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Physical and Functional Interaction of Growth Hormone and Insulin-Like Growth Factor-I Signaling Elements

Department of Medicine (Y.H., S.-O.K., N.Y., J.J., S.J.F.), Division of Endocrinology, Diabetes, and Metabolism, and Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294; and Endocrinology Section (S.J.F.), Medical Service, Veterans Affairs Medical Center, Birmingham, Alabama 35233

Address all correspondence and requests for reprints to: Stuart J. Frank, University of Alabama at Birmingham, 1530 3rd Avenue South, BDB 861, Birmingham, Alabama 35294-0012. E-mail: sjfrank{at}uab.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
GH and IGF-I are critical regulators of growth and metabolism. GH interacts with the GH receptor (GHR), a cytokine superfamily receptor, to activate the cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2), and initiate intracellular signaling cascades. IGF-I, produced in part in response to GH, binds to the heterotetrameric IGF-I receptor (IGF-IR), which is an intrinsic tyrosine kinase growth factor receptor that triggers proliferation, antiapoptosis, and other biological actions. Previous in vitro and overexpression studies have suggested that JAKs may interact with IGF-IR and that IGF-I stimulation may activate JAKs. In this study, we explore interactions between GHR-JAK2 and IGF-IR signaling pathway elements utilizing the GH and IGF-I-responsive 3T3-F442A and 3T3-L1 preadipocyte cell lines, which endogenously express both the GHR and IGF-IR. We find that GH induces formation of a complex that includes GHR, JAK2, and IGF-IR in these preadipocytes. The assembly of this complex in intact cells is rapid, GH concentration dependent, and can be prevented by a GH antagonist, G120K. However, it is not inhibited by the kinase inhibitor, staurosporine, which markedly inhibits GHR tyrosine phosphorylation. Moreover, complex formation does not appear dependent on GH-induced activation of the ERK or phosphatidylinositol 3-kinase signaling pathways or on the tyrosine phosphorylation of GHR, JAK2, or IGF-IR. These results suggest that GH-induced formation of the GHR-JAK2-IGF-IR complex is governed instead by GH-dependent conformational change(s) in the GHR and/or JAK2. We further demonstrate that GH and IGF-I can synergize in acute aspects of signaling and that IGF-I enhances GH-induced assembly of conformationally active GHRs. These findings suggest the existence of previously unappreciated relationships between these two hormones.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
GH IS AN important regulator of growth and me-tabolism that is largely derived from the anterior pituitary gland. All known actions of GH require its specific binding to the GH receptor (GHR), a transmembrane glycoprotein member of the cytokine receptor superfamily (1, 2). The GHR is devoid of intrinsic tyrosine kinase activity, but is physically and functionally coupled to Janus kinase 2 (JAK2), a nonreceptor cytoplasmic tyrosine kinase member of the JAK family (3, 4). GH-induced JAK2 activation results in engagement of several intracellular signaling pathways, including STATs (signal transducers and activators of transcription) –1, –3, and –5, ERKs, and phosphatidylinositol 3-kinase (PI3K) (reviewed in Refs.2 , 5 , and 6). Activation of STAT5b is prominent in GH signaling and is critical for regulation of several GH-responsive hepatic genes, including the IGF-I gene (7, 8, 9). Activation of ERKs has been linked to GH-induced activation of c-fos gene expression (10) and fosters cross-talk between the GHR and epidermal growth factor receptor family signaling pathways (11, 12).

The mechanisms by which GH exerts its somatogenic and metabolic effects have been studied intensively but are still incompletely understood. A major physiological effector of GH is IGF-I (also known as somatomedin-C), the expression and secretion of which is induced by GH in various target tissues. As originally articulated, the somatomedin hypothesis of GH action postulated that GH stimulated the hepatic secretion of IGF-I, which then functioned in an endocrine manner to interact with IGF-I receptors (IGF-IR) in tissues that responded with growth (13, 14). Recent studies of mice with unrestricted targeted deletion of the IGF-I gene validated the importance of IGF-I in growth mediation but also suggested that GH may exert growth-promoting activity in some tissues even in the absence of IGF-I (15, 16). Further, liver-specific knockout of IGF-I, although dramatically lowering serum IGF-I levels, does not prevent normal growth, suggesting that autocrine/paracrine IGF-I (rather than liver-derived IGF-I) may predominate for normal postnatal growth (17, 18). Interestingly, a recent report of the effect of combined knockout of the GHR and IGF-I genes compared with individual knockout of each suggested that GH and IGF-I may independently contribute to growth in the mouse (19).

IGF-I signals through the IGF-IR, a cell surface heterotetramer with similarity to the insulin receptor that has intrinsic kinase activity embedded in its ß-subunit cytoplasmic domains (20, 21). IGF-IR activation engages the ERK and PI3K pathways via Src homology and collagen domain protein and insulin receptor substrate-1 phosphorylation to effect proliferation, antiapoptosis, and other biological actions (22, 23). Recent reports suggest that, in addition to causing activation of their own receptor kinases, IGF-I and insulin may activate members of the JAK family, including JAK1 and JAK2 (24, 25, 26, 27). The impact of JAK activation by these growth factors is not clear in all cases and in most instances has been observed in the setting of overexpression of at least one of the components. In one study, recombinant proteins were used in vitro to demonstrate physical association of JAK1 with insulin and IGF-I receptors, which was dependent on tyrosine phosphorylation of the receptors (28).

We now explore the potential for interaction between GHR-JAK2 and IGF-IR signaling pathway elements. In these studies, we utilize the GH and IGF-I-responsive 3T3-F442A and 3T3-L1 preadipocyte cell lines, which endogenously express both the GHR and IGF-IR. We uncover evidence for the GH-induced formation of a complex that includes GHR, JAK2, and IGF-IR in these preadiopocytes. The assembly of this complex in intact cells can be prevented by a GH antagonist, G120K, but is not inhibited by the kinase inhibitor, staurosporine. Moreover, complex formation does not appear dependent on GH-induced activation of the ERK or PI3K signaling pathways or on the tyrosine phosphorylation of GHR, JAK2, or IGF-IR. These results suggest that GH-induced formation of the GHR-JAK2-IGF-IR complex is governed instead by GH-dependent conformational change(s) in the GHR and/or JAK2. We further demonstrate that GH and IGF-I can synergize in acute aspects of signaling and that IGF-I enhances GH-induced assembly of conformationally active GHRs. These findings suggest the existence of previously unappreciated relationships between these two hormones.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
GH-Induced Formation of a Complex that Includes the IGF-IR and the Tyrosine-Phosphorylated GHR and JAK2
Interactions between JAKs and the IGF-I and insulin receptors have previously been observed in overexpression systems (28). Given the important physiological relationships between GH and IGF-I action, we asked whether cross-talk might also exist between elements of these two signaling systems. The 3T3-F442A preadipocyte is an appealing system in which to study GH signaling, in that it endogenously expresses GHR and responds to GH with detectable activation of multiple pathways (3, 10, 11, 12, 29, 30, 31, 32, 33). Specific binding sites for radioiodinated IGF-I have also been detected on 3T3-F442A preadipocytes, with an estimated 380,000 IGF-I receptors per cell (34). Our initial studies used these cells.

To examine their effects on tyrosine phosphorylation of proximal GH and IGF-I signaling elements, GH, IGF-I, or vehicle (-) were incubated with serum-starved 3T3-F442A cells for 15 min before detergent extraction. Extracted proteins were immunoprecipitated with anti-GHR_cyt-AL47 (Fig. 1A), anti-JAK2_AL33 (Fig. 1B), or anti-IGF-I receptor ß-chain (anti-IGF-IR; Fig. 1C) antibodies, resolved by SDS-PAGE, and immunoblotted with antiphosphotyrosine (anti-pTyr; Fig. 1, A–C, lanes 1–3). As expected, anti-GHR_cyt-AL47 precipitation revealed GH-induced tyrosine phosphorylation of a collection of proteins in the 100- to 125-kDa range, which included a diffuse lower band (100–120 kDa) closely approximated with a sharper band of 125 kDa (Fig. 1A, lane 2 vs. 1; bracket and arrow, respectively). Their migration and appearance in response to GH were consistent with these bands being the tyrosine-phosphorylated GHR and JAK2, as we have previously detected in anti-GHR precipitates from these cells (31, 35). Notably, this complex of tyrosine phosphoproteins was not detected in response to IGF-I (Fig. 1A, lane 3 vs. 1). Reprobing of this blot with anti-GHR_cyt-AL47 revealed the GHR as the diffuse band, the migration of which was retarded in response to GH [as seen previously (31)], but not in response to IGF-I (Fig. 1A, lanes 4–6). Also as expected (11, 31, 35), GH caused tyrosine phosphorylation of JAK2, as detected in the direct anti-JAK2_AL33 precipitates (Fig. 1B, lane 2 vs. 1). IGF-I treatment, in contrast, did not result in JAK2 tyrosine phosphorylation (lane 3 vs. 1). Reprobing with anti-JAK2_AL33 verified that equivalent amounts of JAK2 were precipitated from each sample (Fig. 1B, lanes 4–6).

View larger version (30K): [in this window] [in a new window]   Fig. 1. GH Acutely Induces Appearance of a Tyrosine Phosphoprotein Complex in the Anti-IGF-IR Precipitates A–C, Serum-starved 3T3-F442A cells were stimulated with GH (500 ng/ml), IGF-I (20 ng/ml), or vehicle (–) for 15 min. Detergent extracts (500 µg) were immunoprecipitated with anti-GHRcyt-AL47 (A), anti-JAK2AL33 (B), and anti-IGF-IR (C) antibodies, respectively. Eluted proteins were analyzed by immunoblotting with anti-pTyr (A–C, lanes 1–3), anti-GHRcyt-AL47 (A, lanes 4–6), anti-JAK2AL33 (B, lanes 4–6), and anti-IGF-IR (C, lanes 4–6). Migration of prestained molecular weight markers is indicated. Also indicated are the expected positions of GHR (bracket), JAK2 (arrow), and IGF-IR ß-chain (arrowhead). The experiments shown are representative of six such experiments.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Effects of GH Antagonist and GH Concentration on Formation of anti-IGF-IR-Precipitated Tyrosine Phosphoprotein Complex A, G120K antagonism. Serum-starved 3T3-L1 cells were stimulated with vehicle (lane 1), GH (500 ng/ml, lane 2), IGF-I (20 ng/ml, lane 3), G120K (500 ng/ml (x1), lane 4; 2500 ng/ml (x5), lane 5), or GH (500 ng/ml) plus G120K (2500 ng/ml) (lane 6) for 15 min. Detergent extracts (500 µg) were immunoprecipitated with anti-JAK2AL33 (upper panel) and anti-IGF-IR (lower panel), respectively. Immunoprecipitates were analyzed by immunoblotting with anti-pTyr. B, GH concentration dependence. Serum-starved 3T3-L1 cells were stimulated with various concentrations of GH for 15 min, as indicated. Detergent extracts (500 µg) were subjected to immunoprecipitation with anti-IGF-IR and immunoblotting with anti-pTyr. Note decreased coprecipitation of tyrosine phosphoproteins at 1000 ng/ml GH vs. 500 ng/ml (lane 6 vs. 5). The experiments shown in panels A and B are representative of two such experiments.

View larger version (23K): [in this window] [in a new window]   Fig. 5. Tyrosine Phosphorylation of GHR Is Not Required for GH-Induced GHR-JAK2-IGF-IR Complex Formation A–D, Serum-starved 3T3-L1 cells were pretreated with vehicle (lanes 1 and 2) or 1.25 µM staurosporine (lane 3) for 15 min before stimulation with vehicle (lane 1) or GH (500 ng/ml; lanes 2 and 3) for 15 min, as indicated. Detergent extracts (500 µg) were immunoprecipitated with anti-GHRcyt-AL47 (A and B) or anti-IGF-IR (C and D). The immunoprecipitates were analyzed by immunoblotting with anti-pTyr (A), anti-GHRcyt-AL47 (B and C), and anti-IGF-IR (D), respectively. (Note that the retarded migration of the GHR in GH-treated samples relative to those not treated with GH (B) and those treated with GH in the presence of staurosporine (B and C) is likely related to its tyrosine phosphorylation.) The experiments shown are representative of three such experiments.

View larger version (17K): [in this window] [in a new window]   Fig. 7. LIF, a JAK2 Activator, Does Not Induce the Formation of GHR-JAK2-IGF-IR Complex A and B, Serum-starved 3T3-L1 cells were stimulated with vehicle (lane 1), GH (500 ng/ml; lane 2), or LIF (20 ng/ml; lane 3) for 15 min. Detergent extracts (500 µg) were immunoprecipitated with anti-IGF-IR (A, upper panel), or anti-JAK2AL33 (A, middle and lower panels). Eluted proteins were analyzed by immunoblotting with anti-pTyr (A, upper and middle panels) or anti-JAK2AL33 (A, lower panel). B, A longer exposure of the immunoblot presented as the upper panel of A.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Characterization of GH-Induced Tyrosine Phosphoprotein Complex in the Anti-IGF-IR Precipitates by Enzymatic Deglycosylation Serum-starved 3T3-F442A cells were stimulated with GH (500 ng/ml; lanes 3, 4, and 7–10), IGF-I (20 ng/ml; lanes 5, 6,11, and 12), or vehicle (lanes 1 and 2) for 15 min. Detergent extracts (1000 µg) were immunoprecipitated with anti-IGF-IR (lanes 1–6, 9–12) and anti-GHRcyt-AL47 (lanes 7 and 8). Precipitates were treated with (+) or without (-) endoglycosidase F/N-glycosidase F, as described in Materials and Methods, before resolution by SDS-PAGE and immunoblotting with anti-pTyr (lanes 1–8) and anti-IGF-IR (lanes 9–12). Positions of JAK2 (arrow), glycosylated and deglycosylated GHRs (brackets), and IGF-IRs (arrowheads) are indicated. Note the effects of deglycosylation on the pattern of SDS-PAGE migration of GH-induced tyrosine phosphoproteins in IGF-IR precipitates (lanes 3 and 4) compared with that of the GH-induced phospho-GHR-JAK2 complex in GHR precipitates (lanes 7 and 8). The experiment shown is representative of three such experiments.

The active signaling conformation of the GHR induced by GH is as a dimer bound to a single GH molecule (36). Recent studies suggest that receptor is likely a preformed dimer in the absence of ligand and that the active assemblage arises by a GH-induced conformational change in receptor dimer partners, which somehow allows more productive interaction between the GHR and JAK2 and resultant triggering of signaling (37, 38, 39, 40, 41). A class of recombinant GH antagonists harboring mutations at residues known to be critical for promotion of the active receptor dimer conformation have been developed (42, 43). One of these, hGH-G120K (G120K), does not itself induce GHR and JAK2 tyrosine phosphorylation in 3T3-F442A cells and antagonizes the ability of GH to induce these effects; additionally, GH-induced GHR disulfide linkage, a reflection of the receptor conformational change, is also antagonized by G120K (35).

We used G120K to further characterize the GH-induced accumulation of tyrosine phosphoproteins in IGF-IR immunoprecipitates (Fig. 3A). Like 3T3-F442A, the independently derived 3T3-L1 preadipocyte bears both GHR (33) and IGF-IR (44, 45). In these experiments, we used 3T3-L1, first demonstrating that, as in 3T3-F442A cells, treatment of 3T3-L1 induced tyrosine phosphorylation of JAK2 and coimmunoprecipitation of tyrosine phosphoproteins with anti-IGF-IR ß-chain (Fig. 3A, upper and lower panels, respectively, lanes 2 vs. 1). This illustrates the similarity of these findings in the two preadipoctyes. G120K alone induced neither effect (lanes 4 and 5 vs. 1). Moreover, G120K substantially antagonized both effects of GH at a GH to G120K ratio of 1:5 (lanes 6 vs. 2), strongly suggesting that the coimmunoprecipitated tyrosine phosphoproteins were GHR and JAK2.

The GH concentration dependency of the coimmunoprecipitation was examined in Fig. 3B. The association of tyrosine-phosphorylated GHR and JAK2 with IGF-IR ß-chain was detected with as little as 20 ng/ml GH (lane 2 vs. 1) and declined at a concentration greater than 500 ng/ml (lane 6 vs. 5). This fall off in coprecipitation at high GH concentrations is reminiscent of that observed for other GH-induced phenomena and likely relates to the inability of GH to effectively induce the conformational change(s) necessary for receptor triggering (35, 40, 42).

GH-Induced GHR-JAK2-IGF-IR Complex Formation Does Not Require GHR Tyrosine Phosphorylation
The data in Figs. 1–3 provide evidence for GH-induced association of tyrosine-phosphorylated GHR and JAK2 with the IGF-IR. However, they do not address whether association of the molecules is constitutive or occurs only in response to hormonal stimulation and/or tyrosine phosphorylation. This was initially approached by immunoblotting anti-IGF-IR precipitates from cells treated with vehicle, GH, or IGF-I sequentially with anti-pTyr, anti-GHR_cyt-AL47, anti-JAK2_AL33, and anti-IGF-IR (Fig. 4A). GHR and JAK2 were coprecipitated with anti-IGF-IR only in response to GH treatment (lane 2 vs. 1) and not in response to IGF-I treatment (lane 3 vs. 2). In concert with the data in Fig. 3, these results indicated that the conformational change(s) in GHR and/or JAK2 induced by GH treatment are apparently required for their inclusion in a complex with IGF-IR ß-chain. We note that IGF-IR ß-chain was not detected in immunoprecipitates with our available anti-GHR or anti-JAK2 reagents either in the absence or presence of GH stimulation (data not shown), further suggesting conformational sensitivity for detection of the intact complex.

View larger version (15K): [in this window] [in a new window]   Fig. 4. GH Induces Association of Tyrosine-Phosphorylated GHR and JAK2 with IGF-IR A, GH induces the formation of GHR-JAK2-IGF-IR complex. Serum-starved 3T3-F442A cells were stimulated with vehicle (lane 1), GH (500 ng/ml; lane 2), or IGF-I (20 ng/ml; lane 3) for 15 min. Detergent extracts (500 µg) were immunoprecipitated with anti-IGF-IR. The immunoprecipitates were analyzed by immunoblotting with anti-pTyr, anti-GHRcyt-AL47, anti-JAK2AL33, and anti-IGF-IR, respectively. The experiments shown are representative of five such experiments. B and C, Time course of GH-induced GHR-JAK2-IGF-IR complex formation. Serum-starved cells were stimulated with GH (500 ng/ml) for 0–30 min as indicated. Detergent extracts (500 µg) were immunoprecipitated with anti-JAK2 (B, upper panel), anti-GHRcyt-AL47 (B, lower panel), or anti-IGF-IR (C). The immunoprecipitates were analyzed by immunoblotting with anti-pTyr (B and C, upper panel), anti-GHRcyt-AL47 (C, middle panel), and anti-JAK2AL33 (C, lower panel), respectively. The experiments shown are representative of two such experiments.

To determine whether complex formation was dependent on GH-induced tyrosine phosphorylation, we sought to uncouple GHR engagement from GHR tyrosine phosphorylation. We previously demonstrated that pretreatment of cells with the kinase inhibitor, staurosporine (1.25 µM), markedly decreased GH-induced tyrosine phosphorylation of the JAK2, GHR, and STAT5, but did not prevent GH-induced GHR disulfide linkage (a proxy for GH-induced conformational change of the receptor) or enhancement of the GHR-JAK2 association (35, 46). Cells were treated with (+) or without (–) GH in the presence of staurosporine (+) or its vehicle (–) and evaluated for GH-induced GHR tyrosine phosphorylation (Fig. 5, A and B) and coprecipitation of GHR with anti-IGF-IR (Fig. 5, C and D). Consistent with previous results, staurosporine dramatically inhibited GH-induced GHR tyrosine phosphorylation (Fig. 5A, lane 3 vs. 2). However, this did not prevent GH-induced association of the GHR with IGF-IR (Fig. 5C, lane 3 vs. 2). These data strongly suggest that the formation of the GHR-JAK2-IGF-IR complex does not require GHR tyrosine phosphorylation, but does require GH-induced GHR conformational change, as implicated by the data in Fig. 3.

Specificity of GH-Induced Formation of the GHR-JAK2-IGF-IR Complex
The GH dependency of GHR-JAK2-IGF-IR ß-chain complex formation seen in Figs. 4 and 5 argued that the interaction is specific. However, we pursued further experiments to validate this conclusion. Because GH-induced GHR and JAK2 tyrosine phosphorylation in 3T3-F442A and 3T3-L1 cells is extensive (see Fig. 1, A and B, and Fig. 5 and Refs.3 and 31), their coprecipitation could, in principle, reflect a nonspecific association with the reagents used for immunoprecipitation. As an immunological specificity control, extracted proteins from cells stimulated with GH, IGF-I, or vehicle were precipitated with anti-epidermal growth factor receptor (EGFR) antibody in comparison with anti-IGF-IR antibody (Fig. 6). Both are affinity purified rabbit antibodies, and both were adsorbed by protein A-sepharose. The anti-IGF-IR precipitate of the GH-treated cells again contained the tyrosine-phosphorylated GHR-JAK2 complex (Fig. 6A, lane 2 vs. 1), whereas that from the IGF-I-treated cells contained the tyrosine-phosphorylated IGF-IR ß-chain and coprecipitated tyrosine phosphoproteins, the migration of which was consistent with being insulin receptor substrate-1 and the p66 and p52 isoforms of Src homology and collagen domain protein (lane 3), as expected. In contrast, none of these GH- or IGF-I-induced tyrosine phosphoproteins were detected in anti-EGFR precipitates of the same samples (Fig. 6A, lanes 4–6). In particular, we note that GH-induced tyrosine phosphorylation of the EGFR was observed (lane 4 vs. 6), consistent with previous findings (11, 12, 47), but that the tyrosine-phosphorylated GHR and JAK2 were not detected in this same precipitate. Anti-IGF-IR (Fig. 6B) and anti-EGFR (Fig. 6C) blotting verified that each of these molecules was similarly abundant in its respective specific immunoprecipitates. Thus, the GHR and JAK2 tyrosine phosphorylated in response to GH did not associate with the GH-induced tyrosine-phosphorylated EGFR or the reagents used to specifically immunoprecipitate it. This favors the interpretation that their inclusion in IGF-IR precipitates in response to GH reflects a specific association. This interpretation is even more compelling in that the control antibody used (anti-EGFR) does directly recognize its target (the EGFR) and is thus an even more powerful specificity control than would be a nonimmune antibody.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Specificity of GH-Induced Formation of GHR-JAK2-IGF-IR Complex A–C, Serum-starved 3T3-F442A cells were stimulated with vehicle (lanes 1 and 6), GH (500 ng/ml; lanes 2 and 4), or IGF-I (20 ng/ml; lanes 3 and 5) for 15 min. Detergent extracts (500 µg) were immunoprecipitated with anti-IGF-IR (lanes 1–3) and anti-EGFR (as an immunological specificity control; lanes 4–6), respectively. Eluted proteins were analyzed by immunoblotting with anti-pTyr (A), anti-IGF-IR (B), or anti-EGFR (C).

Although inhibition of GH-induced GHR tyrosine phosphorylation was dramatic (Fig. 5), the inhibition of GH-induced JAK2 tyrosine phosphorylation by staurosporine was incomplete (Ref.35 and data not shown). Thus, we examined whether the formation of the GH-induced GHR-JAK2-IGF-IR complex was dependent on GH-induced JAK2-mediated signals. In particular, we focused on the ERK and PI3K activation pathways. Cells were pretreated with either PD98059 (Fig. 8A) or LY294002 (Fig. 8B) to inhibit the ERK or PI3K pathways, respectively. In neither case did pathway inhibition prevent GH-induced coimmunoprecipitation of tyrosine-phosphorylated GHR and JAK2 with anti-IGF-IR (Fig. 8, A and B, upper panels; lanes 4 vs. 2). Immunoblotting with the anti-pERK and anti-pAkt activation-specific antibodies verified that each inhibitor substantially diminished the activation of the pathways (Fig. 8, A and B, lower panels; lanes 4 vs. 2). Collectively, these findings further supported the conclusion that GH-induced GHR conformational changes, rather than GH-induced signaling, are responsible for allowing formation of the GHR-JAK2-IGF-IR complex.

View larger version (27K): [in this window] [in a new window]   Fig. 8. The GH-Induced Formation of GHR-JAK2-IGF-IR Complex Is Independent of GH-Induced Activation of ERK and PI3K Pathways A and B, Serum-starved 3T3-F442A cells were preincubated with PD98059 (100 µM) for 60 min (A, lanes 3 and 4) or LY294002 (50 µM) for 30 min (B, lanes 3 and 4) before stimulation with vehicle (lanes 1 and 3) or GH (500 ng/ml; lanes 2 and 4) for 15 min. Detergent extracts (500 µg) were immunoprecipitated with anti-IGF-IR (A and B, upper and middle panels). Eluted proteins were immunoblotted with anti-pTyr (A and B, upper panels) or anti-IGF-IR (A and B, middle panels). Detergent extracts (30 µg), as used for immunoprecipitation in A and B, upper and middle panels, were directly resolved by SDS-PAGE and immunoblotted with antiactive ERK (A, lower panel) and anti-pAkt (B, lower panel).

View larger version (16K): [in this window] [in a new window]   Fig. 9. GH and IGF-I Cotreatment Augments GH-Induced Spi-GLE-Luciferase Transactivation A, As detailed in Materials and Methods, 3T3-F442A cells were transfected with Spi-GLE-luc reporter plasmid. Serum-starved cells were then stimulated with vehicle (control), GH (100 ng/ml), IGF-I (20 ng/ml), or GH (100 ng/ml) plus IGF-I (20 ng/ml) in triplicate for 16 h, after which luciferase activity was measured. The data shown in panel A are from a representative experiment, in which the basal luciferase activity (treated with vehicle) was considered as 1 and the mean ± SE of triplicates is indicated for each condition. B, Pooled data from three independent experiments such as those in A are plotted, in which GH-induced transactivation within each experiment was considered as 100%. IGF-I cotreatment augmented GH-induced Spi-GLE-luc transactivation by 53.6 ± 7.6% (P < 0.05).

View larger version (25K): [in this window] [in a new window]   Fig. 10. GH and IGF-I Cotreatment Augments the Activation of ERK 1/2 A, Serum-starved 3T3-L1 cells were treated with vehicle (lane 1), GH (500 ng/ml; lane 2), IGF-I (20 ng/ml; lane 3), or GH (500 ng/ml) plus IGF-I (20 ng/ml) (lane 4) for 5 min. Total cell lysates (extracted in the presence of 1% SDS, 15 µg) were resolved by SDS-PAGE and immunoblotted with antiactive ERK1/2 (upper panel) and anti-ERK1/2 (lower panel). B, Data shown in panel A, along with those obtained from two other experiments, were subjected to densitometric analysis. Comparison of the level of ERK1/2 activation induced by GH and IGF-I cotreatment (referred to as GH/IGF-I) with the sum of those induced by GH alone plus IGF-I alone (referred to as GH alone + IGF-I alone) is shown, in which the sum of ERK1/2 activation induced by GH alone + IGF-I alone within each experiment was considered as 100%. GH and IGF-I cotreatment augmented the ERK1/2 activation by 377 ± 43% (P < 0.01), as indicated.

View larger version (21K): [in this window] [in a new window]   Fig. 11. GH and IGF-I Cotreatment Augments GH-Induced Disulfide Linkage and Tyrosine Phosphorylation of GHR A, Serum-starved 3T3-L1 cells were stimulated with vehicle (lane 1), GH (500 ng/ml; lane 2), IGF-I (20 ng/ml; lane 3), or GH (500 ng/ml) plus IGF-I (20 ng/ml) (lane 4) for 15 min. Detergent extracts (300 µg) were subjected to immunoprecipitation with anti-GHRcyt-AL47 and eluted proteins were analyzed by immunoblotting with anti-pTyr (upper panel) and anti-GHRcyt-AL47 (lower panel). The experiment shown is representative of three such experiments. B, Serum-starved 3T3-L1 cells were stimulated with vehicle (lane 1), GH (100 ng/ml; lanes 2 and 5), IGF-I (20 ng/ml; lanes 3 and 6), or GH (100 ng/ml) plus IGF-I (20 ng/ml) (lanes 4 and 7) for 5 and 30 min, respectively. Detergent extracts (100 µg) were resolved without immnoprecipitation by SDS-PAGE under nonreducing conditions and immunoblotted with anti-GHRcyt-AL37 (upper panel) and anti-pTyr (lower panel). The position of disulfide-linked murine GHR (dsl-GHR) is indicated. C, Enhancement of tyrosine phosphorylation of disulfide-linked GHR. Data from three independent experiments such as those shown in B (lower panel) were subjected to densitometric analysis. In each experiment, the level of GH-induceddsl-GHR tyrosine phosphorylation was considered as 100% for each time point. The data are plotted as a percentage of tyrosine phosphorylation level of dsl-GHR relative to that induced by GH alone and are mean ± SE of three such experiments. The P values for each time point are indicated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
With increasing knowledge of the complexity of signaling pathways and the utilization of common pathways by diverse receptors, cross-talk between receptor signaling systems has emerged as an important and interesting area of signaling research. Our previous work has focused on interactions between GH-induced signaling and signaling mediated by intrinsic tyrosine kinase growth factor receptors, most notably members of the EGF receptor family. We previously showed that GH treatment of murine preadipocytes resulted in phosphorylation of both the EGFR (ErbB-1) and ErbB-2 (also known as c-neu) at a residue(s) recognized by a state-specific antibody that specifically reacts with proteins at ERK consensus phosphorylation sites (11, 12). These GH-induced phosphorylation events were dependent on MAPK kinase 1 (MEK1) activity and were associated with alterations in EGFR and ErbB-2 signaling and/or trafficking.

In the current study, we examined potential cross-talk between GH and IGF-I. We detected the GH-induced formation of a protein complex including the GHR, JAK2, and IGF-IR. GH concentration dependence and GH antagonist experiments suggested that its ability to induce formation of this complex was related to induction by GH of conformational change(s) in its receptor and/or JAK2. The use of the kinase inhibitor staurosporine to inhibit GH-induced GHR tyrosine phosphorylation, although not preventing GH-induced GHR conformational change (35, 46), further indicated that receptor tyrosine phosphorylation was likely not required for the interaction. Likewise, the inability of LIF to promote JAK2-IGF-IR association, despite causing ample JAK2 tyrosine phosphorylation, indicated that JAK2 tyrosine phosphorylation per se is unlikely to be required to support the GH-induced GHR-JAK2-IGF-IR complex. Finally, deglycosylation of the proteins within the complex allowed us to determine that the IGF-IR ß-chain associated with the GHR and JAK2 was also not detectably tyrosine phosphorylated.

Thus, although we cannot completely rule out a requirement for tyrosine phosphorylation of any of the members of the complex for their association, our results do not favor such a requirement. In that respect, our findings differ from those of Gual et al. (28), which suggested that associations between the IGF-IR or insulin receptor and JAKs required tyrosine phosphorylation of the receptors. The associations in that study were dependent on in vitro overexpression of both binding partners and did not include analysis of cytokine receptors with which the JAK can associate in a normal cellular context. Although other studies have indicated that stimulation of insulin or IGF-I receptors can activate JAKs (24, 25, 26, 27), the current report is the first of which we are aware that has studied interaction of cytokine receptors/JAKs with the IGF-IR in cells that do not overexpress any of the components. As such, we find relevant the results of our experiments aimed at probing the specificity of the interactions. In contrast to the GH-induced association of GHR/JAK2 with the IGF-IR, we have observed no such association with the EGFR in the same cellular setting (Fig. 6 and our unpublished observations). GH-induced GHR-EGFR association has been detected by others (47), but again only in the setting of transient overexpression of both proteins. Further studies will be required to understand whether any structural similarities underlie the abilities of GH to promote association of its receptor with the IGF-IR and to engage in cross-talk with the EGFR signaling system. Inherent in such studies will be mapping of the GHR and/or JAK2 region(s) required for each effect. We note, however, that in contrast to the requirement for GH-induced MEK/ERK signaling for cross-talk with the EGFR-family systems (11, 12), GH-induced formation of the GHR-JAK2-IGF-IR complex was not affected by inhibition of the GH-induced MEK/ERK or PI3K pathways.

We probed the potential signaling impact of formation of the complex in response to GH by examining the effects of cotreatment with GH plus IGF-I. Notably, we found that acute induction by GH of GHR tyrosine phosphorylation was augmented when cells were coincubated with IGF-I. Importantly, IGF-I alone had no apparent effect on GHR tyrosine phosphorylation. Further, the GH-induced formation and tyrosine phosphorylation of disulfide-linked GHRs, in particular, were enhanced by cotreatment with IGF-I. These are important observations, in that the disulfide linkage of GHRs reflects for receptor dimers the attainment of conformational competence for signaling (35).

We are intrigued by the mechanistic implications of these findings. Existing data suggest that GH engagement of the GHR causes changes in receptor dimers that allow achievement of an active signaling conformation. Our data suggest that this also allows association of GHR/JAK2 with the IGF-IR, independent of the activation status of IGF-IR. We interpret our findings to further indicate that IGF-I engagement of the IGF-IR in such a complex facilitates or stabilizes the GH-activated dimeric GHR signaling conformation (reflected by GHR disulfide linkage). We do not yet know whether this enhancement of the GHR conformational change induced by IGF-IR engagement depends on IGF-IR tyrosine kinase activation or, rather, kinase-independent changes in the IGF-IR itself induced by IGF-I binding.

Regarding the enhancement of GHR tyrosine phosphorylation observed in the cotreatment situation, several mechanistic possibilities, in addition to solely achievement or stability of the activated conformation, can be considered. The GHR, by virtue of either direct or indirect interactions with the IGF-IR, could, in response to IGF-I, become a better substrate for tyrosine phosphorylation by the GH-activated JAK2 kinase. Alternatively, JAK2, by being in close proximity to the IGF-I-activated IGF-IR kinase, could be rendered a more active kinase and thus more robustly phosphorylate the GHR. Yet another possibility is that the IGF-I-activated IGF-IR kinase, upon being brought into proximity of the GHR in response to GH, itself acts as a GHR kinase, further increasing the abundance of tyrosine phosphorylated GHR. At present, our data do not allow us to discriminate between these or other possible mechanisms. In preliminary experiments, we have yet to detect enhanced JAK2 or IGF-IR kinase activities in the GH plus IGF-I-cotreated vs. individually treated situations; similarly, we have yet to observe synergy in IGF-I-induced Akt activation (our unpublished observations). However, we realize that such changes could be subtle and still produce changes in GHR tyrosine phosphorylation. Conceivably, induction by GH of the GHR-JAK2-IGF-IR complex allows multiple mechanisms to collaborate to produce signaling synergy. A more thorough understanding of the mechanistic details of this interaction will await a better understanding of the structural determinants necessary for GHR-JAK2-IGF-IR complex formation. In particular, reconstitution experiments using kinase-inactive molecules (JAK2 and IGF-IR) and experiments in which JAK2 levels are altered may be useful in discriminating among these possibilities.

Independent of the mechanisms involved, we also observed synergy in acute activation of the ERK pathway for the combination of GH and IGF-I vs. each hormone individually in our cell system. Synergy between GH and IGF-I for signaling responses has also been observed by others. Ashcom et al. (50), examined the abilities of GH and IGF-I to induce c-fos gene expression in 3T3-F442A cells. They observed that treatment of serum-starved cells with GH plus IGF-I induced c-fos mRNA levels nearly 30% greater than the summed response of both individually. Further, run-on transcription experiments suggested nearly 2-fold synergy of c-fos transcription by the combination of hormones compared with the sum of each. Our findings of synergy in ERK activation are particularly noteworthy in light of the established influence of the MEK/ERK pathway on GH-induced c-fos gene activation in preadipocytes (10). Further, we also observe enhanced transactivation of a reporter driven by enhancer elements of Spi 2.1, another GH-regulated gene, suggesting that the enhancement of GH signaling afforded by IGF-I costimulation may affect various aspects of GH action.

Indeed, Edmondson et al. (51) examined the influence of GH and IGF-I on the growth of human melanocytes in vitro. Despite expressing the GHR (58), these cells were unable to grow in response to GH alone. IGF-I or des(1–3)IGF-I (an IGF-I analog with reduced affinity for IGF-binding proteins) alone each caused increased cell numbers, but the addition of GH to IGF-I or des(1–3)IGF-I caused an increase of nearly 1.5-fold or 2-fold, respectively, in the response. The mechanisms responsible for these examples of synergy in c-fos gene induction in preadipocytes and growth responsiveness of melanocytes have not been uncovered and may be quite complicated. However, in neither case is it likely that GH induction of IGF-I gene expression is at the root of the synergy, as GH does not induce IGF-I mRNA within the time frame examined in 3T3-F442A cells (50) and GH treatment alone of the melanocytes had no effect (51). Whether the GH-induced formation of the GHR-JAK2-IGF-IR complex and the synergistic signaling effects of GH and IGF-I that we observe are related to these effects is not yet known.

In conclusion, GH and IGF-I are key somatogenic and metabolic hormones that impact each other in various ways. Our data offer another aspect to the richness of their relationship in that they suggest collaboration between their receptors for initiating and transducing signals. It will be important in future studies to better understand the structural underpinnings for this interaction and the range of downstream signals that are affected. Further, it will be important to determine whether the physical interactions we have uncovered are necessarily related to the functional interactions we demonstrate.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Materials
Recombinant human GH was kindly provided by Eli Lilly & Co. (Indianapolis, IN). Recombinant human IGF-I was purchased from Invitrogen (San Diego, CA). Recombinant human GH-G120K was kindly provided by Sensus Corp. (Austin, TX). Recombinant murine LIF was from Chemicon International (Temecula, CA). The MEK1 inhibitor PD98059 was purchased from Cell Signaling (Beverly, MA). The PI3K inhibitor LY294002, staurosporine, and other routine reagents were from Sigma Chemical Co. (St. Louis, MO), unless otherwise noted.

Antibodies
Polyclonal anti-IGF-IRß and anti-EGFR antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal antiphosphotyrosine antibody 4G10 (pTyr) and affinity-purified polyclonal anti-MAPK antibody (anti-ERK, recognizing both ERK1 and ERK2) were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Affinity-purified polyclonal antiactive MAPK antibody (antiactive ERK, recognizing the dually phosphorylated Thr-183 and Tyr-185 residues corresponding to the active forms of ERK1 and ERK2) was from Promega Corp. (Madison, WI). The rabbit polyclonal antisera, anti-JAK2_AL33 [directed at residues 746-1129 of murine JAK2 (59)], anti-GHR_cyt-AL47 [raised against a bacterially expressed N-terminally His-tagged fusion protein incorporating human GHR residues 271–620 (the entire cytoplasmic domain) (57)], and anti-GHR_cyt-AL37 [directed against a bacterially expressed glutathione-S-transferase fusion with GHR residues 270–620 (59)] have been previously described.

Cells and Cell Culture
3T3-F442A cells (29), kindly provided by Drs. H. Green (Harvard University, Boston, MA) and C. Carter-Su (University of Michigan, Ann Arbor, MI), were cultured in DMEM containing 4.5 g/liter glucose (Cellgro, Inc.), supplemented with 10% calf serum, 50 µg/ml gentamicin sulfate, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Biofluids, Rockville, MD). 3T3-L1 cells were obtained from American Type Culture Collection (Manassas, VA) and grown in the above medium, supplemented with 10% fetal bovine serum (Biofluids) instead of 10% calf serum.

Cell Starvation, Inhibitor Pretreatment, Cell Stimulation, and Protein Extraction
Serum starvation of 3T3-F442A or 3T3-L1 cells was accomplished by substitution of 0.5% (wt/vol) BSA (fraction V, Roche Molecular Biochemicals, Indianapolis, IN) for calf serum or fetal bovine serum in the culture medium (starvation medium) for 16–20 h before experiments. Pretreatments and stimulations were carried out at 37 C in binding buffer [consisting of 25 mM Tris-HCl (pH 7.4), 120 mM NaCl, 5 mM KCl, 1.2 mM MgCl₂, 0.1% (wt/vol) BSA, and 1 mM dextrose]. Serum-starved cells were pretreated with PD98059 (100 µM), LY294002 (50 µM), or vehicle (as a control) for 30 or 60 min before treatment with GH (100 or 500 ng/ml), IGF-I (20 ng/ml), LIF (20 ng/ml), or vehicle, as specified for each experiment. Stimulations were terminated by washing the cells once with ice-cold PBS supplemented with 0.4 mM sodium orthovanadate (PBS-vanadate) before harvesting by scraping in PBS-vanadate. Cells were collected by brief centrifugation and solubilized for 15 min at 4 C in lysis buffer [1% (vol/vol) Triton X-100, 150 mM NaCl, 10% (vol/vol) glycerol, 50 mM Tris-HCl (pH 8.0), 100 mM NaF, 2 mM EDTA, 1 mM PMSF, 1 mM sodium orthovanadate, 10 mM benzamidine, 5 µg/ml aprotinin, and 5 µg/ml leupeptin]. After centrifugation at 15,000 x g for 15 min at 4 C, the detergent extracts (supernatant) were subjected to immunoprecipitation or were directly electrophoresed and immunoblotted, as indicated below.

Immunoprecipitation, Enzymatic Deglycosylation, Electrophoresis, and Immunoblotting
For immunoprecipitation, cell extracts (300–1000 µg) were mixed with 7 µl anti-IGF-IRß, 7 µl anti-EGFR, 3 µl anti-GHR_cyt-AL47, or 3 µl anti-JAK_AL33 antibody and incubated at 4 C for 2 h with continuous agitation. Protein A-Sepharose (Amersham Pharmacia Biotech, Arlington Heights, IL) was added and incubated at 4 C for an additional 1 h. The beads were washed four times with lysis buffer. Laemmli sample buffer eluates were resolved by SDS-PAGE and immunoblotted.

For enzymatic deglycosylation experiments, immunoprecipitation was first performed as above. Precipitated proteins were eluted by boiling the protein A-Sepharose beads in 0.5% sodium dodecyl sulfate (SDS) and 3% 2-mercaptoethanol for 5 min. Deglycosylation in 125 µl was accomplished by adjusting the buffer to include the following: 50 mM NaOAc, pH 5.5; 50 mM EDTA; 0.4% (vol/vol) Nonidet P-40; 6.4 mM phenylmethylsulfonyl fluoride; 0.1% SDS; 0.6% 2-mercaptoethanol; and 0.3 U of endoglycosidase F/N-Glycosidase F (Roche Molecular Biochemicals) at 37 C for 16 h. Nondeglycosylated controls were subjected to the same treatment, but without the addition of the glycosidase mixture. After the addition of Laemmli sample buffer, the proteins were subjected to SDS-PAGE and immunoblotting as indicated below.

Proteins resolved by SDS-PAGE under reducing or nonreducing conditions were transferred to Hybond ECL Nitrocellulose membranes (Amersham Pharmacia Biotech). The membranes were blocked with TBST buffer [20 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% (vol/vol) Tween 20] containing 2% BSA and incubated with primary antibodies (0.5–1 µg/ml or 1:1000 dilution) as specified in each experiment. After three washes with TBST, the membranes were incubated with appropriate secondary antibodies (1:10,000 dilution) and washed. The bound antibodies were detected with SuperSignal chemiluminescent substrate (Pierce Chemical Co., Rockford, IL). Membrane stripping was performed according to manufacturer’s suggestions (Amersham Pharmacia Biotech).

Densitometric Analysis
Immunoblots were scanned using a high-resolution scanner (Hewlett-Packard Co.). Densitomeric quantification of images was performed using a Macintosh II-based image analysis program (Image J 1.30, developed by W. S. Rasband, Research Services Branch, National Institute of Mental Health, Bethesda, MD). Pooled data from several experiments are displayed as mean ± SE. The significance (P value) of differences of pooled results was estimated using unpaired t tests.

Transactivation Assay
3T3-F442A cells (70% confluent in a 100 x 20 mm dish) were transfected with Spi-GLE-luc reporter plasmid (54) (a gift of Dr. W. Lowe, Northwestern University, Chicago, IL) using LipofectAMINE Plus reagents (Invitrogen, San Diego, CA). The cells were split into one 12-well plate 18–20 h after transfection and grown overnight in culture medium containing 10% calf serum. The cells were starved in starvation medium for 8 h and then stimulated with vehicle, GH (100 ng/ml), IGF-I (20 ng/ml), or GH (100 ng/ml) plus IGF-I (20 ng/ml) in triplicate for 16 h. The cells were lysed with luciferase lysis buffer (200 µl per well), and 100 µl of cell lysates were used for luciferase activity assay as described previously (31, 60).

	ACKNOWLEDGMENTS

 
We appreciate helpful conversations with Drs. X. Wang, K. He, K. Loesch, J. Cowan, and X. Li and the generous provision of reagents by those named in the text. We also appreciate critical review of the manuscript by Dr. Joseph L. Messina.

	FOOTNOTES

 
This work was supported by National Institutes of Health (NIH) Grant DK46395 (to S.J.F.) and in part by NIH Grant DK58259 (S.J.F.).

Results from this work were presented in part at the 84th Annual Meeting of The Endocrine Society, San Francisco, CA, 2002.

Abbreviations: EGFR, Epidermal growth factor receptor; GHR, GH receptor; IGF-IR, IGF-I receptor; JAK, Janus kinase; LIF, leukemia-inhibitory factor; MEK, MAPK kinase; PI3K, phosphatidylinositol 3-kinase; pTyr, phosphotyrosine; SDS, sodium dodecyl sulfate; STAT, signal transducer and activator of transcription.

Received for publication October 24, 2003. Accepted for publication March 15, 2004.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

1. Leung DW, Spencer SA, Cachianes G, Hammonds RG, Collins C, Henzel WJ, Barnard R, Waters MJ, Wood WI 1987 Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature 330:537–543[CrossRef][Medline]
2. Frank SJ, Messina JL 2002 Growth hormone receptor. In: Oppenheim JJ, Feldman M, eds. Cytokine reference on-line. London: Academic Press, Harcourt
3. Argetsinger LS, Campbell GS, Yang X, Witthuhn BA, Silvennoinen O, Ihle JN, Carter-Su C 1993 Identification of JAK2 as a growth hormone receptor-associated tyrosine kinase. Cell 74:237–244[CrossRef][Medline]
4. Ihle JN 1995 The Janus protein tyrosine kinase family and its role in cytokine signaling. Adv Immunol 60:1–35[Medline]
5. Carter Su C, Schwartz J, Smit LS 1996 Molecular mechanism of growth hormone action. Annu Rev Physiol 58:187–207[CrossRef][Medline]
6. Waxman DJ, Frank SJ 2000 Growth hormone action: signaling via a JAK/STAT-coupled receptor. In: Conn PM, Means A, eds. Principles of molecular regulation. Totowa, NJ: Humana Press; 55–83
7. Udy GB, Towers RP, Snell RG, Wilkins RJ, Park SH, Ram PA, Waxman DJ, Davey HW 1997 Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression. Proc Natl Acad Sci USA 94:7239–7244[Abstract/Free Full Text]
8. Woelfle J, Billiard J, Rotwein P 2003 Acute control of insulin-like growth factor-1 gene transcription by growth hormone through STAT5B. J Biol Chem 278:22696–22702[Abstract/Free Full Text]
9. Davey HW, Xie T, McLachlan MJ, Wilkins RJ, Waxman DJ, Grattan DR 2001 STAT5b is required for GH-induced liver IGF-I gene expression. Endocrinology 142:3836–3841[Abstract/Free Full Text]
10. Hodge C, Liao J, Stofega M, Guan K, Carter-Su C, Schwartz J 1998 Growth hormone stimulates phosphorylation and activation of elk-1 and expression of c-fos, egr-1, and junB through activation of extracellular signal-regulated kinases 1 and 2. J Biol Chem 273:31327–31336[Abstract/Free Full Text]
11. Kim SO, Houtman JC, Jiang J, Ruppert JM, Bertics PJ, Frank SJ 1999 Growth hormone-induced alteration in ErbB-2 phosphorylation status in 3T3–F442A fibroblasts. J Biol Chem 274:36015–36024[Abstract/Free Full Text]
12. Huang Y, Kim SO, Jiang J, Frank SJ 2003 Growth hormone-induced phosphorylation of epidermal growth factor (EGF) receptor in 3T3–F442A cells. Modulation of EGF-induced trafficking and signaling. J Biol Chem 278:18902–18913[Abstract/Free Full Text]
13. Salmon WD, Daughaday WH 1957 A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. J Lab Clin Med 49:825–836[Medline]
14. Daughaday WH 2000 Growth hormone axis overview—somatomedin hypothesis. Pediatr Nephrol 14:537–540[CrossRef][Medline]
15. Wang J, Zhou J, Powell-Braxton L, Bondy C 1999 Effects of Igf1 gene deletion on postnatal growth patterns. Endocrinology 140:3391–3394[Abstract/Free Full Text]
16. Liu JL, LeRoith D 1999 Insulin-like growth factor I is essential for postnatal growth in response to growth hormone. Endocrinology 140:5178–5184[Abstract/Free Full Text]
17. Yakar S, Liu JL, Stannard B, Butler A, Accili D, Sauer B, LeRoith D 1999 Normal growth and development in the absence of hepatic insulin-like growth factor I. Proc Natl Acad Sci USA 96:7324–7329[Abstract/Free Full Text]
18. Sjogren K, Liu JL, Blad K, Skrtic S, Vidal O, Wallenius V, LeRoith D, Tornell J, Isaksson OG, Jansson JO, Ohlsson C 1999 Liver-derived insulin-like growth factor I (IGF-I) is the principal source of IGF-I in blood but is not required for postnatal body growth in mice. Proc Natl Acad Sci USA 96:7088–7092[Abstract/Free Full Text]
19. Lupu F, Terwilliger JD, Lee K, Segre GV, Efstratiadis A 2001 Roles of growth hormone and insulin-like growth factor 1 in mouse postnatal growth. Dev Biol 229:141–162[CrossRef][Medline]
20. Favelyukis S, Till JH, Hubbard SR, Miller WT 2001 Structure and autoregulation of the insulin-like growth factor 1 receptor kinase. Nat Struct Biol 8:1058–1063[CrossRef][Medline]
21. Ullrich A, Gray A, Tam AW, Yang-Feng T, Tsubokawa M, Collins C, Henzel W, Le Bon T, Kathuria S, Chen E, Jacobs S, Francke U, Ramachandran J, Fujita-Yamaguchi Y 1986 Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J 5:2503–2512[Medline]
22. LeRoith D 2000 Insulin-like growth factor I receptor signaling—overlapping or redundant pathways? Endocrinology 141:1287–1288[Free Full Text]
23. Nakae J, Kido Y, Accili D 2001 Distinct and overlapping functions of insulin and IGF-I receptors. Endocr Rev 22:818–835[Abstract/Free Full Text]
24. Zong CS, Chan J, Levy DE, Horvath C, Sadowski HB, Wang LH 2000 Mechanism of STAT3 activation by insulin-like growth factor I receptor. J Biol Chem 275:15099–15105[Abstract/Free Full Text]
25. Peraldi P, Filloux C, Emanuelli B, Hilton DJ, Van Obberghen E 2001 Insulin induces suppressor of cytokine signaling-3 tyrosine phosphorylation through janus-activated kinase. J Biol Chem 276:24614–24620[Abstract/Free Full Text]
26. Velloso LA, Carvalho CR, Rojas FA, Folli F, Saad MJ 1998 Insulin signalling in heart involves insulin receptor substrates-1 and –2, activation of phosphatidylinositol 3-kinase and the JAK 2-growth related pathway. Cardiovasc Res 40:96–102[Abstract/Free Full Text]
27. Giorgetti-Peraldi S, Peyrade F, Baron V, Van Obberghen E 1995 Involvement of Janus kinases in the insulin signaling pathway. Eur J Biochem 234:656–660[Medline]
28. Gual P, Baron V, Lequoy V, Van Obberghen E 1998 Interaction of Janus kinases JAK-1 and JAK-2 with the insulin receptor and the insulin-like growth factor-1 receptor. Endocrinology 139:884–893[Abstract/Free Full Text]
29. Green H, Kehinde O 1976 Spontaneous heritable changes leading to increased adipose conversion in 3T3 cells. Cell 7:105–113[CrossRef][Medline]
30. Winston LA, Bertics PJ 1992 Growth hormone stimulates the tyrosine phosphorylation of 42- and 45-kDa ERK-related proteins. J Biol Chem 267:4747–4751[Abstract/Free Full Text]
31. Kim SO, Jiang J, Yi W, Feng GS, Frank