©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Growth Hormone-dependent Phosphorylation of Tyrosine 333 and/or 338 of the Growth Hormone Receptor (*)

(Received for publication, March 8, 1995; and in revised form, May 30, 1995)

Joyce A. VanderKuur (1)(§) Xueyan Wang (1)(¶) Liying Zhang (1) Giovanna Allevato (2) Nils Billestrup (2) Christin Carter-Su (1)(**)

From the  (1)Department of Physiology, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0622 and the (2)Hagedorn Research Laboratory, Neils Steensenevej 6, DK-2820 Gentofte, Denmark

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Many signaling pathways initiated by ligands that activate receptor tyrosine kinases have been shown to involve the binding of SH2 domain-containing proteins to specific phosphorylated tyrosines in the receptor. Although the receptor for growth hormone (GH) does not contain intrinsic tyrosine kinase activity, GH has recently been shown to promote the association of its receptor with JAK2 tyrosine kinase, to activate JAK2, and to promote the tyrosyl phosphorylation of both GH receptor (GHR) and JAK2. In this work, we examined whether tyrosines 333 and/or 338 in GHR are phosphorylated by JAK2 in response to GH. Tyrosines 333 and 338 in rat full-length (GHR) and truncated (GHR) receptor were replaced with phenylalanines and the mutated GHRs expressed in Chinese hamster ovary cells. These substitutions caused a loss of GH-dependent tyrosyl phosphorylation of truncated receptor and a reduction of GH-dependent phosphorylation of the full-length receptor. Consistent with Tyr and/or Tyr serving as substrates of JAK2, these substitutions resulted in a loss of tyrosyl phosphorylation of truncated receptor in an in vitro kinase assay using substantially purified GHbulletGHRbulletJAK2 complexes. The Tyr to Phe substitutions did not substantially alter GH-dependent JAK2 association with GHR or tyrosyl phosphorylation of JAK2. These results suggest that Tyr and/or Tyr in GHR are phosphorylated in response to GH and may therefore serve as binding sites for SH2 domain-containing proteins in GH signal transduction pathways.


INTRODUCTION

Ligand binding to membrane receptors with intrinsic tyrosine kinase activity has been shown to result in the phosphorylation of multiple tyrosines in the receptors themselves (reviewed in (1) ). Once phosphorylated, these tyrosines have been shown to bind the Src homology 2 (SH2) domain (a region of 100 amino acids) of both enzymes and noncatalytic proteins involved in signal transduction (2, 3, 4, 5) . Thus, phosphorylated tyrosines in receptors are believed to serve a vital role linking extracellular stimuli and cellular responses. Although GHR (^1)itself is not a tyrosine kinase, GH has recently been shown to promote the association of GHR with the tyrosine kinase JAK2, to activate JAK2, and to promote phosphorylation of tyrosyl residues in both GHR and JAK2(6, 7) . Since JAK2 is activated (and thus phosphorylated) in response to multiple ligands that bind to members of the cytokine receptor superfamily(6, 8, 9, 10) , it has been presumed that specificity in ligand response resides at least in part in the tyrosines that are phosphorylated in individual receptors. We were therefore interested in determining which tyrosines in GHR are phosphorylated in response to GH and thereby could potentially serve as binding sites for SH2 domain-containing signaling molecules that mediate responses to GH.

Previous studies showed that rat GHR (numbering system of (11) ), which lacks approximately half of the cytoplasmic domain of GHR, is phosphorylated on tyrosines in response to GH in intact cells(7) . Furthermore, this truncated receptor is phosphorylated on tyrosines when GHbulletGHRbulletJAK2 complexes are substantially purified from GH-treated cells and incubated with ATP. These results indicate that one or more of the 4 intracellular tyrosines (Tyr, Tyr, Tyr, Tyr) present in this truncated GHR is phosphorylated in response to GH, presumably by JAK2. Based upon predictions of secondary structure, hydrophilicity, chain flexibility, and surface probability(12, 13, 14, 15, 16, 17) , Tyr of GHR was judged to be the most likely of the 10 cytoplasmic tyrosines in the receptor to be phosphorylated. It is also the only tyrosine in the truncated receptor that is conserved among species (cow, human, rabbit, sheep, rat, mouse, chicken, pig)(18, 19, 20, 21, 22, 23, 24, 25) , suggesting that Tyr may play an important role in GH action. In this work, we use site-directed mutagenesis to examine whether Tyr and/or its close neighbor Tyr is phosphorylated in response to GH. Our findings provide strong evidence that one or both of these tyrosines is phosphorylated in response to GH, serves as a substrate for JAK2, and is not required for association of the receptor with JAK2 or GH activation of JAK2. The accompanying paper (42) provides evidence that Tyr and/or Tyr may be involved in at least some actions of GH.


EXPERIMENTAL PROCEDURES

Materials

Recombinant DNA-derived 22,000-dalton hGH was a gift of Lilly. Recombinant protein A-agarose was from Repligen, and the protein assay (BCA) was from Pierce. Triton X-100, aprotinin, and leupeptin were purchased from Boehringer Mannheim. Molecular weight standards (unstained) and ovalbumin were purchased from Sigma, prestained molecular weight standards were from Life Technologies, Inc., nitrocellulose membranes were from Schleicher & Schuell, and [-P]ATP (6000 Ci/mmol) was from DuPont NEN. The enhanced chemiluminescence detection system and x-ray film were from Amersham Corp.

Antisera

Antibody to GH (alphaGH) (NIDDK-anti-hGH-IC3, lot C11981) came from the National Institute of Diabetes and Digestive and Kidney Diseases/National Hormone and Pituitary Program. Anti-phosphotyrosine antibody (alphaPY) (4G10) was purchased from Upstate Biotechnology, Inc. Antibody to JAK2 (alphaJAK2) was prepared either in our laboratory in conjunction with Pel-Freez Biologicals or the laboratory of J. Ihle (St. Jude Children's Research Hospital, Memphis, TN) in rabbits against a synthetic peptide corresponding to amino acids 758-776 as described previously(9) . Antibody to GHR (alphaGHBP), kindly provided by W. Baumbach (American Cyanamid, Princeton, NJ), was produced in rabbits using recombinant rat GH-binding protein produced in Escherichia coli(26) .

Mutagenesis, Transfection, and Cell Culture

CHO cells were co-transfected with plasmids pLM108 and pIBP-1(27, 28) . Plasmid pIBP-1 contains a thymidine kinase promoter fused to the bacterial neomycin phosphotransferase gene conferring G418 resistance. Plasmid pLM108 contains the simian virus 40 enhancer and the Zn-inducible human metallothionein IIa promoter driving the expression of the cDNA coding for full-length rat liver GHR (GHR) (gift of G. Norstedt, Center for Biotechnology, Karolinska Institute, Huddinge, Sweden), the same cDNA with the termination codon replacing the lysine codon 455 (GHR) (gift of G. Norstedt); the same cDNA with phenylalanines replacing tyrosines at positions 333 and 338 (GHRY333F,Y338F) and the same cDNA with the termination codon replacing the lysine codon 455 and with phenylalanines replacing tyrosines at positions 333 and 338 (GHRY333F,Y338F) (Fig. 1). The cDNAs encoding the truncated and mutated GHR were generated by polymerase chain reaction (28, 29, 7) . CHO cells were cultured and screened for GHR expression as described previously(7, 30, 31) . Two cell lines expressing different levels of GHRY333F,Y338F were used in these studies, designated clones 23 (level of expression similar to cells expressing GHR) and 3 (level of expression only 20% that of cells expressing GHR). Experiments were performed using clone 23 unless noted otherwise.


Figure 1: Wild-type and mutated GHRs expressed in CHO cells. Denoted are the extracellular domain, the transmembrane (hatched area), and the cytoplasmic domain of the mutated rat liver GHR. Tyrosyl residues in the cytoplasmic domain are denoted by Y. Phenylalanine residues that were substituted for tyrosyl residues are denoted by F. Binding data were determined as described under ``Experimental Procedures.''



Immunoprecipitation and Western Blotting

Confluent CHO cells were incubated in the absence of serum overnight(32) . Cells were incubated for the indicated times with hGH at 37 °C in 95% air, 5% CO(2), rinsed with three changes of ice-cold PBSV (10 mM sodium phosphate, pH 7.4, 137 mM NaCl, 1 mM Na(3)VO(4)) and scraped on ice in lysis buffer (50 mM Tris, pH 7.5, 0.1% Triton X-100, 137 mM NaCl, 2 mM EGTA, 1 mM Na(3)VO(4), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin). Cell lysates were centrifuged at 12,000 times g for 10 min, and the resulting supernatants were incubated on ice for 2 h with the indicated antibody. Immune complexes were collected on protein A-agarose during a 1-h incubation at 8 °C, washed three times with wash buffer (50 mM Tris, pH 7.5, 0.1% Triton X-100, 137 mM NaCl, 2 mM EGTA), and boiled for 5 min in a mixture (80:20) of lysis buffer and SDS sample buffer (250 mM Tris, pH 6.8, 5% SDS, 10% beta-mercaptoethanol, 40% glycerol). The immunoprecipitates and lysates were subjected to SDS-PAGE (with the amount of sample normalized to protein) followed by Western blot analysis with the indicated antibody using the enhanced chemiluminescence detection system(33) . In some experiments, the blots were rinsed and reprobed with a second antibody.

Formation of Cross-linked I-hGH Receptor Complexes and GH Binding Assay

Human GH was labeled with I to an estimated specific activity of 90 µCi/µg using chloramine T by the University of Michigan Reproductive Sciences Training Grant Core Facility. As described previously(34) , cells were incubated in serum-free medium overnight and then washed with Krebs-Ringer phosphate buffer (KRP) containing 1% bovine serum albumin. I-hGH (12 times 10^6 counts/min/100-mm dish, 8-40 ng/ml) in KRP, 1% bovine serum albumin was added to the cells in the presence or absence of 1 µg/ml unlabeled hGH and incubated for 1 h at 25 °C. After extensive washing with KRP, 1% bovine serum albumin, disuccinimidyl suberate (0.4 mM final concentration) dissolved in dimethyl sulfoxide was added, and cells were incubated for 15 min at 8 °C. Cells were solubilized using HVTDP buffer (25 mM HEPES, 0.1% Triton X-100, 2 mM Na(3)VO(4), 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, pH 7.4, 10 µg/ml aprotinin, 10 µg/ml leupeptin) and mixed (80:20) with SDS sample buffer (32) and analyzed by SDS-PAGE. Relative affinities of the GHR for I-hGH were estimated as described previously(28) . Because the affinities of the different GHR appeared not to differ (data not shown), receptor numbers were compared by incubating cell monolayers with I-hGH (1-6 ng/ml) for 1 h at 25 °C. Cells were washed with ice-cold KRP and lysed with 1 N NaOH. Radioactivity associated with the cells was determined by counting cell lysates in a counter. Results were normalized to protein content and corrected for nonspecific binding, which was determined by incubating cell monolayers with I-hGH in the presence of 2 µg/ml unlabeled hGH. The relative abilities of the different cell lines to bind I-hGH were similar when binding was determined by incubating the cells with I-hGH for 1 h at 25 °C or overnight at 4 °C. (^2)

In Vitro Kinase Assay

Cells were grown to confluence and deprived of serum overnight as described previously(35) . Cells were then incubated in the absence or presence of 100 ng/ml (4.5 nM) GH at 25 °C for 1 h (Fig. 3) or 100 ng/ml GH at 37 °C for 15 min (Fig. 4) and lysed in HVTDP buffer. Cell lysates were centrifuged at 230,000 times g for 1 h. Supernatants were then incubated with alphaGH (1:10,000) for 2 h at 8 °C. Immune complexes were precipitated with immobilized protein A, and immunomatrices were extensively washed with NHT (50 mM HEPES, 150 mM NaCl, 0.1% Triton X-100, pH 7.6) plus 0.5 mM dithiothreitol. The immunomatrices were washed in buffer C (50 mM HEPES, 100 mM NaCl, 6.25 mM MnCl(2), 0.5 mM dithiothreitol, 0.1% Triton X-100, pH 7.6) and resuspended in 200 µl of buffer C containing 250 µg/ml aprotinin and 250 µg/ml leupeptin. In vitro phosphorylation was carried out by adding 20 µl of 50 mM HEPES, 0.1% Triton X-100, pH 7.6, containing vehicle, unlabeled ATP (5 µM), or unlabeled ATP (5 µM) plus [-P]ATP (200 µCi) and incubating for 10 min at 30 °C as described previously (35, 36) . The reaction was stopped by the addition of 12 ml of ice-cold NHT buffer containing 10 mM EDTA, pH 7.6, followed by extensive washing. Immunoprecipitated proteins were eluted from the immunomatrices by boiling in 200 µl of 150 mM Tris, pH 6.8, 3% SDS, 3% beta-mercaptoethanol, 30% glycerol, and 0.03 mg/ml bromphenol blue and analyzed by SDS-PAGE followed by either autoradiography or Western blotting using alphaPY as described above.


Figure 3: Ability of mutated GHR to be phosphorylated in an in vitro kinase assay. a, CHO cells expressing GHR (lanes A and B) and GHRY333F,Y338F (lanes C-F) (four 100-mm dishes/condition) were incubated for 1 h at 25 °C with 4.5 nM (100 ng/ml) hGH (+GH, lanes B, D, and F) or without hGH (-GH, lanes A, C, and E) and lysed with HVTDP buffer. Solubilized proteins were immunoprecipitated using alphaGH (1:10,000). Immunoprecipitates were incubated with [-P]ATP for 10 min and analyzed by SDS-PAGE. The migration of molecular weight standards (times 10) are indicated on the right. The migration of JAK2 and GHR are indicated on the left. b, CHO cells expressing GHR (lanes A-C) or GHRY333F,Y338F (lanes D-I) were incubated at 37 °C for 15 min with 4.5 nM hGH (+GH, lanes B, C, E, F, H, and I) or without hGH (-GH, lanes A, D, and G). Solubilized proteins were immunoprecipitated with alphaGH (1:10,000). Samples were incubated without (-ATP, lanes A, B, D, E, G, and H) or with (+ATP) (lanes C, F, and I) 5 µM unlabeled ATP for 10 min. Samples were eluted with SDS-PAGE sample buffer and Western-blotted with alphaPY. The migration of molecular weight standards (times 10) is indicated on the right. The migration of JAK2 and truncated receptor is indicated on the left.




Figure 4: 125I-hGH affinity labeling of mutated GHRs expressed in CHO cells. One 100-mm dish each of CHO cells expressing GHR (lanes A, B, and G), GHR (lanes C and D), GHR Y333F,Y338F (lanes E and F), and GHRY333F,Y338F (lane H) were incubated for 1 h at 25 °C with I-hGH in the absence (TOT, lanes B, D, F, G, and H) or presence (NS, lanes A, C, and E) of 1 µg/ml unlabeled hGH. Disuccinimidyl suberate (0.4 mM) was then added and the incubation continued for 15 min at 8 °C. Samples were analyzed by SDS-PAGE followed by autoradiography. The migration of molecular weight standards (times 10) is indicated between lanes F and G. The migration of GHR, GHR, and degraded GHRY333F, Y338F is indicated.



SDS-PAGE and Densitometry

SDS-PAGE was performed using 3-10% gradient gels (30:0.5, acrylamide:bisacrylamide) as described previously(36) . SDS-PAGE gels described in Fig. 2, Fig. 6, and Fig. 7contained prestained molecular weight standards: ovalbumin (43,600), bovine serum albumin (70,800), phosphorylase b (105,000), and myosin (203,000). SDS-PAGE gels in Fig. 3Fig. 4Fig. 5contained unstained standards: ovalbumin (48,000), bovine serum albumin (66,000), phosphorylase b (97,400), beta-galactosidase (116,000), and myosin (205,000). Densitometry was performed using a BioMed Instruments laser scanning densitometer attached to an Apple IIE computer (Bio-Med Instruments Videophoresis II data analysis computer program). For averaged results, mean ± S.E. are shown.


Figure 2: GH-dependent tyrosyl phosphorylation of mutated GHR. CHO cells expressing various GHR as noted were incubated for 5 min at 37 °C without (-GH) (lanes A, C, E, and G) or with 23 nM (500 ng/ml) hGH (+GH) (lanes B, D, F, and H). Cellular proteins were immunoprecipitated with alphaGH (1:8000) and Western-blotted with alphaPY (1:7500). The migration of JAK2 and GHR are indicated on the right and prestained molecular weight standards (times 10) on the left.




Figure 6: The ability of mutated GHR to elicit GH-dependent tyrosyl phosphorylation of JAK2. CHO cells expressing various GHR as noted were incubated for 5 min at 37 °C without (-GH) (lanes A, C, E, G, I, and K) or with 23 nM (500 ng/ml) hGH (+GH) (lanes B, D, F, H, J, and L) as described for Fig. 2. Proteins were immunoprecipitated with alphaJAK2 (1:1000) and Western-blotted with alphaPY (1:7500). The migration of JAK2 is indicated. Lanes A-H are directly comparable with lanes A-H in Fig. 2, since samples were prepared using aliquots from the same cells and were separated on the same gel and Western-blotted together.




Figure 7: The ability of GH to stimulate tyrosyl phosphorylation of cellular proteins in CHO cells expressing mutated GHR. CHO cells expressing various GHR as noted were incubated at 37 °C with 23 nM (500 ng/ml) hGH for the times indicated and then lysed with boiling SDS sample buffer diluted (20:80) with lysis buffer. Proteins were separated by SDS-PAGE and Western-blotted with alphaPY. The migration of prestained molecular weight standards (times 10) are indicated between lanes F and G. The migration of p121, p97, p42, and p39 is indicated by arrows on the left.




Figure 5: The ability of mutated GHR to associate with JAK2. CHO cells expressing GHR as noted were incubated for 5 min at 37 °C without (lanes B, D, F, and H) or with 23 nM (500 ng/ml) hGH (lanes A, C, E, and G). Cellular proteins were immunoprecipitated with alphaGH (1:8000) and Western-blotted with alphaJAK2 (1:7500). The migration of JAK2 is indicated.




RESULTS

Ability of GH to Promote Tyrosyl Phosphorylation of GHR Lacking Tyrand Tyr

Previous results indicated that GHR is tyrosyl-phosphorylated in response to GH(7) , indicating that 1 or more of the 4 tyrosines present in the cytoplasmic domain of this truncated receptor is a substrate of the GH-activated, GHR-associated JAK2 tyrosine kinase. To determine which of the 4 tyrosines present in this truncated receptor is phosphorylated in response to GH, we replaced Tyr and Tyr in both wild-type (GHR) and GHR with phenylalanines (Fig. 1), expressed the mutated receptors in CHO cells, compared the relative levels of I-hGH binding in the different cell lines, and examined whether GH stimulates tyrosyl phosphorylation of these mutated receptors. I-hGH binding to cells expressing GHRY333F,Y338F, GHR, and GHRY333F,Y338F was 88 ± 2% (n = 2), 71 ± 4% (n = 12), 41 ± 5% (n = 10), respectively, that of cells expressing wild-type GHR (Fig. 1). GHbulletGHRbulletJAK2 complexes were prepared from GH-treated cells using alphaGH. Because GHR is not phosphorylated in the absence of GH(6, 7) , the amount of tyrosyl phosphorylated GHR observed in the alphaGH precipitates from GH-treated cells reflects the amount of GH-dependent phosphorylation. When GHbulletGHRbulletJAK2 complexes are precipitated from CHO cells expressing truncated receptor and Western-blotted with alphaPY, two tyrosyl phosphorylated proteins are detectable (Fig. 2, lane F) as reported previously(7) . The lower, relatively diffuse band (M(r) 80,000) has been identified as receptor and the upper, relatively narrow band as JAK2 because of their sizes and their ability to be recognized in Western blots by alphaGHR and alphaJAK2, respectively(7) . In contrast, when GHbulletGHR complexes are precipitated from CHO cells expressing the truncated Y333F,Y338F receptor and blotted with alphaPY, a band corresponding to JAK2 is observed, but no band corresponding in size to the truncated receptor is observed (Fig. 2, lane H), even in experiments in which the signal for GHR is 20 times that in Fig. 2(data not shown). Although binding was reduced (by 41 ± 5%, see Fig. 1) in the cells expressing the mutated, truncated receptor compared with cells expressing its unmutated counterpart, it was not sufficiently reduced to account for the total absence of a band corresponding to GHR. Thus, this result is consistent with Tyr and/or Tyr being the tyrosine in the N-terminal half of the cytoplasmic domain of GHR that is phosphorylated in response to GH.

When GHbulletGHRbulletJAK2 complexes are precipitated using alphaGH from GH-treated CHO cells expressing wild-type receptor and Western-blotted with alphaPY, a broad band migrating with M(r) 120,000-130,000 is observed (Fig. 2, lane B), as reported previously(7) . Western blotting with alphaGHR and alphaJAK2 indicates that this band contains both GHR and JAK2, with JAK2 migrating as a rather narrow band (M(r) 130,000) (see Fig. 6) and GHR migrating as a diffuse band (M(r) 120,000) with and just below JAK2(6, 7) . In alphaPY blots of alphaGH precipitates from GH-treated CHO cells expressing Y333F,Y338F full-length receptor, a diffuse band migrating with a M(r) appropriate for both GHR and JAK2 is also observed (Fig. 2, lane D). The diffuseness of the band indicates that the mutated receptor is phosphorylated, suggesting that tyrosines other than 333 and/or 338 in GHR are phosphorylated in response to GH. However, the significantly reduced intensity of this band (by 76 ± 2%, n = 3) compared with that obtained with wild-type receptor suggests that phosphorylation of Tyr and/or Tyr contributes to the level of GHR phosphorylation observed in wild-type receptor.

Ability of Substantially Purified GHbulletGHR Complexes to Incorporate Phosphate in an in Vitro Kinase Assay

To examine whether Tyr and/or Tyr are likely to be phosphorylated by the GHR-associated JAK2 kinase, GHbulletGHRbulletJAK2 complexes were substantially purified from GH-treated CHO cells using alphaGH and incubated with [-P]ATP. Fig. 3a illustrates that P is incorporated into proteins migrating with molecular weights appropriate for both GHR and JAK2 when GHbulletGHRbulletJAK2 complexes are prepared from CHO cells expressing GHR(Fig. 3a, lane B), as reported previously(7) . When a similar experiment is performed using the Y333F,Y338F mutated receptor, P is incorporated almost exclusively into a band corresponding in size to JAK2 (Fig. 3a, lane D). The amount of mutated receptor is assumed to be less (by 40%) than the amount of unmutated receptor based upon I-hGH binding data (Fig. 1). To ensure that a 40% reduction in the number of GHR could not account for the inability to detect phosphorylated Tyr Phe truncated receptor, lanes C and D were exposed to film for a longer period of time sufficient to almost triple the phosphorylation signal for GHR. Even with the longer exposure, no band co-migrating with phosphorylated truncated receptor was observed (Fig. 3a, lane F). A faint band of unknown origin may be seen migrating slightly ahead of where truncated GHR would be (Fig. 3a, lane F).

To confirm that in the in vitro kinase assay, there is a difference between mutated and unmutated receptor in the amount of phosphate incorporated into tyrosyl, as opposed to seryl and threonyl, residues, GHbulletGHRbulletJAK2 complexes were prepared from CHO cells treated with 100 ng/ml (4.5 nM) GH for 15 min at 37 °C and incubated in the absence and presence of unlabeled ATP at the same concentration of ATP (5 µM) used in the [-P]ATP experiment. Kinase assay-dependent changes in the amount of tyrosyl phosphorylation of GHR were assessed by Western blotting with alphaPY. An ATP-dependent tyrosyl phosphorylation of a protein migrating with appropriate M(r) was observed when GHR was prepared from CHO cells expressing truncated receptor (Fig. 3b, compare lanes B and C), but not when it was prepared from cells expressing truncated receptor with the Tyr to Phe substitution (Fig. 3b, compare lanes E and F). As in Fig. 3a, the intensity of the JAK2 band from mutated versus unmutated cells was reduced approximately to the same extent as binding of I-hGH (Fig. 1). Even when lanes E and F were exposed to film for a substantially longer period of time (Fig. 3b, lanes H and I) to compensate for the 40% decrease in GH binding in the cells expressing mutated receptor and making the JAK2 signal comparable for the mutated and unmutated receptors, no band corresponding to the mutated receptor was detectable. (^3)

Size of GHR Lacking Tyrand Tyr

The experiments described for Fig. 2and Fig. 3provide evidence that the amount of phosphate incorporated into GHR lacking Tyr and Tyr both in vivo and in the in vitro kinase assay is reduced compared with nonmutated GHR by more than can be accounted for by differences in the amount of GHR expressed in the corresponding cell lines. This is consistent with GH promoting the tyrosyl phosphorylation of Tyr and/or Tyr and with one or both of these tyrosines serving as a substrate of a GHR-associated kinase, presumably JAK2. However, the reduced level of phosphorylation observed for GHR lacking Tyr and Tyr could also potentially arise if in comparison to their nonmutated counterparts, the mutated receptors: 1) were more susceptible to proteolysis so that they lacked tyrosines other than 333 and 338 that are sites of phosphorylation or 2) had a substantially reduced ability to associate with or to activate JAK2.

To verify that mutation of Tyr and Tyr to Phe did not result in adventitious proteolysis of full-length and truncated receptors to the extent that potential alternative phosphorylation sites were deleted, CHO cells expressing the various GHRs were incubated with I-hGH, followed by the cross-linking reagent disuccinimidyl suberate. Fig. 4illustrates that when the molecular weight of hGH (22,000, (37) ) is taken into account, the various cross-linked I-hGHbulletGHR complexes migrate as proteins of the appropriate size (M(r) 134,000 for full-length, M(r) 95,000 for truncated receptor). In Fig. 4, a significantly greater portion of the I-hGHbulletGHRY333F, Y338F complexes compared with other I-hGHbulletGHR complexes appeared to be degraded, migrating as if the receptor was truncated at amino acid 415. However, this large difference was not reproducible. In the three cross-linking experiments performed, the amount of I-hGH cross-linked to full-length mutated GHR was only 26 ± 37% less than the amount of I-hGH cross-linked to full-length GHR. This suggests that the reduced (by 80%) phosphorylation observed for GHRY333F,Y338FbulletJAK2 complexes compared with GHRbulletJAK2 complexes cannot be attributed to a comparable reduction in the amount of intact receptor. Despite the presence of substantial amounts of degraded I-hGHbulletGHR complexes corresponding in size to GHR in Fig. 5, one does not see in Fig. 2a phosphorylated band corresponding in size to this truncated receptor. This provides additional evidence that Tyr (predicted to be the only tyrosine present in Tyr Phe mutated GHR) is not phosphorylated in response to GH, supporting our overall conclusion that Tyr and/or Tyr, but not Tyr (or Tyr) are phosphorylated in response to GH.

Ability of GHR Mutants to Mediate GH-promoted Association of JAK2 with GHR and JAK2 Activation

The results of Fig. 2and Fig. 3showing the presence of a tyrosyl-phosphorylated protein migrating with a molecular weight appropriate for JAK2 in alphaGH immunoprecipitates from GH-treated cells indicate that JAK2 co-precipitates with the mutated full-length and truncated GHR. This suggests that JAK2 is capable of associating with these mutant receptors. To provide more direct evidence of the ability of JAK2 to associate with these mutant receptors, and to compare the ability of the mutated and nonmutated receptors to bind JAK2, GHbulletGHRbulletJAK2 complexes were immunoprecipitated using alphaGH and Western-blotted with alphaJAK2 (Fig. 5). JAK2 was found to associate with both the nonmutated (lanes C and E) and mutated (lanes A and G) receptors. Although the amount of JAK2 associated with GHRY333F,Y338F compared with GHR in Fig. 5appeared to be substantially reduced, this finding was not reproducible. In three experiments, the amount of JAK2 associated with GHRY333F,Y338F was 115 ± 31% the amount associated with GHR. The amount associated with mutated, full-length GHR was 67 ± 1% the amount associated with wild-type GHR. Thus, JAK2 association with GHR does not depend upon Tyr and/or Tyr. Furthermore, the substantial reduction in the amount of phosphorylated receptor observed when Tyr and Tyr are mutated to Phe cannot be attributed to a comparable decreased level of JAK2 associated with these GHR.

To determine whether Tyr and/or Tyr are required for activation of JAK2, we compared the abilities of the mutated and unmutated GHR to mediate GH-dependent tyrosyl phosphorylation of JAK2. Tyrosyl phosphorylation of JAK kinases is thought to be due to autophosphorylation and thus to reflect JAK activation(6, 8) , a hypothesis supported by the finding that tyrosyl phosphorylation of JAK2 correlates well with JAK2 activation by GH. (^4)JAK2 was precipitated from CHO cells expressing the various GHRs using alphaJAK2 and tyrosyl phosphorylation of JAK2 was assessed by Western blotting with alphaPY. Fig. 6illustrates that for CHO cells expressing the four different GHR, including two CHO cell lines expressing different levels of GHR used in the accompanying paper (42) (clone 23 used in all other experiments (relatively high) and clone 3 (relatively low)), GH stimulates tyrosyl phosphorylation of JAK2, roughly in proportion to the amount of GH binding detected in the different cell lines. Phosphorylation of JAK2 was 129 ± 29% (n = 2) for mutated versus unmutated full-length receptor and 48 ± 20% (n = 2) for mutated versus unmutated truncated receptor. The corresponding ratios for I-hGH binding were 88 ± 2% and 60 ± 6% (n = 10), respectively. alphaJAK2 Western blots of the alphaJAK2 immunoprecipitates revealed similar amounts of JAK2 expressed in the different cell lines (data not shown), indicating that differences in JAK2 phosphorylation in response to GH cannot be attributed to differences in levels of JAK2 expressed in the different cell lines.

Ability of GHR Mutants to Stimulate Tyrosyl Phosphorylation of Cellular Proteins

To provide additional evidence of the ability of the various mutant GHR to mediate GH-dependent JAK2 activation, we examined the ability of the mutated GHR to stimulate tyrosyl phosphorylation of cellular proteins, a phenomenon thought to be dependent upon JAK2 activation(33) . Consistent with previous results(7, 35) , when lysates from CHO cells that express wild-type GHR are lysed with boiling SDS lysis buffer and then analyzed by alphaPY Western blot, GH-dependent tyrosyl phosphorylation of four proteins (p121, p97, p42, and p39, designated by the arrows on the left of Fig. 7) is observed (Fig. 7, lanes A-C). p121 has been identified as JAK2 ((6) ; data not shown). The identity of p97, which sometimes appears as a doublet (lanes B and C) and may actually represent two proteins, is currently unknown. p42 and p39 have been tentatively identified as the mitogen-activated protein kinase isoforms designated extracellular signal-regulated kinases (ERKs) 1 and 2(27, 35, 38, 39) . p121, p97, p42, and p39 were all phosphorylated in response to GH in CHO cells expressing GHRY333F,Y338F (Fig. 7, lanes D-F). In contrast, in CHO cells expressing truncated receptor, GH stimulates tyrosyl phosphorylation of p121, p42, and p39 but not p97 (Fig. 6, lanes G-I) as shown previously(7) . The same three proteins are tyrosyl phosphorylated in response to GH in CHO cells when Tyr and Tyr in the truncated receptor are mutated. Phosphorylation was reduced, consistent with the reduced number of receptors in these cells. These results provide evidence that Tyr and Tyr are not required for the phosphorylation of p97, ERK 1, and ERK 2. They also provide additional evidence that GH-dependent JAK2 association and activation occurs with the full-length or half-truncated GHR in the absence of Tyr and Tyr.


DISCUSSION

The results presented in this work provide strong evidence that Tyr and Tyr are not required for JAK2 association with GHR or for JAK2 activation. This is consistent with the finding using human GHR expressed in FDC-P1 cells (published while the present paper was under review) that no tyrosines in GHR are required for JAK2 phosphorylation in response to GH(41) . The results of this study also provide evidence that Tyr and/or Tyr are phosphorylated in response to GH, since receptors lacking these tyrosines are phosphorylated to a significantly reduced extent (full-length) or not at all (truncated receptor) compared with the same sized receptors retaining these tyrosines. Presumably, it is the GHR-associated JAK2 tyrosine kinase that phosphorylates these tyrosine(s), since the truncated receptor is phosphorylated when GHbulletGHRbulletJAK2 complexes are precipitated with alphaGH and incubated with [-P]ATP, whereas the truncated receptor lacking Tyr and Tyr is not. Although Tyr seems the most likely candidate based upon sequence analysis, additional studies will be required to determine which of the two tyrosines (333 or 338) is phosphorylated. Whether or not these are the only tyrosines in GHR that are phosphorylated is not known, since it is possible that phosphorylation of Tyr and/or Tyr is required for the subsequent phosphorylation of Tyr and Tyr. Although it seems unlikely given the conservative nature of the amino acid substitution, our results cannot rule out the alternative possibility that Tyr and Tyr are not themselves phosphorylated but rather, mutating Tyr and Tyr to Phe alters the ability of Tyr and/or Tyr to be phosphorylated.

Tyr and/or Tyr appear not to be the only tyrosines phosphorylated in response to GH, since GH appears to stimulate the tyrosyl phosphorylation of the full-length, Tyr Phe mutated receptor. The 6 tyrosines between amino acids 454-638 are the most likely candidates because tyrosines other than 333 and/or 338 present in GHR appear not to be phosphorylated to any great extent. Multiple phosphorylated tyrosines in GHR would be consistent with multiple sites of phosphorylation in receptors with intrinsic tyrosine kinase activity (e.g. receptors for insulin, epidermal growth factor, platelet-derived growth factor) (reviewed in (1) ). Multiple sites of phosphorylation with differing affinities for various SH2 domains would provide a mechanism by which GH could initiate several signaling pathways simultaneously.

The finding that Tyr and/or Tyr are likely to be phosphorylated in response to GH raises the question of whether either or both serve as binding sites for specific SH2 domains. Neither tyrosine, with its surrounding amino acids, closely resembles a high affinity binding site (as currently defined) of the SH2 domains of Csk, SHC, Syk, Vav, Grb2, 3BP2, HCP, or fps/fes(5) . Consistent with this, neither tyrosine appears to be required for GH-dependent SHC phosphorylation(40) . Since SHC lies downstream of GHR and upstream of mitogen-activated protein kinases, the SHC data are consistent with the data presented here and in the accompanying paper (42) that Tyr and Tyr are not required for GH activation of the mitogen-activated protein kinases ERKs 1 and 2. Tyr and Tyr also appear not be required for GH stimulation of insulin receptor substrate-1 (^5)or for activation of signal transducer and activator of transcriptions (Stats) 1 and 3 , (^6)although the data do not exclude the possibility that one or more of these SH2 domain-containing proteins binds to Tyr and/or Tyr. However, as described in the accompanying paper(42) , Tyr and/or Tyr do seem to be required for other actions of GH (lipid synthesis, protein synthesis), cellular responses for which the signaling pathways are less well defined. Verification that Tyr and/or Tyr are tyrosyl-phosphorylated using phosphopeptide analysis and identification of the signaling molecules that bind to the corresponding phosphorylated tyrosine(s) is likely to provide important information about signaling pathways used not only by GH but by other ligands that signal via tyrosine kinases.


FOOTNOTES

*
This work was supported by Research Grants DK34171 and DK48283 from the National Institutes of Health (to C. C.-S.). Computer studies were supported in part by the General Clinical Research Center at The University of Michigan, funded by Grant M01 RR00042 from The National Center for Research Resources, National Institutes of Health, United States Public Health Services. 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.

§
Recipient of Postdoctoral Fellowships from the National Institutes of Health (5T32-DK07245) and the Arthritis Foundation.

Recipient of a Rackham Dissertation Grant from The University of Michigan.

**
To whom correspondence should be addressed: Dept. of Physiology, The University of Michigan Medical School, Ann Arbor, MI 48109-0622. Fax: 313-936-8813.

(^1)
The abbreviations used are: GHR, growth hormone receptor; GH, growth hormone; hGH, human growth hormone; rGHR, rat growth hormone receptor; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; KRP, Krebs-Ringer phosphate buffer.

(^2)
T. King and C. Carter-Su, unpublished observation.

(^3)
In contrast to Fig. 2, in vivo tyrosyl phosphorylation of truncated receptor is not evident in Fig. 3b (i.e. no 80-kDa band is detected in lane B). This apparent discrepancy results from the difference in GH concentrations used in the two experiments. In Fig. 2, cells were incubated with a maximally stimulatory concentration of GH (500 ng/ml, 22 nM) to maximize the amount of GHR phosphorylation. For Fig. 3b, cells were incubated with a submaximal concentration of GH (100 ng/ml, 4.5 nM) to enable additional phosphorylation of these proteins to occur in the in vitro kinase assay. The result was the same whether the incubation with GH was for 15 min for 37 °C as in Fig. 3b or for 1 h at 25 °C as for Fig. 3a (data not shown).

(^4)
E. Adkins, G. Campbell, and C. Carter-Su, unpublished observation.

(^5)
Argetsinger, L. S., Hsu, G. W., Myers, M. G., Jr., Billestrup, N., White, M. F., and Carter-Su, C. (1995) J. Biol. Chem.270, 14685-14692.

(^6)
L. S. Smit, D. J. Meyer, N. Billestrup, J. Schwartz and C. Carter-Su, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. L. S. Argetsinger and G. Hsu for their helpful comments and review of the manuscript and D. Kim for help with the cell culture. We are grateful to Drs. W. R. Baumbach for antibody to GHR, J. Ihle for antibody to JAK2, and G. Norstedt for CHO cells expressing GHR and GHR.


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