; F51.5, cell expressing the D351Stop/Y314F GH receptor.



FDC-P1 cells expressing the three forms of the GH receptor were assayed for GH-dependent proliferation in the presence of saturating concentrations of ligand (Fig. 3). No response was found without GH or IL-3. All three receptor expressing lines, including the line expressing the GH receptor without tyrosine residues in the cytoplasmic domain (F51.5), show a proliferation response that equals that for IL-3. A full proliferative response was also found for FDC-P1 cells expressing the Y314F mutation in the full-length receptor (data not shown). Thus, tyrosine phosphorylation of the cytoplasmic domain of the human GH receptor is not required to signal a growth response in this system.


Figure 3: Proliferation of FDC-P1 cells expressing mutants of the GH receptor. Parental FDC-P1 cells and the GH receptor-expressing cell lines were assayed for GH-dependent growth as described under ``Materials and Methods.'' The standard deviation of triplicates is shown.



To test whether some of the intracellular signaling events coupled to receptor activation are affected by the lack of tyrosine phosphorylation of the GH receptor, we examined Jak2 tyrosine phosphorylation (Fig. 4) and transcription factor activation (Fig. 5) in response to GH binding. The GH-stimulated tyrosine phosphorylation of Jak2, and the band shifts induced with the DNA elements SIE or GRR, are unaffected in the D351Stop/Y314F mutant (F51.5). Thus, these two intracellular signals also do not require tyrosine phosphorylation of the GH receptor.


Figure 4: Jak2 tyrosine phosphorylation in GH receptor expressing FDC-P1 cell lines. Whole cell extracts prepared from cells treated without(-) or with (+) 10 nM human GH were immunoprecipitated with anti-Jak2 antiserum and analyzed by phosphotyrosine immunoblot as described under ``Materials and Methods.''




Figure 5: Transcription factor complex formation in GH receptor expressing FDC-P1 cell lines. The binding of whole cell extracts to labeled DNA elements was performed as described under ``Materials and Methods.'' Binding was performed with extracts from lines treated without(-) or with (+) 10 nM human GH (GH) or unlabeled oligonucleotide (Cold Oligo). A, binding to the element SIE. B, binding to the element GRR.




DISCUSSION

Following dimerization of the GH receptor by a single molecule of GH, there is a rapid tyrosine phosphorylation of a number of proteins in the cytoplasm, including the GH receptor itself(15, 17) . Recently, Jak2 has been identified as the tyrosine kinase likely to be responsible for these phosphorylation events(14) . Jak2 associates with the receptor following ligand binding and is activated by tyrosine phosphorylation(14) . The activation of Jak2 and the related kinases, Jak1 and Tyk2, have been implicated in the intracellular signaling pathways for many if not all of the receptors of the GH/cytokine receptor family(9) . Direct evidence that the Jak family kinases are an essential component of the signaling pathway has been provided for the IFN-alpha and IFN- receptors where cell lines lacking these kinases or STAT proteins fail to respond to ligand stimulation(20, 21, 22) .

We find that the full-length GH receptor when expressed in FDC-P1 cells is rapidly tyrosine phosphorylated in response to GH stimulation, as has been found for other systems(16, 17, 18, 19) . In order to determine whether this tyrosine phosphorylation is an essential step in the signaling of proliferation and in the activation of the Jak-STAT signaling pathway, we have expressed a truncated form of the human GH receptor that lacks tyrosine residues in the cytoplasmic domain. This receptor is able to signal a hormone-dependent biological response (e.g. proliferation), as well as stimulate the phosphorylation of Jak2 and the activation of STAT protein(s). Thus, at least in FDC-P1 cells, tyrosine phosphorylation of the GH receptor is not an essential step in these signaling events.

Work is in progress to identify which STAT protein or proteins are activated by growth hormone in FDC-P1 cells. Published data from other systems indicate that p91 and STAT3 may be the specific STAT proteins activated by the GH receptor(11, 32) .

Recently described experiments show that Tyr of the alpha chain of the IFN- receptor is required for signal transduction in a mouse fibroblast cell line, SCC16-5(23) . Furthermore, activation of the STAT protein, p91 (as assayed by electrophoretic mobility shift assay with homogenates of the human cell line, Colo-205), can be specifically inhibited by a 9-amino acid phosphorylated peptide having the sequence surrounding Tyr; the unphosphorylated peptide fails to block p91 activation(24) . Although no direct binding of p91 to the phosphorylated IFN- receptor has been reported, the docking of p91 to Tyr of the receptor has been proposed as a key step in its activation that is followed by its tyrosine phosphorylation, dissociation, and translocation to the nucleus(24) . Recent, phosphorylated peptide inhibition experiments with the IL-4 receptor and the newly identified IL-4 STAT protein support a similar model for IL-4 STAT binding to two phosphotyrosine-containing sequences of the IL-4 receptor cytoplasmic domain(33) . On the other hand, mutants of the IL-4 receptor lacking tyrosine residues in the cytoplasmic domain are able to signal proliferation in Ba/F3 cells, although STAT activation was not determined(34) . Although additional studies will be required to resolve these apparently divergent actions, perhaps there are phosphotyrosine-containing STAT binding sites on proteins other than the receptors (for example on ancillary molecules, on the Jak proteins themselves, or on receptors for other ligands) that can be used as alternative sites for STAT docking and activation. Such a hypothesis would predict that deletion of any one site would have little or no effect, whereas competition for all the sites would inhibit STAT activation and signaling.


FOOTNOTES

*
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.

§
To whom correspondence should be addressed. Tel.: 415-225-1221; Fax: 415-225-6127.

(^1)
The abbreviations used are: GH, growth hormone; G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocyte-macrophage colony stimulating factor; Jak, Janus kinase; STAT, signal transducers and activators of transcription; IFN, interferon; IL, interleukin; PBS, phosphate-buffered saline; SIE, c-sis-inducible element; GRR, interferon- response region of the fgr1 gene.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.