Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rui, L.
Right arrow Articles by Carter-Su, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rui, L.
Right arrow Articles by Carter-Su, C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

J Biol Chem, Vol. 275, Issue 4, 2885-2892, January 28, 2000


Platelet-derived Growth Factor and Lysophosphatidic Acid Inhibit Growth Hormone Binding and Signaling via a Protein Kinase C-dependent Pathway*

Liangyou Rui, Stephen F. ArcherDagger , Lawrence S. Argetsinger, and Christin Carter-Su§

From the Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Growth hormone (GH) regulates body growth and metabolism. GH exerts its biological action by stimulating JAK2, a GH receptor (GHR)-associated tyrosine kinase. Activated JAK2 phosphorylates itself and GHR, thus initiating multiple signaling pathways. In this work, we demonstrate that platelet-derived growth factor (PDGF) and lysophosphatidic acid (LPA) down-regulate GH signaling via a protein kinase C (PKC)-dependent pathway. PDGF substantially reduces tyrosyl phosphorylation of JAK2 induced by GH but not interferon-gamma or leukemia inhibitory factor. PDGF, but not epidermal growth factor, decreases tyrosyl phosphorylation of GHR (by approximately 90%) and the amount of both total cellular GHR (by approximately 80%) and GH binding (by approximately 70%). The inhibitory effect of PDGF on GH-induced tyrosyl phosphorylation of JAK2 and GHR is abolished by depletion of 4beta -phorbol 12-myristate 13-acetate (PMA)-sensitive PKCs with chronic PMA treatment and is severely inhibited by GF109203X, an inhibitor of PKCs. In contrast, extracellular signal-regulated kinases 1 and 2 and phosphatidylinositol 3-kinase appear not to be involved in this inhibitory effect of PDGF. LPA, a known activator of PKC, also inhibits GH-induced tyrosyl phosphorylation of JAK2 and GHR and reduces the number of GHR. We propose that ligands that activate PKC, including PDGF, LPA, and PMA, down-regulate GH signaling by decreasing the number of cell surface GHR through promoting GHR internalization and degradation and/or cleavage of membrane GHR and release of the extracellular domain of GHR.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Growth hormone (GH)1 is a circulating peptide hormone secreted from the anterior pituitary. It is the primary hormone known to stimulate postnatal longitudinal bone growth and increase body mass (1, 2). GH deficiency or dysfunction of the GH receptor (GHR) leads to dwarfism, whereas GH in excess results in gigantism or, in adults, acromegaly. In addition to stimulating body growth, GH regulates a variety of other biological functions, including body metabolism and the immune response (2). Clinically, GH has long been used to treat children with short stature. More recently, it has been used to prevent muscle wasting in AIDS patients (3, 4). Clinical studies indicate it greatly increases donor site wound healing in human patients (5).

GH exerts its diverse biological functions via GHR. Upon GH binding, GHR binds and activates JAK2, a cytoplasmic tyrosine kinase (6, 7). Activated JAK2 then phosphorylates itself and GHR on multiple tyrosines, generating binding sites for other signaling proteins containing Src homology 2 or phosphotyrosine interacting domains, including signal transducers and activators of transcription (8-13), Shc (14), and SH2-B (15, 16). Recruitment of these signaling molecules to GHR·JAK2 complexes and the subsequent tyrosyl phosphorylation of these proteins by JAK2 initiate a variety of signaling pathways, leading to the multiple cellular responses responsible for the diverse actions of GH (7, 17).

In contrast to GHR that is a member of the cytokine receptor family, the PDGF receptor is a receptor tyrosine kinase. A variety of signaling pathways, including the MEK/MAP kinase, the phosphatidylinositol 3 (PI-3)-kinase, and the protein kinase C (PKC) pathways (7, 18-21), has been shown to be activated by both GH and PDGF. Despite some shared signaling pathways, PDGF and GH are thought to regulate different functions even in the same cell type. Whereas GH has been implicated as a differentiation factor for a variety of cells (22-24), PDGF has been shown to be a competence factor that causes 3T3 cells to progress from a quiescent G0 state to the G1 phase of the cell cycle (25, 26). In 3T3-F442A cells, GH has been shown to dampen the mitogenic effect of PDGF and insulin (27). Because PDGF and GH can have opposing effects, even in the same cell, and receptors for GH and PDGF are coexpressed in many tissues and cell types, including brain (28-32), liver (29-34), bone (35-37), kidney (29-31, 38-40), and muscle (29, 30, 41-45), we hypothesized that PDGF and GH cross-talk and modulate each others' action at the cellular level. Cross-talk among different hormones, cytokines, and growth factors has been shown to be an important mechanism regulating the magnitude and specificity of cellular responses and to be involved in many physiological and/or pathological processes. For instance, tumor necrosis factor-alpha (TNF-alpha ) causes insulin resistance by inhibiting insulin signaling at the cellular level (46, 47).

In this study, we demonstrate that PDGF, but not epidermal growth factor (EGF), dramatically inhibits GH-stimulated tyrosyl phosphorylation of JAK2 and GHR and rapidly reduces the number of both total and cell surface GHR. Depletion or inhibition of PMA-sensitive PKCs blocks the action of PDGF. In contrast, the MEK/MAP kinase and the PI 3-kinase pathways appear not to be involved in this action of PDGF. LPA, a G protein-coupled receptor that, like PDGF, activates PKC (20, 21, 48), similarly inhibits GH-stimulated tyrosyl phosphorylation of JAK2 and GHR and reduces the number of GHR by a PKC-dependent pathway. We conclude that PDGF and LPA down-regulate GH signaling via a PKC-dependent pathway by at least in part reducing the level of GHR. It is likely that other ligands that activate PMA-sensitive PKCs down-regulate GH signaling in a similar fashion.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Recombinant hGH was a gift of Lilly. hGH was iodinated by the Reproductive Sciences Training Grant Core Facility at the University of Michigan Medical School to a specific activity of ~2,000 µCi/nmol. Recombinant murine EGF was from Collaborative Biomedical Products. Recombinant human PDGF-BB was from Intergen. Recombinant murine LIF was from R & D Systems. Recombinant murine IFN-gamma , LPA, and PMA were from Sigma. Protein A-agarose was from Repligen. Aprotinin, leupeptin, and Triton X-100 were purchased from Roche Molecular Biochemicals. Enhanced chemiluminescence (ECL) detection system was from Amersham Pharmacia Biotech. Wortmannin and bisindolylmaleimide I (GF109203X) were from Calbiochem. Anti-JAK2 antiserum (alpha JAK2) was raised in rabbits against a synthetic peptide corresponding to amino acids 758-776 (49) and was used at a dilution of 1:500 for immunoprecipitation and 1:15,000 for immunoblotting. Polyclonal anti-GHBP (alpha GHBP) recognizing the extracellular domain of GHR (50) was a gift of Dr. W. R. Baumbach (American Cyanamid, Princeton, NJ) and was used at a dilution of 1:250 for immunoprecipitation and 1:5,000 for immunoblotting. Polyclonal anti-phosphorylated (Ser-473), active Akt (alpha pAkt) was from New England Biolabs Inc. and was used at a dilution of 1:1,000 for immunoblotting. Polyclonal anti-phosphorylated (Thr-183 and Tyr-185), active MAP kinase (alpha active MAP kinase) was from Promega and was used at a dilution of 1:5,000 for immunoblotting. Monoclonal anti-phosphotyrosine antibody 4G10 (alpha PY) was purchased from Upstate Biotechnology Inc. and was used at a dilution of 1:7,500 for immunoblotting. The stock of 3T3-F442A cells was provided by H. Green (Harvard University, Cambridge, MA).

Immunoprecipitation and Immunoblotting-- 3T3-F442A fibroblasts were grown on 100-mm tissue culture dishes in Dulbecco's modified Eagle's medium supplemented with 8% calf serum, 1 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. Cells were deprived of serum overnight in the same growth medium except that 1% bovine serum albumin (BSA) was substituted for the calf serum. The deprived cells were treated for various times with the indicated drugs and/or ligands at 37 °C and then rinsed three times with 10 mM sodium phosphate, pH 7.4, 150 mM NaCl, 1 mM Na3VO4. The cells were solubilized in lysis buffer (50 mM Tris, pH 7.5, 0.1% Triton X-100, 150 mM NaCl, 2 mM EGTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin), and centrifuged at 14,000 × g for 10 min at 4 °C. Proteins in the supernatant were quantified using BCATM Protein Assay Reagent (Pierce). The supernatant was incubated with the indicated antibody on ice for 2 h. The immune complexes were collected on protein A-agarose (50 µl) during 1 h incubation at 4 °C. The beads were washed 3 times with washing buffer (50 mM Tris, pH 7.5, 0.1% Triton X-100, 150 mM NaCl, 2 mM EGTA) and boiled for 5 min in a mixture (80:20) of lysis buffer and SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 10% beta -mercaptoethanol, 40% glycerol, 0.01% bromphenol blue). The eluted proteins were separated by SDS-PAGE (5-12% gradient gel) followed by immunoblotting with the indicated antibody using the ECL detection system.

To assess the total amount of GHR, 3T3-F442A cells were solubilized in the above lysis buffer supplemented with 1% SDS and boiled for 5 min. The concentration of proteins in the cell lysates was determined using BCATM Protein Assay Reagent. Proteins (50 µg) in the lysates were boiled for 5 min in a mixture (80:20) of lysates and SDS-PAGE sample buffer, separated by SDS-PAGE, and subjected to immunoblotting using alpha GHBP.

125I-hGH Binding Assay-- Confluent 3T3-F442A fibroblasts in 6-well plates were deprived of serum overnight and stimulated with the indicated ligands for 45 min. The cells were washed with Krebs-Ringer phosphate (KRP) buffer (128 mM NaCl, 6.7 mM KCl, 1 mM CaCl2, 2.6 mM MgSO4, and 10 mM Na2HPO4, pH 7.4) containing 1% BSA and then incubated in KRP, 1% BSA containing 125I-hGH (80,000 cpm/well) overnight at 4 °C. The cells were then washed with cold KRP and solubilized in 1 ml of 1 M NaOH for counting radiation. The concentration of protein in the cell lysates was determined using BCA Protein Assay Reagent. 125I-hGH binding activity was normalized to the protein level and expressed as mean ± S.E.

For quantification, immunoblots were scanned using an Agfa ArcusII scanner and Fotolook SA software (Mortsel, Belgium). The resulting image was analyzed using multi-analyst image analysis software from Bio-Rad.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PDGF Rapidly Inhibits GH-induced Tyrosyl Phosphorylation of JAK2 and GHR and Reduces the Amount of GHR-- To examine whether PDGF cross-talks with GH, 3T3-F442A cells, which express endogenous receptors for PDGF (51, 52) and GH (53), were deprived of serum overnight, preincubated with 25 ng/ml PDGF for 20 min, and stimulated with 50 ng/ml GH for an additional 10 min. JAK2 was immunoprecipitated with alpha JAK2 and immunoblotted with alpha PY. The level of tyrosyl phosphorylation of JAK2 was used to assess the extent of activation of JAK2 by GH (6, 16). PDGF substantially inhibits GH-induced tyrosyl phosphorylation of JAK2 (Fig. 1A, upper panel). Densitometric analysis revealed that PDGF reduced GH-induced tyrosyl phosphorylation of JAK2 by approximately 90%. PDGF does not significantly change the amount of JAK2 (Fig. 1A, lower panel).


View larger version (60K):
[in this window]
[in a new window]
  		Fig. 1.   PDGF, but not EGF, specifically inhibits GH-induced tyrosyl phosphorylation of JAK2 and GHR and reduces the amount of GHR. A and B, 3T3-F442A cells were pretreated with or without PDGF (25 ng/ml) for 20 min prior to GH (50 ng/ml) stimulation for 10 min. Proteins in cell lysates were immunoprecipitated (IP) with alpha JAK2 (A) or alpha GHBP (B) and immunoblotted (IB) with alpha PY (upper panel). The blots were reprobed with alpha JAK2 (A, lower panel) or alpha GHBP (B, lower panel). C and D, 3T3-F442A cells were pretreated with or without PDGF (25 ng/ml) for 20 min and then stimulated for 10 min with IFN-gamma (10 ng/ml) (C) or LIF (25 ng/ml) (D). JAK2 was immunoprecipitated with alpha JAK2 and immunoblotted with alpha PY (upper panel) and reprobed with alpha JAK2 (lower panel). E, 3T3-F442A cells were pretreated with PDGF (25 ng/ml) or EGF (125 ng/ml) for 20 min prior to GH (50 ng/ml) stimulation for 10 min. Proteins in cell lysates were immunoprecipitated with alpha JAK2 (lanes 1-4) or alpha GHBP (lanes 5-8) and immunoblotted with alpha PY (upper panel). The same blots were reprobed with alpha JAK2 (lanes 1-4, lower panel) or alpha GHBP (lanes 5-8, lower panel).

Because GHR is a physiological substrate of JAK2, we tested whether PDGF alters tyrosyl phosphorylation of GHR in response to GH. 3T3-F442A cells were deprived of serum overnight and pretreated with PDGF for 20 min prior to stimulation with 50 ng/ml GH for 10 min. GHR was immunoprecipitated with alpha GHBP that recognizes the extracellular domain of GHR and immunoblotted with alpha PY or alpha GHBP. PDGF dramatically inhibits GH-induced tyrosyl phosphorylation of GHR (Fig. 1B, upper panel). Densitometric analysis of Fig. 1B indicates that PDGF inhibited tyrosyl phosphorylation of GHR by approximately 90%. PDGF by itself does not stimulate tyrosyl phosphorylation of GHR (Fig. 1B, lane 3, upper panel). Interestingly, PDGF appears to decrease total cellular GHR (Fig. 1B, lower panel, compare lanes 1 and 3, 2 and 4). GH stimulates an upward shift in mobility of GHR (Fig. 1B, lanes 1 and 2, lower panel). We believe the mobility shift is caused by phosphorylation of GHR, presumably on tyrosines (6, 54, 55).

Because JAK2 is also activated by interferon-gamma (IFN-gamma ) and leukemia inhibitory factor (LIF) in 3T3-F442A cells (56), we examined whether PDGF inhibits signaling by those ligands. 3T3-F442A cells were deprived of serum overnight and pretreated with 25 ng/ml PDGF for 20 min prior to treatment for 10 min with 10 ng/ml IFN-gamma or 25 ng/ml LIF. JAK2 was immunoprecipitated with alpha JAK2 and immunoblotted with alpha PY (Fig. 1, C and D, upper panel) or alpha JAK2 (Fig. 1, C and D, lower panel). IFN-gamma (Fig. 1C, lane 2) and LIF (Fig. 1D, lane 2) stimulate tyrosyl phosphorylation of JAK2, as reported previously (56). Surprisingly, PDGF does not inhibit tyrosyl phosphorylation of JAK2 induced by either IFN-gamma (Fig. 1C) or LIF (Fig. 1D). These data suggest that PDGF does not directly inhibit JAK2 but rather inhibits specifically GH signaling by a mechanism involving GHR activation and/or the coupling of GHR to JAK2.

To address further the specificity of the inhibition of GH signaling by PDGF, we examined whether EGF inhibits GH signaling. The receptors for both PDGF and EGF are receptor tyrosine kinases. 3T3-F442A cells, which express endogenous EGF receptors (51), were deprived of serum overnight and incubated with 125 ng/ml EGF for 20 min prior to 50 ng/ml GH for 10 min. Proteins in cell lysates were immunoprecipitated with alpha JAK2 (Fig. 1E, lanes 1-4) or alpha GHBP (lanes 5-8) and immunoblotted with alpha PY (Fig. 1E, upper panel), alpha JAK2 (Fig. 1E, lanes 1-4, lower panel), or alpha GHBP (Fig. 1E, lanes 5-8, lower panel). Consistent with the data in Fig. 1A, PDGF dramatically reduces GH-induced tyrosyl phosphorylation of JAK2 (Fig. 1E, lane 3, upper panel) and GHR (Fig. 1E, lane 7, upper panel). PDGF reduced the amount of GHR by approximately 80% (Fig. 1E, lower panel). In contrast, EGF neither inhibits GH-induced tyrosyl phosphorylation of JAK2 (Fig. 1E, lane 4, upper panel) and GHR (Fig. 1E, lane 8, upper panel) nor reduces the amount of GHR (Fig. 1E, lane 8 versus 6, lower panel). These results suggest that signaling events that cause down-regulation of GH signaling by PDGF are not shared by EGF.

PDGF-induced inhibition of GH signaling is very rapid. Pretreatment with PDGF for 2 min results in significant inhibition of GH-dependent tyrosyl phosphorylation of JAK2 (Fig. 2A, lane 3) and GHR (Fig. 2B, lane 3). The inhibition of tyrosyl phosphorylation of GHR and JAK2 reaches a maximal level within 15 min pretreatment with PDGF (Fig. 2, A and B). This rapid onset makes it unlikely that PDGF-induced inhibition of GH signaling involves new gene expression or synthesis of new proteins. PDGF-induced inhibition of GH-dependent tyrosyl phosphorylation of JAK2 (Fig. 2C) and GHR (Fig. 2D) is dependent on the dose of PDGF. It is detectable at 1 ng/ml and reaches a maximum with 25 ng/ml PDGF (Fig. 2, C and D).


View larger version (49K):
[in this window]
[in a new window]
  		Fig. 2.   PDGF rapidly inhibits GH-induced tyrosyl phosphorylation of JAK2 and GHR and reduces the amount of GHR in a dose-dependent manner. A and B, 3T3-F442A cells were pretreated with PDGF (25 ng/ml) for the indicated times prior to GH (50 ng/ml) stimulation for 10 min. Proteins in cell lysates were immunoprecipitated (IP) with alpha JAK2 (A) or alpha GHBP (B) and immunoblotted (IB) with alpha PY (A and B, upper panel). The blots were reprobed with alpha JAK2 (A, lower panel) or alpha GHBP (B, lower panel). C and D, 3T3-F442A cells were pretreated for 20 min with the indicated concentrations of PDGF prior to GH (50 ng/ml) stimulation for 10 min. Proteins in cell lysates were immunoprecipitated with alpha JAK2 (C) or alpha GHBP (D) and immunoblotted with alpha PY (C and D, upper panel). The blots were reprobed with alpha JAK2 (C, lower panel) or alpha GHBP (D, lower panel).

PDGF also reduces the level of GHR in a time- and dose-dependent manner. The reduction is substantial at 2 min, and maximal within 15 min stimulation with PDGF (Fig. 2B, lower panel). The reduction of GHR is detected at a concentration as low as 1 ng/ml PDGF (Fig. 2D, lane 3, lower panel).2 Thus, the PDGF-induced inhibition of GH-stimulated tyrosyl phosphorylation of JAK2 (Fig. 2, A and C, upper panel) and GHR (Fig. 2, B and D, upper panel) correlates in magnitude, time, and dose with the PDGF-induced reduction of GHR (Fig. 2, B and D, lower panel). This correlation suggests that PDGF inhibits GH signaling by reducing the amount of GHR present in cells.

PDGF Decreases the Number of GHR on the Cell Surface Available to Bind to GH-- Because PDGF does not interfere with the activation of JAK2 by IFN-gamma (Fig. 1C) or LIF (Fig. 1D), and PDGF-induced inhibition of tyrosyl phosphorylation of JAK2 and GHR correlates with the reduction in the level of cellular GHR (Fig. 2), we reasoned that PDGF does not directly inhibit JAK2 but rather reduces the number of GHR on the cell surface available to bind GH. To examine whether PDGF decreases the number of GHR on the cell surface available to bind GH, 3T3-F442A cells were deprived of serum overnight, treated for 45 min with 25 ng/ml PDGF, 125 ng/ml EGF, or 200 nM PMA, and subjected to a GH binding assay at 4 °C as described under "Experimental Procedures." PMA has been shown to reduce GH binding in 3T3-F442A cells (57) and in IM9 cells (58) and was used as a positive control. PDGF reduced GH binding by approximately 70% (Fig. 3). PMA decreased GH binding to a slightly greater extent (by approximately 75%). In contrast, EGF did not affect GH binding.


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 3.   PDGF, but not EGF, reduces GH binding. 3T3-F442A cells were pretreated for 45 min with or without PDGF (25 ng/ml) or PMA (200 nM) and then were subjected to a GH binding assay as described under "Experimental Procedures" (n = 3, ± S.E.).

The PDGF-induced Reduction of GHR Requires neither the ERK Cascade nor PI 3-Kinase-- To verify the reduction of total cellular GHR by PDGF described in Figs. 1 and 2, 3T3-F442A cells were deprived of serum overnight and treated with 25 ng/ml PDGF or 125 ng/ml EGF for 40 min. Cells were lysed in lysis buffer containing 1% SDS and boiled for 5 min. No residual cell pellet was observed following this treatment. The whole cell lysates were subjected to SDS-PAGE. Proteins in the cell lysates were immunoblotted with alpha GHBP (Fig. 4A, lanes 2-4). To verify the migration of GHR, alpha GHBP immunoprecipitate was also analyzed on the same SDS-PAGE gel (Fig. 4A, lane 1). PDGF reduced the amount of total cellular GHR by approximately 75% (Fig. 4A, lanes 3 versus 2). In contrast, EGF does not affect the level of GHR (Fig. 4A, lane 4).


View larger version (47K):
[in this window]
[in a new window]
  		Fig. 4.   ERKs 1 and 2 and PI 3-kinase are not involved in PDGF-induced reduction of the amount of GHR and tyrosyl phosphorylation of JAK2 and GHR. A, 3T3-F442A cells were treated with PDGF (25 ng/ml) or EGF (125 ng/ml) for 40 min and solubilized in lysis buffer containing 1% SDS as described under "Experimental Procedures." GHR in cell lysates was immunoprecipitated (IP) and immunoblotted (IB) with alpha GHBP (lane 1). Proteins (50 µg) in the whole cell lysates (lanes 2-4) were immunoblotted with alpha GHBP. B, 3T3-F442A cells were pretreated with PDGF (25 ng/ml) or EGF (125 ng/ml) for 40 min. Proteins (50 µg) in cell lysates were immunoblotted with alpha PY (top panel), anti-active MAP kinase (alpha active MAP kinase) (middle panel), and antibody recognizing specifically phosphorylated, active Akt (alpha pAkt) (bottom panel). The migration of molecular weight standards (× 103), receptors for EGF (EGFR) and PDGF (PDGFR), PLCgamma , ERKs 1 and 2, and activated Akt are indicated. C-E, 3T3-F442A cells were pretreated with or without wortmannin (Wort) (500 nM) for 25 min and then treated with PDGF (25 ng/ml) for 20 min. The cells were then stimulated with GH (50 ng/ml) for 10 min. Proteins in cell lysates were immunoprecipitated with alpha JAK2 (C) or alpha GHBP (D) and immunoblotted with alpha PY (C and D, upper panel). The same blots were reprobed with alpha JAK2 (C, lower panel) and alpha GHBP (D, lower panel), respectively. Proteins (50 µg) in cell lysates were immunoblotted with alpha pAkt (E).

PDGF activates multiple signaling molecules and pathways, including the MEK/ERK cascade, the PI 3-kinase/Akt pathway, and the PLCgamma /PKC pathway. To determine which molecules and pathways are involved in PDGF-induced inhibition of GH signaling, 3T3-F442A cells were treated with 25 ng/ml PDGF or 125 ng/ml EGF for 40 min, and proteins in cell lysates were immunoblotted with alpha PY (Fig. 4B, top panel) antibody to the activated form of mitogen-activated protein kinase which recognizes the activated, dually phosphorylated form of ERKs 1 and 2 (Fig. 4B, middle panel) or antibody to the activated form of Akt phosphorylated on Ser-473 which is the site phosphorylated by PI 3-kinase (Fig. 4B, bottom panel). PDGF stimulates tyrosyl phosphorylation of two proteins migrating with apparent molecular weights of approximately 175,000 and 145,000 (Fig. 4B, lane 2, top panel), sizes appropriate for the PDGF receptor and PLCgamma , respectively. In contrast, EGF stimulates tyrosyl phosphorylation of a protein migrating with apparent molecular weight 160,000, a size appropriate for the EGF receptor (Fig. 4B, lane 3, top panel). PDGF activates both ERKs 1 and 2 (Fig. 4B, lane 2, middle panel) and Akt (Fig. 4B, lane 2, bottom panel). EGF activates ERKs 1 and 2 to an extent similar to that observed with PDGF (Fig. 4B, lane 3, middle panel) but does not activate Akt (Fig. 4B, lane 3, bottom panel). Because PDGF but not EGF reduces the amount of GHR and inhibits GH-induced tyrosyl phosphorylation of JAK2 and GHR, it seems unlikely that ERKs 1 and 2 are involved in the inhibition of GH signaling by PDGF.

Because PDGF appears to be a more potent activator of Akt than EGF (Fig. 4B, bottom panel), a downstream effector of PI 3-kinase (59), we tested whether PI 3-kinase plays a role in the inhibition of GH signaling by PDGF. 3T3-F442A cells were pretreated for 20 min with 200 nM wortmannin, a potent inhibitor of PI 3-kinase, incubated with 25 ng/ml PDGF for 20 min, and then stimulated for 10 min with 50 ng/ml GH. Proteins in cell lysates were immunoprecipitated with alpha JAK2 or alpha GHBP and immunoblotted with alpha PY, alpha JAK2, or alpha GHBP. Wortmannin is unable to inhibit the effect of PDGF on GH-induced tyrosyl phosphorylation of JAK2 (Fig. 4C, upper panel) or GHR (Fig. 4D, upper panel). To verify the inhibition of PI 3-kinase by wortmannin, proteins in cell lysates were immunoblotted with antibody recognizing specifically the phosphorylated, active form of Akt. GH stimulates phosphorylation of Akt (Fig. 4E, lane 2). PDGF and GH together stimulate phosphorylation of Akt to a much greater extent (Fig. 4E, lane 3). Wortmannin completely blocks phosphorylation of Akt by GH and PDGF (Fig. 4E, lane 4). These data indicate that PI 3-kinase and signaling molecules downstream of PI 3-kinase are unlikely to be involved in the inhibition of GH signaling by PDGF.

PKCs Are Required for Down-regulation of GH Signaling by PDGF-- In 3T3-F442A cells, PDGF stimulates a more robust and sustained tyrosyl phosphorylation of PLCgamma compared with EGF (Fig. 4B, top panel, and data not shown). Because PLCgamma is an upstream activator of PKC, and activation of the PKC pathway by PMA has been shown to decrease GH binding (57, 58, 60), we hypothesized that PKC-initiated signaling events play a role in the inhibition of GH signaling by PDGF. To test this hypothesis, PMA-sensitive isoforms of PKC were depleted from 3T3-F442A cells by incubating cells with PMA (200 nM) for 25 h. The treated cells were then incubated with 25 ng/ml PDGF for 20 min, followed by 50 ng/ml GH for 10 min. JAK2 was immunoprecipitated with alpha JAK2 and immunoblotted with alpha PY (Fig. 5A, upper panel) or alpha JAK2 (Fig. 5A, lower panel). PDGF and PMA dramatically inhibit the tyrosyl phosphorylation of JAK2 induced by GH (Fig. 5A, lanes 3 and 4), consistent with the data in Figs. 1 and 2. Depletion of PMA-sensitive PKCs abolishes the ability of either PDGF or PMA to inhibit GH-stimulated tyrosyl phosphorylation of JAK2 (Fig. 5A, lanes 7 and 8, upper panel). Treatment with PMA for 25 h does not increase basal tyrosyl phosphorylation of JAK2 (Fig. 5A, lane 5, upper panel) but slightly increases the amount of JAK2 (Fig. 5A, lower panel). Similarly, when PMA-sensitive PKCs are depleted, GH-stimulated tyrosyl phosphorylation of GHR is not affected by either PDGF or PMA (Fig. 5B, lanes 7 and 8).


View larger version (42K):
[in this window]
[in a new window]
  		Fig. 5.   Depletion of PKCs blocks the inhibition of GH-induced tyrosyl phosphorylation of JAK2 and GHR by PDGF and PMA. 3T3-F442A cells were pretreated with (A and B, lanes 5-8) or without (A and B, lanes 1-4) PMA (200 nM) for 25 h. Cells were then preincubated with or without PDGF (25 ng/ml) or PMA (200 nM) for 20 min prior to GH (50 ng/ml) stimulation for 10 min. A, JAK2 was immunoprecipitated (IP) with alpha JAK2 and immunoblotted (IB) with alpha PY (upper panel). The same blot was reprobed with alpha JAK2 (lower panel). B, GHR was immunoprecipitated with alpha GHBP and immunoblotted with alpha PY.

To confirm further a role for PKCs in the inhibition of GH signaling by PDGF, 3T3-F442A cells were incubated with GF109203X, a potent inhibitor of PKCs, prior to the treatment with PDGF and GH. Proteins in cell lysates were immunoprecipitated with alpha JAK2 (Fig. 6A) or alpha GHBP (Fig. 6B) and immunoblotted with alpha PY (Fig. 6, A and B, upper panel), alpha JAK2 (Fig. 6A, lower panel), or alpha GHBP (Fig. 6B, lower panel). GF109203X blocks the inhibitory effect of PMA on GH-dependent tyrosyl phosphorylation of JAK2 (Fig. 6A, lane 4 versus 6, upper panel) and GHR (Fig. 6B, lane 4 versus 6, upper panel). It also substantially reduces the inhibitory effect of PDGF on GH-stimulated tyrosyl phosphorylation of JAK2 (Fig. 6A, lane 3 versus 5, upper panel) and GHR (Fig. 6B, lane 3 versus 5, upper panel).


View larger version (32K):
[in this window]
[in a new window]
  		Fig. 6.   GF109203X inhibits PDGF- and PMA-induced reduction of GH-dependent tyrosyl phosphorylation of JAK2 and GHR. 3T3-F442A cells were pretreated with or without GF109203X (GF, 500 nM) for 20 min and then treated for 20 min with PDGF (25 ng/ml) or PMA (200 nM). The cells were then stimulated with GH (50 ng/ml) for 10 min. Proteins in cell lysates were immunoprecipitated (IP) with alpha JAK2 (A) or alpha GHBP (B) and immunoblotted (IB) with alpha PY (A and B, upper panel). The blots were reprobed with alpha JAK2 (A, lower panel) and alpha GHBP (B, lower panel), respectively.

LPA Down-regulates GH Signaling-- Many hormones and growth factors that activate G protein-coupled receptors activate the PKC pathway. We hypothesized that these ligands would down-regulate GH signaling in a fashion similar to PDGF. We tested whether LPA, whose receptor is widely expressed in tissues and cell lines and activates PKCs via Gq (61), alters GH-induced tyrosyl phosphorylation of JAK2 and GHR. 3T3-F442A cells were preincubated for 20 min with the indicated concentrations of LPA prior to 50 ng/ml GH stimulation for an additional 10 min. Proteins in cell lysates were immunoprecipitated with alpha JAK2 (Fig. 7A) and alpha GHBP (Fig. 7, B and C) and immunoblotted with alpha PY (Fig. 7, A-C, upper panel), alpha JAK2 (Fig. 7A, lower panel), and alpha GHBP (Fig. 7C, lower panel). LPA substantially inhibits GH-stimulated tyrosyl phosphorylation of JAK2 (Fig. 7A) and GHR (Fig. 7, B and C, upper panel). LPA also significantly reduces the amount of total cellular GHR (Fig. 7C, lower panel) and GH binding (data not shown). Depletion of PMA-sensitive PKCs blocks the inhibitory effect of LPA on GH-stimulated tyrosyl phosphorylation of JAK2 and GHR (data not shown).


View larger version (36K):
[in this window]
[in a new window]
  		Fig. 7.   LPA reduces total cellular GHR and inhibits GH-induced tyrosyl phosphorylation of JAK2 and GHR. 3T3-F442A cells were pretreated for 20 min with the indicated concentration of LPA prior to GH (50 ng/ml) stimulation for 10 min. A, JAK2 was immunoprecipitated (IP) with alpha JAK2 and immunoblotted (IB) with alpha PY (upper panel). The blot was reprobed with alpha JAK2 (lower panel). B and C, GHR was immunoprecipitated with alpha GHBP and immunoblotted with alpha PY (B and C, upper panel). The blot was reprobed with alpha GHBP (C, lower panel).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We report in this work that PDGF and LPA are potent inhibitors of GH signaling. Pretreating cells with PDGF or LPA dramatically inhibits GH-dependent tyrosyl phosphorylation of the tyrosine kinase JAK2, a key enzyme in GH signaling. Because tyrosyl phosphorylation of JAK2 correlates with its activation (6), PDGF and LPA most likely strongly inhibit GH-dependent activation of JAK2. Consistent with this idea, GH-induced tyrosyl phosphorylation of GHR, a physiological substrate of JAK2, is also severely inhibited by PDGF and LPA. Because activation of JAK2 and tyrosyl phosphorylation of JAK2 and GHR are early obligatory steps in GH signaling (7, 62), it is likely that most, if not all, downstream signaling events are inhibited by PDGF and LPA.

Another important finding of this work is that PDGF and LPA decrease both GH binding and total cellular GHR. PDGF substantially reduces both GH binding and total cellular GHR. The decrease in GH binding and number of GHR roughly correlates with the inhibition of GH-induced tyrosyl phosphorylation of JAK2 and GHR, suggesting that the reduction of GHR is the primary cause of down-regulation of GH signaling by PDGF and LPA. In agreement with this idea, PDGF does not inhibit the activation of JAK2 by LIF or IFN-gamma , suggesting that PDGF and LPA do not directly inhibit JAK2.

When PMA-sensitive isoforms of PKC are depleted by preincubating cells for 25 h with PMA, neither PDGF nor LPA inhibits GH-induced tyrosyl phosphorylation of JAK2 and GHR, indicating that PMA-sensitive PKCs are required for PDGF- and LPA-induced inhibition of GH action. In support of this hypothesis, GF109203X, a potent inhibitor of PKCs, substantially blocks the inhibition of GH-induced tyrosyl phosphorylation of JAK2 and GHR and the reduction in GHR by PDGF. In agreement with an essential role of PKC in down-regulation of GH signaling by PDGF and LPA, activation of PKC by PMA is sufficient to inhibit GH-induced tyrosyl phosphorylation of JAK2 and GHR and reduce the number of GHR. Interestingly, depletion of PMA-sensitive PKCs blocks the ability of both PDGF and PMA to decrease GH signaling, whereas GF109203X, which is capable of almost completely blocking the ability of PMA to inhibit GH signaling, only partially blocks the inhibitory effect of PDGF on GH signaling. One explanation for this apparent discrepancy is that PDGF stimulates a subset of PKCs that are less sensitive to GF109203X. Different PKCs are known to have different sensitivities to GF109203X (63, 64).

It is unlikely that the MEK/ERK cascade and the PI 3-kinase pathway contribute to the down-regulation of GH signaling by PDGF and LPA. EGF, which activates ERKs 1 and 2 to a similar extent as PDGF, is unable to either inhibit GH-induced tyrosyl phosphorylation of JAK2 and GHR or reduce the number of GHR. In addition, when ERKs 1 and 2 are inhibited by PD98059, a potent inhibitor of MEK, or partially inhibited by 1 µM wortmannin, the inhibitory effect of PDGF on GH signaling is not affected (data not shown). Similarly, inhibition of PI 3-kinase activity with wortmannin completely blocks GH- and PDGF-induced phosphorylation and activation of Akt but does not affect PDGF-induced inhibition of GH signaling.

Although it has been reported previously that PMA can regulate levels of GH binding (57, 58, 60), this is the first report of physiological ligands that down-regulate GH binding and GHR signaling by a PKC-dependent mechanism. Two models have been proposed to explain the inhibition of GH signaling by PMA-sensitive PKCs (57, 58, 60). In the first model (57, 58), activation of PKCs by PMA leads to a redistribution of GHR within the cell, resulting in a reduction of cell surface GHR and an increase in cytoplasmic GHR. Our data do not support the second half of this model (i.e. cytoplasmic GHR are increased in number). However, we cannot exclude the possibility that PMA activation of PKCs increases the rate of internalization of GHR which are then rapidly degraded, resulting in a decrease in the overall number of GHR.

In the second model (60), activation of PKCs by PMA leads to the activation of a protease that cleaves cell surface GHR. This results in the formation of GHR lacking its extracellular domain and release of GH-binding protein (GHBP, the extracellular domain of GHR) from the cell (shedding). Recent evidence suggests that the PKC-regulated protease may be tumor necrosis factor (TNF)-alpha -converting enzyme (TACE) or a TACE-like metalloprotease (65). Consistent with a TACE-like metaolloprotease cleaving GHR and producing GHBP, PMA activation of TACE family members has been shown to lead to ectodomain cleavage of the receptors for TNF-alpha and transforming growth factor-alpha , the adhesion protein L-selectin, and beta -amyloid protein precursor (66, 67). Also consistent with this second model is the finding that the PMA-induced decrease in GHR binding is dependent upon a region of GHR composed of the extracellular and transmembrane domains (57). The best evidence for the production of GHBP as a by-product of GHR cleavage comes from studies with GHR from humans and rabbits (60, 68-70), species in which proteolysis is thought to be the major source of GHBP (71). However, a preliminary report indicates that formation of GHBP as a result of proteolytic cleavage of GHR also occurs with GHR from mice (65), a species for which GHBP is also synthesized from the gene for GHR as an alternative splice product (71). If proteolysis accounts for at least some of the PKC-induced decrease in GH binding, then it seems likely that PDGF, LPA, or any other ligand that activates PMA-sensitive PKCs will stimulate the production of GHBP in cells that express GHR. Up to 50% of human serum GH is believed to bind to GHBP (72). The interaction of GH with GHBP is proposed to increase the stability of GH. GHBP has also been reported to have inhibitory (73) as well as potentiating roles in GH signaling (74-76). Thus, in addition to regulating the ability of an individual cell to respond to GH, ligands such as PDGF and LPA could also increase the local production of GHBP and thereby modulate GH signaling in neighboring cells. In addition, because PMA is known to activate members of the TACE family of proteases, it seems likely that PDGF and LPA might stimulate the cleavage of the receptors for TNF-alpha and transforming growth factor-alpha , the adhesion protein L-selectin, and/or the beta -amyloid protein precursor.

The finding that PDGF and LPA inhibit GH signaling via a PKC-dependent pathway may have important therapeutic implications. Receptors for PDGF and LPA are members of the receptor tyrosine kinase family and the G protein-coupled receptor family, respectively. Many ligands, including numerous chemical compounds and a variety of hormones, cytokines, and growth factors, are able to activate PKCs. It seems likely that any ligand that activates PMA-sensitive PKCs will also down-regulate GH action. Receptors for a variety of hormones, growth factors, and cytokines that activate PKC, including LPA and PDGF, are expressed in GH target tissues and cells. It is likely that these ligands constantly modulate GH action in vivo by regulating the abundance of GHR on the plasma membrane through a PKC-dependent pathway. Therefore, it is extremely important during GH replacement therapy to be cognizant of the fact that agents that stimulate PKCs will render a given dose of GH much less effective. Similarly, it is important to be cognizant that continuous treatment with agents that stimulate PKCs may inhibit growth.

In summary, we have shown that PDGF and LPA inhibit GH-induced tyrosyl phosphorylation of JAK2 and GHR. PDGF and LPA appear to down-regulate GH signaling by decreasing the number of GHR. PMA-sensitive PKCs, but not ERKs 1 and 2 nor PI 3-kinase, are required for these actions of PDGF and LPA. We propose that any ligand that actives PMA-sensitive isoforms of PKC inhibits GH signaling in a similar fashion.

    ACKNOWLEDGEMENTS

We thank X. Wang for technical assistance and B. Hawkins for assistance with the manuscript.

    FOOTNOTES

* This work was supported in part by National Institutes of Health (NIH) Grant DK 34171. Iodination of hGH was performed at the Reproductive Sciences Program core facility. This facility was funded by NIH Grant P30 HD 18258.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by National Institutes of Health Predoctoral Fellowship GM07315.

§ To whom correspondence should be addressed: Dept. of Physiology, University of Michigan Medical School, Ann Arbor, MI 48109-0622. Tel.: 734-763-2561; Fax: 734-647-9523; E-mail: cartersu@umich.edu.

2 Proteins in the tight band migrating with apparent Mr ~90,000 in Fig. 2, B and D, lower panel, are believed not to be functional GHR, because they are not tyrosyl-phosphorylated in response to GH (Fig. 2, B and D, upper panel).

    ABBREVIATIONS

The abbreviations used are: GH, growth hormone; GHR, GH receptor; GHBP, GH-binding protein; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; LPA, lysophosphatidic acid; IFN-gamma , interferon-gamma ; LIF, leukemia inhibitory factor; ERK, extracellular signal-regulated kinase; PI 3-kinase, phosphatidylinositol 3-kinase; PKC, protein kinase C; PMA, 4beta -phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; MAP, mitogen-activated protein; MEK, MAP kinase/ERK kinase; BSA, bovine serum albumin; hGH, human growth hormone; TNF-alpha , tumor necrosis factor-alpha ; TACE, TNF-alpha -converting enzyme; PLC, phospholipase C; alpha PY, anti-phosphotyrosine antibody 4G10.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Isaksson, O. G., Eden, S., and Jansson, J. O. (1985) Annu. Rev. Physiol. 47, 483-499[CrossRef][Medline] [Order article via Infotrieve]
2. Strobl, J. S., and Thomas, M. J. (1994) Pharmacol. Rev. 46, 1-34[Abstract]
3. Tritos, N. A., and Mantzoros, C. S. (1998) Am. J. Med. 105, 44-57[CrossRef][Medline] [Order article via Infotrieve]
4. Windisch, P. A., Papatheofanis, F. J., and Matuszewski, K. A. (1998) Ann. Pharmacother. 32, 437-445[Abstract]
5. Herndon, D. N., Hawkins, H. K., Nguyen, T. T., Pierre, E., Cox, R., and Barrow, R. E. (1995) Ann. Surg. 221, 649-656[Medline] [Order article via Infotrieve]
6. Argetsinger, L. S., Campbell, G. S., Yang, X., Witthuhn, B. A., Silvennoinen, O., Ihle, J. N., and Carter-Su, C. (1993) Cell 74, 237-244[CrossRef][Medline] [Order article via Infotrieve]
7. Argetsinger, L. S., and Carter-Su, C. (1996) Physiol. Rev. 76, 1089-1107[Abstract/Free Full Text]
8. Campbell, G. S., Meyer, D. J., Raz, R., Levy, D. E., Schwartz, J., and Carter-Su, C. (1995) J. Biol. Chem. 270, 3974-3979[Abstract/Free Full Text]
9. Meyer, D. J., Campbell, G. S., Cochran, B. H., Argetsinger, L. S., Larner, A. C., Finbloom, D. S., Carter-Su, C., and Schwartz, J. (1994) J. Biol. Chem. 269, 4701-4704[Abstract/Free Full Text]
10. Smit, L. S., VanderKuur, J. A., Stimage, A., Han, Y., Luo, G., Yu-lee, L.-Y., Schwartz, J., and Carter-Su, C. (1997) Endocrinology 138, 3426-3434[Abstract/Free Full Text]
11. Sotiropoulos, A., Perrot-Applanat, M., Dinerstein, H., Pallier, A., Postel-Vinay, M. C., Finidori, J., and Kelly, P. A. (1994) Endocrinology 135, 1292-1298[Abstract]
12. Gronowski, A. M., Zhong, Z., Wen, Z., Thomas, M. J., Darnell, J. E., Jr., and Rotwein, P. (1995) Mol. Endocrinol. 9, 171-177[Abstract/Free Full Text]
13. Yi, W., Kim, S. O., Jiang, J., Park, S. H., Kraft, A. S., Waxman, D. J., and Frank, S. J. (1996) Mol. Endocrinol. 10, 1425-1443[Abstract/Free Full Text]
14. VanderKuur, J., Allevato, G., Billestrup, N., Norstedt, G., and Carter-Su, C. (1995) J. Biol. Chem. 270, 7587-7593[Abstract/Free Full Text]
15. Rui, L., Mathews, L. S., Hotta, K., Gustafson, T. A., and Carter-Su, C. (1997) Mol. Cell. Biol. 17, 6633-6644[Abstract]
16. Rui, L., and Carter-Su, C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 7172-7177[Abstract/Free Full Text]
17. Carter-Su, C., King, A. P. J., Smit, L. S., VanderKuur, J. A., Argetsinger, L. S., Campbell, G. S., and Huo, W.-H. (1997) J. Anim. Sci. 75 Suppl. 2, 1-10[Abstract/Free Full Text]
18. Claesson-Welsh, L. (1994) J. Biol. Chem. 269, 32023-32026[Free Full Text]
19. Claesson-Welsh, L. (1996) Int. J. Biochem. Cell Biol. 28, 373-385[CrossRef][Medline] [Order article via Infotrieve]
20. Moolenaar, W. H. (1995) Curr. Opin. Cell Biol. 7, 203-210[CrossRef][Medline] [Order article via Infotrieve]
21. Moolenaar, W. H. (1995) J. Biol. Chem. 270, 12949-12952[Free Full Text]
22. Lindahl, A., Nilsson, A., and Isaksson, O. G. (1987) J. Endocrinol. 115, 263-271[Abstract/Free Full Text]
23. Maor, G., Hochberg, Z., von der Mark, K., Heinegard, D., and Silbermann, M. (1989) Endocrinology 125, 1239-1245[Abstract/Free Full Text]
24. Nixon, B. T., and Green, H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3429-3432[Abstract/Free Full Text]
25. Stiles, C. D., Capone, G. T., Scher, C. D., Antoniades, H. N., Van Wyk, J. J., and Pledger, W. J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 1279-1283[Abstract/Free Full Text]
26. Smith, J. C., and Stiles, C. D. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4363-4367[Abstract/Free Full Text]
27. Guller, S., Sonenberg, M., Wu, K.-Y., Szabo, P., and Corin, R. E. (1989) Endocrinology 125, 2360-2367[Abstract/Free Full Text]
28. Burton, K. A., Kabigting, E. B., Clifton, D. K., and Steiner, R. A. (1992) Endocrinology 131, 958-963[Abstract/Free Full Text]
29. Tiong, T. S., and Herington, A. C. (1991) Endocrinology 129, 1628-1634[Abstract/Free Full Text]
30. Tiong, T. S., Freed, K. A., and Herington, A. C. (1989) Biochem. Biophys. Res. Commun. 158, 141-148[CrossRef][Medline] [Order article via Infotrieve]
31. Mertani, H. C., and Morel, G. (1995) Mol. Cell. Endocrinol. 109, 47-61[CrossRef][Medline] [Order article via Infotrieve]
32. Hu, Y., Schett, G., Zou, Y., Dietrich, H., and Xu, Q. (1998) Brain Res. Mol. Brain Res. 53, 252-259[Medline] [Order article via Infotrieve]
33. Kimura, M., and Ogihara, M. (1998) Jpn. J. Pharmacol. 76, 165-174[CrossRef][Medline] [Order article via Infotrieve]
34. Kimura, M., and Ogihara, M. (1998) Biol. & Pharm. Bull. 21, 22-28[Medline] [Order article via Infotrieve]
35. Hock, J. M., and Canalis, E. (1994) Endocrinology 134, 1423-1428[Abstract/Free Full Text]
36. Ohlsson, C., and Vidal, O. (1998) Eur. J. Clin. Invest. 28, 184-186[CrossRef][Medline] [Order article via Infotrieve]
37. Ohlsson, C., Bengtsson, B. A., Isaksson, O. G., Andreassen, T. T., and Slootweg, M. C. (1998) Endocr. Rev. 19, 55-79[Abstract/Free Full Text]
38. Chin, E., Zhou, J., and Bondy, C. A. (1992) Endocrinology 131, 3061-3066[Abstract/Free Full Text]
39. Alpers, C. E., Seifert, R. A., Hudkins, K. L., Johnson, R. J., and Bowen-Pope, D. F. (1993) Kidney Int. 43, 286-294[Medline] [Order article via Infotrieve]
40. Van Zoelen, E. J., Van Rotterdam, W., Van de Wetering, R. A., and Heldin, C. H. (1993) Growth Factors 9, 329-339[Medline] [Order article via Infotrieve]
41. Shoba, L., An, M. R., Frank, S. J., and Lowe, W. L., Jr. (1999) Mol. Cell. Endocrinol. 152, 125-136[CrossRef][Medline] [Order article via Infotrieve]
42. Yablonka-Reuveni, Z., Balestreri, T. M., and Bowen-Pope, D. F. (1990) J. Cell Biol. 111, 1623-1629[Abstract/Free Full Text]
43. Tidball, J. G., and Spencer, M. J. (1993) J. Cell Biol. 123, 627-635[Abstract/Free Full Text]
44. Albrecht, D. E., and Tidball, J. G. (1997) J. Biol. Chem. 272, 2236-2244[Abstract/Free Full Text]
45. Florini, J. R., Ewton, D. Z., and Coolican, S. A. (1996) Endocr. Rev. 17, 481-517[Abstract/Free Full Text]
46. Hotamisligil, G. S., Murray, D. L., Choy, L. N., and Spiegelman, B. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4854-4858[Abstract/Free Full Text]
47. Hotamisligil, G. S., Peraldi, P., Budavari, A., Ellis, R., White, M. F., and Spiegelman, B. M. (1996) Science 271, 665-668[Abstract]
48. Goetzl, E. J., and An, S. (1998) FASEB J. 12, 1589-1598[Abstract/Free Full Text]
49. Silvennoinen, O., Witthuhn, B., Quelle, F. W., Cleveland, J. L., Yi, T., and Ihle, J. N. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8429-8433[Abstract/Free Full Text]
50. Sadeghi, H., Wang, B. S., Lumanglas, A. L., Logan, J. S., and Baumbach, W. R. (1990) Mol. Endocrinol. 4, 1799-1805[Abstract/Free Full Text]
51. Campbell, G. S., Christian, L. J., and Carter-Su, C. (1993) J. Biol. Chem. 268, 7427-7434[Abstract/Free Full Text]
52. Rui, L., and Carter-Su, C. (1998) J. Biol. Chem. 273, 21239-21245[Abstract/Free Full Text]
53. Nixon, T., and Green, H. (1983) J. Cell. Physiol. 115, 291-296[CrossRef][Medline] [Order article via Infotrieve]
54. Silva, C. M., Day, R. N., Weber, M. J., and Thorner, M. O. (1993) Endocrinology 133, 2307-2312[Abstract/Free Full Text]
55. Silva, C. M., Weber, M. J., and Thorner, M. O. (1993) Endocrinology 132, 101-108[Abstract/Free Full Text]
56. Argetsinger, L. S., Hsu, G. W., Myers, M. G., Jr., Billestrup, N., Norstedt, G., White, M. F., and Carter-Su, C. (1995) J. Biol. Chem. 270, 14685-14692[Abstract/Free Full Text]
57. King, A. P. J., Tseng, M. J., Logsdon, C. D., Billestrup, N., and Carter-Su, C. (1996) J. Biol. Chem. 271, 18088-18094[Abstract/Free Full Text]
58. Suzuki, K., Suzuki, S., Saito, Y., Ikebuchi, H., and Terao, T. (1990) J. Biol. Chem. 265, 11320-11327[Abstract/Free Full Text]
59. Franke, T. F., Kaplan, D. R., and Cantley, L. C. (1997) Cell 88, 435-437[CrossRef][Medline] [Order article via Infotrieve]
60. Alele, J., Jiang, J., Goldsmith, J. F., Yang, X., Maheshwari, H. G., Black, R. A., Baumann, G., and Frank, S. J. (1998) Endocrinology 139, 1927-1935[Abstract/Free Full Text]
61. Seewald, S., Schmitz, U., Seul, C., Ko, Y., Sachinidis, A., and Vetter, H. (1999) Am. J. Hypertens. 12, 532-537[CrossRef][Medline] [Order article via Infotrieve]
62. Carter-Su, C., Schwartz, J., and Smit, L. S. (1996) Annu. Rev. Physiol. 58, 187-207[CrossRef][Medline] [Order article via Infotrieve]
63. Martiny-Baron, G., Kazanietz, M. G., Mischak, H., Blumberg, P. M., Kochs, G., Hug, H., Marme, D., and Schachtele, C. (1993) J. Biol. Chem. 268, 9194-9197[Abstract/Free Full Text]
64. Gschwendt, M., Dieterich, S., Rennecke, J., Kittstein, W., Mueller, H. J., and Johannes, F. J. (1996) FEBS Lett. 392, 77-80[CrossRef][Medline] [Order article via Infotrieve]
65. Zhang, Y., Guan, R., Jiang, J., Maheshwari, H. G., Baumann, C. A., Black, R. A., Baumann, G., and Frank, S. J. (1999) Proceeding of the 81st Annual Meeting of the Endocrine Society, San Diego, CA, June 12-15, 1999 , p. 154, Endocrine Society Press, Bethesda, MD
66. Peschon, J. J., Slack, J. L., Reddy, P., Stocking, K. L., Sunnarborg, S. W., Lee, D. C., Russell, W. E., Castner, B. J., Johnson, R. S., Fitzner, J. N., Boyce, R. W., Nelson, N., Kozlosky, C. J., Wolfson, M. F., Rauch, C. T., Cerretti, D. P., Paxton, R. J., March, C. J., and Black, R. A. (1998) Science 282, 1281-1284[Abstract/Free Full Text]
67. Buxbaum, J. D., Liu, K. N., Luo, Y., Slack, J. L., Stocking, K. L., Peschon, J. J., Johnson, R. S., Castner, B. J., Cerretti, D. P., and Black, R. A. (1998) J. Biol. Chem. 273, 27765-27767[Abstract/Free Full Text]
68. Sotiropoulos, A., Goujon, L., Simonin, G., Kelly, P. A., Postel-Vinay, M. C., and Finidori, J. (1993) Endocrinology 132, 1863-1865[Abstract/Free Full Text]
69. Baumann, G. (1994) J. Endocrinol. 141, 1-6[Abstract/Free Full Text]
70. Barnard, R., and Waters, M. J. (1997) J. Endocrinol. 153, 1-14[Abstract/Free Full Text]
71. Waters, M. J. (1999) in Handbook of Physiology (Kostyo, J. L., ed), Vol. V , pp. 397-444, Oxford University Press, New York
72. Baumann, G., Amburn, K., and Shaw, M. A. (1988) Endocrinology 122, 976-984[Abstract/Free Full Text]
73. Hansen, B. S., Hjorth, S., Welinder, B. S., Skriver, L., and De Meyts, P. (1993) Endocrinology 133, 2809-2817[Abstract/Free Full Text]
74. Lim, L., Spencer, S. A., McKay, P., and Waters, M. J. (1990) Endocrinology 127, 1287-1291[Abstract/Free Full Text]
75. Mannor, D. A., Winer, L. M., Shaw, M. A., and Baumann, G. (1991) J. Clin. Endocrinol. & Metab. 73, 30-34[Abstract/Free Full Text]
76. Clark, R. G., Mortensen, D. L., Carlsson, L. M., Spencer, S. A., McKay, P., Mulkerrin, M., Moore, J., and Cunningham, B. C. (1996) Endocrinology 137, 4308-4315[Abstract]

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 EndocrinologyHome page
K. He, K. Loesch, J. W. Cowan, X. Li, L. Deng, X. Wang, J. Jiang, and S. J. Frank
Janus Kinase 2 Enhances the Stability of the Mature Growth Hormone Receptor
Endocrinology, November 1, 2005; 146(11): 4755 - 4765.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. Duan, M. Li, and L. Rui
SH2-B Promotes Insulin Receptor Substrate 1 (IRS1)- and IRS2-mediated Activation of the Phosphatidylinositol 3-Kinase Pathway in Response to Leptin
J. Biol. Chem., October 15, 2004; 279(42): 43684 - 43691.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Huang, S.-O. Kim, J. Jiang, and S. J. Frank
Growth Hormone-induced Phosphorylation of Epidermal Growth Factor (EGF) Receptor in 3T3-F442A Cells: MODULATION OF EGF-INDUCED TRAFFICKING AND SIGNALING
J. Biol. Chem., May 23, 2003; 278(21): 18902 - 18913.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
V. Beauloye, B. Willems, V. de Coninck, S. J. Frank, M. Edery, and J.-P. Thissen
Impairment of Liver GH Receptor Signaling by Fasting
Endocrinology, March 1, 2002; 143(3): 792 - 800.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
R. Guan, Y. Zhang, J. Jiang, C. A. Baumann, R. A. Black, G. Baumann, and S. J. Frank
Phorbol Ester- and Growth Factor-Induced Growth Hormone (GH) Receptor Proteolysis and GH-Binding Protein Shedding: Relationship to GH Receptor Down-Regulation
Endocrinology, March 1, 2001; 142(3): 1137 - 1147.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rui, L.
Right arrow Articles by Carter-Su, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rui, L.
Right arrow Articles by Carter-Su, C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2000 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement