Functional Collaboration of Insulin-Like Growth Factor-1 Receptor (IGF-1R), but Not Insulin Receptor (IR), With Acute GH Signaling in Mouse Calvarial Cells

Yujun Gan; Andrew J Paterson; Yue Zhang; Jing Jiang; Stuart J Frank

doi:10.1210/en.2013-1732

. 2013 Dec 3;155(3):1000–1009. doi: 10.1210/en.2013-1732

Functional Collaboration of Insulin-Like Growth Factor-1 Receptor (IGF-1R), but Not Insulin Receptor (IR), With Acute GH Signaling in Mouse Calvarial Cells

Yujun Gan ¹, Andrew J Paterson ¹, Yue Zhang ¹, Jing Jiang ¹, Stuart J Frank ^1,^✉

PMCID: PMC3929739 PMID: 24302626

Abstract

GH signals through the GH receptor (GHR), a cytokine receptor linked to Janus kinase 2 (JAK2). GH activates signal transducer and activator of transcription 5 (STAT5), causing expression of genes including IGF-I. IGF-I binds IGF-I receptor (IGF-IR), a heterotetrameric (α₂-β₂) tyrosine kinase growth factor receptor similar to insulin receptor (IR). In addition to this GH -> GHR -> IGF-I -> IGF-IR pathway, GH induces a complex including GHR, JAK2, and IGF-IR and deletion of floxed IGF-1R in primary murine calvarial cells with Cre-recombinase-expressing adenovirus (Ad-Cre) desensitizes cells to GH for STAT5 activation and IGF-I mRNA accumulation. Diminished GH-induced STAT5 phosphorylation in Ad-Cre-treated cells is rescued by adenoviruses encoding either IGF-IR or IGF-IR lacking the β-chain intracellular domain. Reasoning that IGF-IR's extracellular portion (α or extracellular β) mediates functional interaction with GH signaling, we pursued reconstitution studies. Although structurally related to IGF-IR, IR expressed adenovirally did not rescue GH-induced STAT5 phosphorylation in Ad-Cre-treated cells. We thus created chimeras, swapping homologous IR extracellular regions into IGF-IR. IR and IGF-IR possess N-terminal L1, cysteine-rich (CR), and L2 α-chain domains. We created Ad-IGF-IR/IR-L1 and Ad-IGF-IR/IR-L1-CR-L2, in which L1 alone or L1, CR, and L2 of IR replace corresponding IGF-IR regions, respectively. Ad-IGF-IR/IR-L1, but not Ad-IGF-IR/IR-L1-CR-L2, rescued GH-induced STAT5 phosphorylation in Ad-Cre-treated cells. Additionally, medium containing a soluble IGF-IR (including only L1-CR-L2) dampened GH-induced STAT5 phosphorylation in calvarial cells and two other GH-responsive cell lines. Thus, an extracellular determinant(s), likely in CR-L2, specifically allows IGF-IR to collaborate with GHR and JAK2 for robust GH-induced acute STAT5 phosphorylation.

GH is a pituitary-derived peptide hormone with various biological actions (1, 2). Anabolic effects of GH include enhanced protein synthesis, proliferation and antiapoptosis, muscle accretion, and longitudinal bone growth. GH's anabolic effects are best appreciated in states of GH deficiency (3) or GH resistance (4), in which growth is stunted, or in states of GH excess (5), in which bony and connective tissue overgrowth are seen. In addition, experimental models suggest that ablation of the GH axis may lessen cancer formation and/or progression (6 –9). GH also has metabolic effects, profoundly influencing lipid and carbohydrate metabolism (1). Although studied for at least 7 decades, molecular mechanisms of GH action are only partially understood. GH binds the cell surface GH receptor (GHR), causing activation of the Janus kinase 2 (JAK2) tyrosine kinase and triggering of downstream pathways including signal transducer and activator of transcription 5 (STAT5) phosphorylation and nuclear translocation and gene expression (10 –12).

IGF-I is a powerful anabolic peptide produced in multiple tissues, in part stimulated by GH via STAT5 activation (13, 14). IGF-I binds the cell surface IGF-I receptor (IGF-IR), a heterotetameric tyrosine kinase growth factor receptor with several key substrates (15 –17). Thus, IGF-I functions as both a GH effector and in part independently of GH; likewise, GH actions in some situations are direct, rather than IGF-I-dependent (18 –23).

Our recent findings add further complexity to the rich interrelationship between these two major hormones and their receptors. In addition to the GH -> GHR -> IGF-I -> IGF-IR paradigm (analogous to a series circuit), we have made three observations that suggest IGF-IR may also be a key participant in proximal steps of GH signaling: 1) cotreatment with GH plus IGF-I can result in synergistic (greater than additive) signaling compared with either GH or IGF-I alone (24, 25); 2) GH, in the absence of IGF-I, can promote formation of a coimmunoprecipitable complex that includes GHR, JAK2, and IGF-IR (24, 25); and 3) silencing of IGF-IR results in marked reduction of GH-induced proximal signaling and downstream gene expression (25 –27). This implied functional collaboration of IGF-IR with GHR/JAK2/STAT5 signaling may be related to (unliganded) IGF-IR's ability to prevent GH-induced negative regulation by the protein tyrosine phosphatase (PTP)-1B (27) and, interestingly, can be conferred even by an IGF-IR that lacks much of its intracellular domain. In the current study, we examine determinants in IGF-IR's extracellular domain that foster its specific functional contribution to GH signaling.

Materials and Methods

Materials

Recombinant human GH was kindly provided by Eli Lilly & Co. Routine reagents were from Sigma-Aldrich Co, unless otherwise noted. Cell culture media, α-MEM, and RPMI 1640, were obtained from Cellgro-Mediatech, and fetal bovine serum was from Atlanta Biologicals.

Antibodies

Polyclonal anti-STAT5, anti-IGF-IRα, anti-IGF-IRβ, and anti-IRβ antibodies were purchased from Santa Cruz Biotechnology, Inc. Polyclonal antiphospho-STAT5 was purchased from Cell Signaling Technology. Anti-FLAG monoclonal antibody was from Sigma-Aldrich.

Cells and cell culture

Calvarial cells (previously referred to as osteoblasts) were isolated from calvaria of newborn Igf1r^flox/flox mice and maintained in culture as described previously (26 –29). This method has produced cultures highly enriched in osteoblasts. In general, a single newborn mouse calvaria preparation produced a yield of primary cells sufficient for approximately 10 samples of 1 × 10⁶ cells each in experiments outlined below. LNCaP cells (30) and 3T3-F442A cells (31) were maintained in culture as described previously.

Generation of recombinant adenoviruses

Adenoviruses encoding the full-length IR and IR-IGF-1R chimeras were generated as previously described for other adenoviruses (26). In brief, appropriate cDNAs were subcloned into the pAdTrack shuttle vector, which was linearized with PmeI and transformed into Escherichia coli BJ5138 cells containing the pAdEasy-1 viral DNA. Colonies harboring recombinants were selected by virtue of kanamycin resistance. Linearized (PacI) recombinant plasmid was transfected into human embryonic kidney (HEK)-293 cells and high titer viral stock was obtained. Ad-IGF-1R_1–482 was previously described (32). Ad-IR_1–474 with a C-terminal FLAG tag was constructed by PCR. Primer sequences are available upon request. Desired mutations and the lack of unwanted mutations were confirmed by DNA sequencing.

Adenovirus preparation and infection

Adenoviruses were amplified by infecting HEK-293 cells (26, 33). Cells were harvested when cytopathic effects became apparent. After lysis by five freeze/thaw cycles, cell debris was pelleted by centrifugation and subjected to further cesium chloride purification procedures. Concentration of purified virus was calculated by measuring the value of OD₂₆₀. For deletion of the IGF-IR, calvarial cells containing floxed IGF-IR alleles were cultured to 70% confluence and then, in the absence of serum, were infected with an adenovirus encoding Cre recombinase (Ad-Cre) or, as a control, an adenovirus encoding green fluorescent protein (Ad-GFP), at 800 multiplicity of infection, unless otherwise noted (26, 28). After 1 hour, culture medium containing 10% fetal bovine serum was added, and the cells were allowed to recover for 48 hours prior to stimulation. Coinfection with other adenoviruses in calvarial cells was at a multiplicity of infection of 400 and accomplished as previously described (26). Conditioned medium (CM) from HEK-293 cells infected with Ad-IGF-IR_1–482, Ad-IR_1–474, or Ad-GFP was collected either in complete medium or in 2% serum-containing medium (with indistinguishable results) and used undiluted.

Cell starvation, cell stimulation, and protein extraction

As previously described (26), serum starvation of primary calvarial cells (∼1 × 10⁶ cells per 6 cm² dish per sample) was accomplished by substitution of 0.5% (wt/vol) bovine serum albumin (fraction V; Roche Molecular Biochemicals) for serum in their respective culture media for 16–20 hours prior to experiments. Stimulations were performed at 37°C. The cells were stimulated in starvation medium, and stimulations were terminated by washing the cells once with and then harvesting by scraping in ice-cold PBS in the presence of 0.4 mM sodium orthovanadate. Pelleted cells were collected by brief centrifugation and solubilized for 30 minutes at 4°C in fusion 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 phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 mM benzamidine, and 10 μg/mL aprotinin]. After centrifugation at 15 000 × g for 15 minutes at 4°C, the detergent extracts were electrophoresed under reducing conditions. LNCaP and 3T3-F442A cells were stimulated and extracted as described previously (30, 31).

Immunoprecipitation, electrophoresis, and immunoblotting

For analysis of detergent cell extracts, proteins resolved by SDS-PAGE were transferred to Hybond ECL nitrocellulose membranes (Amersham Biosciences). The membranes were blocked with a buffer of 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% (vol/vol) Tween 20 containing 2% (wt/vol) BSA and incubated with primary antibodies (0.5–1 μg/mL) as specified in each experiment. After three washes with the buffer of 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% (vol/vol) Tween 20, the membranes were incubated with appropriate secondary antibodies (1:7500 dilution) and washed. The bound antibodies were detected with SuperSignal chemiluminescent substrate (Pierce Chemical Co). Membrane stripping was performed according to the manufacturer's suggestions (Amersham Biosciences).

Figure presentation and densitometric analysis

Immunoblots shown are in all instances representative of at least three experiments, as indicated in the figure legends. In generating the figures, irrelevant intervening lanes from original immunoblots have been cropped for clarity of presentation. In these cases, a space is maintained where intervening lanes were cropped. In all cases, only data from the same original blots are incorporated in figures with consistent brightness/contrast adjustment made across each blot. Immunoblots were obtained digitally using a Biospectrum 500 imaging system (UVP, LLC). Densitometric quantitation of immunoblots was performed using the ImageJ 1.30 program (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. In each experiment, the maximum signal was considered as 100%. The significance of differences (P value) of pooled results was estimated using paired t tests. In all cases, the signal obtained for phosphorylated STAT5 was normalized by that obtained for total (nonshifted and shifted) STAT5.

Results

Expression of IR does not rescue diminished GH-induced STAT5 phosphorylation in the setting of IGF-IR deletion in calvarial cells

Using primary calvarial cells from mice with a loxP-flanked IGF-IR that could be deleted in vitro by infection with Ad-Cre, we previously identified IGF-IR as a likely proximal contributor to GH-induced STAT5 activation and subsequent IGF-I gene expression (26). In those experiments, the reduced GH-induced STAT5 activation observed in cells infected with Ad-Cre and lacking endogenous mouse IGF-IR was rescued by coinfection with Ad-IGF-IR, which encodes the wild-type human IGF-IR (26), Because of similarities between IGF-IR and IR (Figure 1 and more below), we asked whether expression of IR could also rescue GH-induced STAT5 activation in Ad-Cre-infected cells.

Figure 1. — Diagram of structural features of IGF-IR, IR, and IGF-IR/IR chimeras. The L1, CR, and L2 domains within the extracellular subdomains of the IGF-IR and IR α subunits are indicated. Other subdomains within the α and β subunits of each receptor are delineated, but not labeled. S-S indicates positions of interchain disulfide linkages within the heterotetrameric assemblages. Double horizontal lines denote the position of the plasma membrane, which is traversed by the β subunits, but not the α subunits, of each receptor. See text for further details.

Primary cells harboring the floxed IGF-IR were infected with Ad-GFP (as a control) or with Ad-Cre plus Ad-GFP vs Ad-Cre plus Ad-IR. When detergent cell extracts were resolved by SDS-PAGE and immunoblotted with anti-IGF-IRα (Figure 2A) and anti-IGF-IRβ (Figure 2B), Ad-Cre infection resulted in loss of endogenous IGF-IR, as expected, and coinfection with Ad-IR yielded no detectable signal with these two antibodies, verifying the lack of cross-reactivity of the antibodies, as expected. In distinction, immunoblotting with anti-IRβ (Figure 2C) revealed that Ad-Cre infection did not diminish endogenous IR levels [as previously observed with this system (29)] and coinfection with Ad-IR resulted in enhanced abundance of the IR.

Figure 2. — Ad-Cre-mediated deletion of endogenous IGF-IR in primary calvarial cells and specific expression of IR or IGF-IR/IR chimeras by adenoviral infection. A–C, Primary calvarial cells were harvested from newborn mice bearing lox-P sites flanking both IGF-IR alleles. Calvarial cells were infected with Ad-Cre vs Ad-GFP plus Ad-IR, Ad-IGF-IR/IR-L1CRL2, Ad-IGF-IR/IR-L1, or Ad-GFP as a control, as indicated, as in *Materials and Methods*. Serum-starved cells were harvested and detergent cell extracts were resolved by SDS-PAGE and immunoblotted, as indicated with anti-IGF-IRα (A), anti-IGF-IRβ (B), or anti-IRβ (C), as indicated to specifically detect each protein. Note the Cre-mediated deletion of endogenous IGF-IR (A and B) and the specific detection of adenovirally reconstituted IGF-IR/IR chimeras, which are not detected with anti-IGF-IRα (A) but are detected with anti-IGF-IRβ, as anticipated. Note also continued expression of endogenous IR in Ad-Cre-infected cells and greater abundance of IR in Ad-Cre- plus Ad-IR-infected cells (C). WB, Western blotting.

We tested the effect of IGF-IR depletion on acute GH signaling by treating Ad-GFP-infected vs Ad-Cre-infected cells with GH (250 ng/mL, 10 minutes) vs vehicle. GH-induced phosphorylated STAT5 in cell extracts was assayed by immunoblotting and revealed the expected reduction (>50% diminished on average) in Ad-Cre-infected cells; this was not accounted for by changes in the levels of immunodetectable STAT5 (Figure 3, A and B). Notably, expression of IR driven by Ad-IR infection of Ad-Cre-infected cells did not rescue the reduced GH-induced STAT5 activation engendered by Cre-mediated lessening of IGF-IR abundance.

Figure 3. — Reduction of GH-induced STAT5 phosphorylation in Ad-Cre-infected primary calvarial cells and its rescue by coinfection with Ad-IGF-IR/IR-L1 but not Ad-IGF-IR/IR-L1CRL2. A, Primary calvarial cells from newborn mice bearing lox-P sites flanking both IGF-IR alleles were infected with Ad-Cre vs Ad-GFP plus Ad-IR, Ad-IGF-IR/IR-L1CRL2, Ad-IGF-IR/IR-L1, or Ad-GFP as a control, as indicated, as in *Materials and Methods* and Figure 2. Serum-starved cells were treated with GH (+; 250 ng/mL) or vehicle (−) for 10 minutes. Detergent cell extracts were resolved by SDS-PAGE and serially immunoblotted with anti-pSTAT5 and anti-STAT5. Immunoblots of a representative experiment are shown in A. pSTAT5, phosphorylated STAT5; WB, Western blotting. B, Densitometric quantitation of pSTAT5/STAT5 signals from GH-treated samples from nine independent experiments (including that shown in panel A). In each experiment, the maximum signal was considered 100%. Data are plotted as mean ± SE. *, P < .05 for comparison of the indicated group with any other GH-treated group without an asterisk. pSTAT5, phosphorylated STAT5; WB, Western blotting.

Construction and characterization of adenovirally driven IGF-IR/IR chimeras

We previously found that expression of an IGF-IR lacking most of its intracellular domain (IGF-IR_Δ950), like wild-type IGF-IR, substantially rescued the reduced GH-induced STAT5 activation observed with deletion of endogenous IGF-IR (26). Combined with our current findings that IR could not replace IGF-IR for such rescue, we reasoned that important determinants of the IGF-IR's participation in GH signaling might reside in its extracellular and/or transmembrane domain. The IR and IGF-IR genes each encode a single mRNA including both the α- and β-chains. For both receptors, cleavage of these α-β-precursors and disulfide linkage between the two chains occurs posttranslationally such that the mature α-chain is completely extracellular and the β-chain spans the membrane. Disulfide linkages between α-chains allow formation of the mature α₂β₂-heterotetrameric assemblage that is the mature receptor (diagrammed in Figure 1). The β-chains of both IGF-IR and IR harbor two extracellular fibronectin III domains (FNIII-2 and FNIII-3), a transmembrane domain, and an intracellular domain that features the tyrosine kinase moiety and a C-terminal tail. In contrast to the β-chains, the IGF-IR and IR α-chains are entirely extracellular. In their C-termini, the α-chains have the FNIII-1 and FNIII-2; beginning at their N termini, the α-chains have three domains that have been studied intensively (34 –42), largely because of their roles in ligand binding. These are the L1, cysteine-rich (CR), and L2 domains. The L1 domains are 70% identical and the L2 domains are also similar. A substantial difference resides in the CR domain, wherein the sequence identity is only 47% and IR contains an insertion and intrachain disulfide bond not found in IGF-IR.

To enable testing of how extracellular domain components from IGF-IR might be required for IGF-IR to optimally cooperate with proximal GHR signaling elements to augment GH-induced STAT5 activation, we constructed two chimeric molecules (diagrammed in Figure 1) and inserted them into adenoviruses to allow cellular expression. The two mutants are chimeras in which the IGF-IRβ-chain and α-chain fibronectin domains remain intact, but the more N-terminal α-chain extracellular domains of IGF-IR are swapped out and replaced by analogous IR domains. In IGF-IR/IR-L1CRL2, the L1, CR, and L2 domains of IRα-chain replace the analogous domains in the α-chain of IGF-IR. Likewise, IGF-IR/IR-L1 has the IRα-chain L1 domain in place of the IGF-IRα L1 domain at the N terminus of protein.

We tested the intactness of these chimeras by coinfecting floxed-IGF-IR-containing primary calvarial cells with Ad-Cre plus either Ad-IGF-IR/IR-L1CRL2 or Ad-IGF-IR/IR-L1. Immunoblotting of cell extracts revealed the expected lack of reactivity with anti-IGF-IRα (Figure 2A), which reacts with the N terminus of IGF-IR that is replaced by IR domains in both chimeras. However, both chimeras were detected by anti-IGF-IRβ (Figure 2B) and migrated at an M_r (apparent molecular mass) indistinguishable from endogenous IGF-IR (detected in cells infected with Ad-GFP alone), verifying that the chimeras retained their integrity and that the reading frames and α-β-cleavage patterns that yield the mature proteins were not disrupted, despite the molecular manipulations used to create them.

The two chimeras were tested for their abilities to rescue acute GH-induced STAT5 activation in floxed-IGF-IR cells (Figure 3, A and B). As described above, Ad-Cre infection alone rendered the cells less responsive to GH in terms of STAT5 activation, as expected. When coinfected with Ad-IGF-IR/IR-L1CRL2, the diminished GH-induced STAT5 activation related to Cre-mediated endogenous IGF-IR deletion was unaffected. In contrast, coinfection with Ad-IGF-IR/IR-L1 resulted in a 71% rescue, on average, of the Cre-mediated loss of GH-induced STAT5 activation.

Exposure of GH-responsive cells to a soluble fragment of the IGF-IR extracellular domain blunts subsequent GH-induced STAT5 phosphorylation

Our previous findings and those described above suggest that the ability of an intact IGF-IR to augment acute GH signaling (even in the absence of an IGF-IR binding ligand) depends on the presence and intactness of the IGF-IRα L1-CR-L2 region, perhaps by virtue of its ability to interact with GHR or a GHR-associated protein (24, 26). To pursue further the involvement of this IGF-IR extracellular domain region, we used an adenovirus that encodes IGF-IR_1–482, diagrammed in Figure 4A in comparison with wild-type IGF-IR. This IGF-IR fragment includes the L1-CR-L2 region and is secreted by cells that express it, consistent with its lack of transmembrane domain or other membrane-anchoring moiety (32). We first tested whether expression of Ad-IGF-IR_1–482 could rescue the diminished GH-induced STAT5 activation observed in floxed IGF-IR calvarial cells by Ad-Cre infection (Figure 4B). Although a high level of IGF-IR_1–482 was detected by immunoblotting of detergent extracts of cells infected with Ad-Cre and Ad-IGF-IR_1–482, expression of this protein was insufficient to allow more than 29% rescue of GH-induced STAT5 phosphorylation in cells lacking endogenous IGF-IR. The inability of the soluble IGF-IR_1–482 to completely mimic the ability of the membrane-anchored form of IGF-IR that lacks its intracellular domain (IGF-IR_Δ950) to rescue GH-induced STAT5 activation in IGF-IR-deficient cells (26) may relate to either its inability to efficiently functionally interact with cell surface GHR and/or its inability to translate such an interaction into an intracellular effect because of its lack of a transmembrane domain.

Figure 4. — Reduction of GH-induced STAT5 phosphorylation in Ad-Cre-infected primary calvarial cells is not rescued by coinfection with Ad-IGF-IR_1–482. A, Diagram of structural features of IGF-IR_1–482 compared with wild-type (WT) IGF-IR. Note that IGF-IR_1–482 contains only the L1, CR, and L2 subdomains of the IGF-IRα chain and has no transmembrane domain. B, Primary calvarial cells from newborn mice bearing lox-P sites flanking both IGF-IR alleles were infected with Ad-Cre vs Ad-GFP plus Ad-IGF-IR_1–482, as indicated. Serum-starved cells were treated with GH (+; 250 ng/mL) for 10 minutes. Detergent cell extracts were resolved by SDS-PAGE and serially immunoblotted with anti-pSTAT5, anti-STAT5, and anti-IGF-IRα. Immunoblots of a representative experiment of n = 4 are shown. Positions of IGF-IR precursor (prec) and α-chain (arrowheads) and IGF-IR_1–482 (bracket) are shown. pSTAT5, phosphorylated STAT5; WB, Western blotting.

To further pursue this, we tested the effect of IGF-IR_1–482 on acute GH signaling when incubated with intact (non-IGF-IR depleted) calvarial cells (Figure 5). HEK-293 cells were infected with Ad-IGF-IR_1–482 or, as a control, Ad-GFP, and CM was collected from each of the infected cells. Anti-IGF-IRα immunoblotting verified the presence of the soluble IGF-IR in the CM of Ad-IGF-IR_1–482-, but not Ad-GFP-infected HEK-293 cells (Figure 5A). Aliquots of primary calvarial cells from IGF-IR-floxed mice (but not Cre treated) were grown as usual in medium. Growth medium was then removed and replaced by CM from either Ad-GFP-infected or Ad-IGF-IR_1–482-infected HEK-293 cells, and 1 hour later the cells were stimulated with GH (0–250 ng/mL for 10 minutes) (Figure 5B). STAT5 phosphorylation induced by each concentration of GH was diminished in cells incubated with CM containing Ad-IGF-IR_1–482 compared with CM from Ad-GFP-infected cells. Densitometric quantitation of multiple experiments demonstrated greater than 20% inhibition of STAT5 phosphorylation induced by GH, resulting from incubation with IGF-IR_1–482-enriched CM (Figure 5C). Notably, although stimulated cells were washed prior to extraction, immunoblotting demonstrated that the soluble IGF-IR_1–482 remained present in the extract and was recovered in a GH-dependent fashion (Figure 5D). These experiments suggest that the presence of a soluble protein including the IGF-IRα L1-CR-L2 extracellular domain region affected GH-dependent signaling, perhaps via GH-dependent interaction with the cell surface.

Figure 5. — Reduction of GH-induced STAT5 phosphorylation by preincubation with medium enriched in soluble IGF-IR_1–482 in mouse primary calvarial cells. A, Equal aliquots of CM from HEK-293 cells infected with either Ad-GFP or Ad-IGF-IR_1–482 were resolved by SDS-PAGE and immunoblotted with anti-anti-IGF-IRα. Note specifically detected soluble IGF-IR in CM. B and C, Primary calvarial cells from newborn mice were incubated for 1 hour with CM from HEK-293 cells infected with Ad-IGF-IR_1–482 or, as a control, Ad-GFP. Cells were treated with GH (50, 125, or 250 ng/mL) or vehicle (0) for 10 minutes. Detergent cell extracts were resolved by SDS-PAGE and serially immunoblotted with anti-pSTAT5 and anti-STAT5. Immunoblots of a representative experiment are shown in A. B, Densitometric quantitation of pSTAT5/STAT5 signals from samples treated with 50 ng/mL (n = 7), 125 ng/mL (n = 3), and 250 ng/mL (n = 9) GH for 10 minutes (including that shown in A). In each experiment, the maximum signal was considered 100%. Data are plotted as mean ± SE. *, P = .07, **, P = .04, ***, P = .003 for comparisons of cells pretreated with CM from Ad-IGF-IR_1–482-infected HEK-293 cells compared with CM from Ad-GFP-infected HEK-293 cells. D, Primary calvarial cells were incubated for 1 hour with CM from HEK-293 cells infected with Ad-IGF-IR_1–482 or, as a control, Ad-GFP. Cells were treated with GH (50 or 250 ng/mL) or vehicle (0) for 10 minutes. Detergent cell extracts were resolved by SDS-PAGE and immunoblotted with anti-IGF-IRα. Note GH-augmented detection of IGF-IR_1–482 in cell extracts exposed to with Ad-IGF-IR_1–482 CM. pSTAT5, phosphorylated STAT5; WB, Western blotting.

To probe the generalizability of this finding, we also examined the effect of incubation with IGF-IR_1–482-enriched CM on acute GH-induced STAT5 activation in two other GH-responsive cell lines. We have demonstrated GH-dependent phosphorylation of STAT5 in the human LNCaP prostate cancer cell line (30); similarly, the mouse 3T3-F442A preadipocyte fibroblast is a cell line that we and others have shown is highly responsive to GH in terms of STAT5 phosphorylation (24). Both cell lines endogenously express GHR, JAK2, and STAT5 as well as IGF-IR [(24) and data not show]. As seen in Figure 6, A and B, and C and D, respectively, GH-induced STAT5 phosphorylation was blunted in both LNCaP and 3T3-F442A cells by more than 30% by preincubation with CM from Ad-IGF-IR_1–482-infected HEK-293 cells compared with CM from Ad-GFP-infected HEK-293 cells. This suggests that the effect of the soluble extracellular domain fragment of the IGF-IR on acute GH signaling was neither cell type nor species specific.

Figure 6. — Effect of soluble IGF-IR preincubation on GH-induced acute STAT5 phosphorylation in human prostate cancer cells and mouse preadipocytes. A–D, Representative immunoblots (A and C) and densitometric analysis of multiple experiments (B and D) with LNCaP (A and B) and 3T3-F442A (C and D) cells are shown, using the same methods as for Figure 5, B and C. In B, data for GH concentrations of 25 ng/mL (n = 3), 50 ng/mL (n = 5), 125 ng/mL (n = 3), and 250 ng/mL (n = 5) are shown. *, P = .16, **, P = .06, ***, P < .04 for comparisons of cells pretreated with CM from Ad-IGF-IR_1–482-infected HEK-293 cells compared with CM from Ad-GFP-infected HEK-293 cells. In D, asterisk indicates P < .08 for comparison of cells pretreated with CM from Ad-IGF-IR_1–482-infected HEK-293 cells compared with CM from Ad-GFP-infected HEK-293 cells (n = 3). pSTAT5, phosphorylated STAT5; WB, Western blotting.

We further pursued the specificity of this IGF-IR extracellular domain region by preparing an adenovirus encoding IR_1–474, the soluble form of IR containing the L1-CR-L2 regions of the IR analogous to that expressed by Ad-IGF-IR_1–482. As expected, CM of HEK-293 cells infected with either Ad-IGF-IR_1–482 or Ad-IR_1–474 was enriched in the respective proteins that each migrated at expected similar M_r and were detected appropriately by immunoblotting (Figure 7A). Consistent with data in Figures 5 and 6, preincubation with CM from Ad-IGF-IR_1–482-infected HEK-293 cells resulted in reduced acute GH-induced STAT5 phosphorylation in calvarial cells; however, preincubation with CM from Ad-IR_1–474-infected HEK-293 cells did not inhibit subsequent acute GH-induced STAT5 phosphorylation (Figure 7B). Likewise, we have not detected cell-associated IR_1–474 in the extracts of the CM-preincubated cells (data not shown). Thus, the effects of the soluble IGF-1R_1–482 on GH signaling appear specific, in concert with our findings with the chimeric receptor rescue experiments.

Figure 7. — Preincubation with medium enriched in IR_1–474 does not reduce GH-induced STAT5 phosphorylation in mouse primary calvarial cells. A, Equal aliquots of CM from HEK-293 cells infected with Ad-GFP, Ad-IGF-1R_1–482, or Ad-IR_1–474 were resolved by SDS-PAGE and immunoblotted sequentially with anti-anti-IGF-1Rα (to detect IGF-IR_1–482) or anti-FLAG (to detect Ad-IR_1–474). Note specifically detected soluble IGF-IR and IR in CM. B, Primary calvarial cells from newborn mice were incubated for 1 hour with CM from HEK-293 cells infected with Ad-GFP, Ad-IGF-IR_1–482, or Ad-IR_1–474. Cells were treated with GH (250 ng/mL; +) or vehicle (−) for 10 minutes. Detergent cell extracts were resolved by SDS-PAGE and serially immunoblotted with anti-pSTAT5 and anti-STAT5. pSTAT5, phosphorylated STAT5; WB, Western blotting.

Discussion

The original somatomedin hypothesis of GH action proposed that pituitary-derived GH elicits IGF-I (somatomedin-C) secretion from the liver and that this circulating IGF-I mediates the growth-promoting actions of GH by interacting with relevant tissues (18, 19). Although IGF-I is undoubtedly in part an effector of GH action, it is now clear that: GH can have direct effects at multiple tissues; IGF-I has endocrine, paracrine, and autocrine effects that are, in part, GH-independent; and GH and IGF-I likely have overlapping, counteractive, and/or collaborative effects (20, 22, 24, 43 –46). We have identified a novel physical and functional interaction between IGF-IR and GHR and have proposed that IGF-IR, in addition to mediating IGF-I and IGF-II effects, serves as a proximal contributor to efficient GH signaling (24 –27). In particular, we showed that GH induces formation of a GHR-JAK2-IGF-IR complex (24, 25) and that deletion of IGF-IR in several cellular systems reduces acute GH-induced STAT5 activation and subsequent gene expression (25, 26). Additionally, in some systems, we demonstrated that GH plus IGF-I cotreatment synergistically augments GH signaling (24, 25). These observations suggest an even richer interaction between GH/GHR and IGF-I/IGF-IR than encompassed by the original somatomedin hypothesis or subsequent modifications thereof.

In the current study, we add substantially to our understanding of IGF-IR's influence on GH signaling. We confirmed previous observations that deletion of IGF-IR in mouse calvarial cells blunted GH-induced STAT5 phosphorylation. Although reexpression of IGF-IR normalized GH signaling (26), we showed herein that neither endogenous IR nor forced overexpression of IR by adenoviral infection could fulfill this role of IGF-IR; thus, the IGF-1R effect is specific in that the structurally similar IR cannot suffice for IGF-IR. We extended this reasoning by testing whether IGF-IR/IR chimeras could replace IGF-IR to rescue acute GH signaling. We found that an IGF-IR harboring the IR L1 region rescued GH signaling but a chimera in which the IR L1, CR, and L2 regions replaced those of IGF-IR failed to restore GH sensitivity. This furthered the notion that a specific IGF-IRα extracellular domain region(s) is needed to allow IGF-IR to foster full GH signaling.

Although GH promotes a coimmunoprecipitable complex that includes GHR, JAK2, and IGF-IR, our findings do not yet prove that (unliganded) IGF-IR facilitates acute GH signaling by virtue of directly or indirectly associating with GHR. However, our chimera findings are consistent with our previous observations that the IGF-IR intracellular domain is not required for the receptor's ability to (at least partially) rescue GH signaling upon deletion of endogenous IGF-IR. Furthermore, our previous finding in preadipocytes that occupation of IGF-IR by IGF-I could even further augment acute GH signaling in that system suggests that the presence of the IGF-IRα region(s) that also includes determinants for ligand binding is in some way important for the augmentative role in GH signaling.

Along these lines, the results of our experiments with the soluble IGF-IR fragment, IGF-IR_1–482, are interesting. IGF-IR_1–482 encodes the L1-CR-L2 region of IGF-IRα that in the chimera setting was implicated in rescuing acute GH-induced STAT5 activation upon endogenous IGF-IR deletion. Yet infection with Ad-IGF-IR_1–482 failed to fully rescue GH-induced STAT5 activation, suggesting that membrane anchoring of IGF-IR is necessary for this effect. Notably, in the setting of normal endogenous IGF-IR levels, exposure of three separate GH-responsive cell lines (mouse primary calvarial cells, mouse preadipocytes, and human prostate cancer cells) to medium enriched in IGF-IR_1–482 blunted responsiveness to subsequent GH stimulation in terms of acute STAT5 phosphorylation. However, CM containing an analogous soluble IR (IR_1–474) did not diminish acute GH signaling, consistent with the conclusions drawn from the chimera reconstitution data about the specificity of IGF-IR in functional interaction with acute GH signaling. Although other potential explanations for these findings exist, our working hypothesis is that (nonmembrane anchored) IGF-IR_1–482 interacted with GHR or a cell surface GHR-associated molecule to prevent endogenous cell surface IGF-IR from productively interacting with the proximal GH signaling apparatus to exert its augmentative effect. [We note that we have as yet been unsuccessful in coimmunoprecipitating IGF-IR_1–482 with GHR; however, the GH dependence of augmented association of this soluble IGF-IR (but not the soluble IR) with the cells suggests GHR or a GHR-associated protein may allow this specific association.] Further studies dissecting soluble IGF-IRα structural determinants required for this inhibitory effect may be profitable for gaining a better mechanistic understanding of whether its putative association is directly with GHR vs indirect, perhaps via another molecule(s) that is itself GHR-associated, which may be further defined proteomically.

We note that the α-chain L1-CR-L2 regions of IGF-IR and IR differ most within the CR domains (discussed in Reference 35), wherein in module 6 there exists a four-residue (NSRR) insertion in IR that is not present in IGF-IR. Immediately surrounding this insertion in the IR is an additional intrachain disulfide linkage compared with the analogous IGF-IR CR module 6. Furthermore, structural analysis predicts that the CR domains of the two receptors differ substantially, with the CR of IR being mostly positive and the CR of IGF-IR being highly negative (35). These differences likely underlie differential binding to insulin vs IGF-I; however, in addition, it is intriguing to consider whether they also underlie the ligand-independent effects of IGF-IR vs IR on acute GH signaling that we observe. Because our chimera experiments suggest that the L1 domain of either receptor allows IGF-IR to functionally collaborate with GH signaling and the L2 domains of the two receptors are overall quite similar, future experiments targeting analysis of the CR domain of IGF-IR might be most worthwhile.

In recent work (27), we found that the reduced GH-induced STAT5 phosphorylation that results from deletion of IGF-IR in primary calvarial cells is rescued by inhibition of the phosphatase activity of PTP-1B. Yet PTP-1B inhibition had no effect on GH signaling in cells in which endogenous IGF-IR expression was preserved. Furthermore, a PTP-1B substrate trapping mutant bound to GH-induced tyrosine phosphorylated JAK2 only in the cells in which IGF-IR was deleted. Because PTP-1B is an entirely intracellular protein, these findings suggested that the presence of IGF-IR or, perhaps, an IGF-IR- (and GHR)-associated transmembrane protein was required to reduce PTP-1B's access to the GH-induced activated JAK2.

It will be critical in future studies to determine whether the findings in the current study provide complementary mechanistic information to the PTP-1B studies to allow us to generate a comprehensive and testable model of IGF-IR's involvement in proximal GH signaling. Such a model may invoke the existence of GHR- and IGF-IR-associated proteins that allow GH binding and signal initiation in the extracellular environment to be modulated in intensity and/or duration by intracellular factors. Because GH and IGF-I actions are intricately interrelated physiologically, it may be envisioned that the presence of IGF-IR may play an important modulatory role in governing GH responsiveness at particular GH-responsive tissues and under particular physiological and/or pathophysiological circumstances.

Acknowledgments

The authors appreciate the helpful conversations with Drs Y. Huang, D. Sun, A. Buckels, Y. Liu, P. Berry, J. Messina, L. Deng, and J. Xu and the generous provision of reagents by those named in the text.

This work was supported by National Institutes of Health Grant R01 DK46395 (to S.J.F.).

Parts of this work were presented at the 97th Annual Meeting of The Endocrine Society, San Francisco, CA, June 15–18, 2013.

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:

Ad-Cre: Cre-recombinase-expressing adenovirus
Ad-GFP: adenovirus encoding green fluorescent protein
CM: conditioned medium
CR: cysteine rich
FNIII: fibronectin III
GHR: GH receptor
HEK: human embryonic kidney
IGF-IR: IGF-I receptor
JAK2: Janus kinase 2
PTP: protein tyrosine phosphatase
STAT5: signal transducer and activator of transcription 5.

References

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2. Waters MJ, Hoang HN, Fairlie DP, Pelekanos RA, Brown RJ. New insights into growth hormone action. J Mol Endocrinol. 2006;36:1–7 [DOI] [PubMed] [Google Scholar]
3. Hernandez LM, Lee PD, Camacho-Hubner C. Isolated growth hormone deficiency. Pituitary. 2007;10:351–357 [DOI] [PubMed] [Google Scholar]
4. David A, Hwa V, Metherell LA, et al. Evidence for a continuum of genetic, phenotypic, and biochemical abnormalities in children with growth hormone insensitivity. Endocr Rev. 2011;32:472–497 [DOI] [PubMed] [Google Scholar]
5. Duncan E, Wass JA. Investigation protocol: acromegaly and its investigation. Clin Endocrinol (Oxf). 1999;50:285–293 [DOI] [PubMed] [Google Scholar]
6. Shen Q, Lantvit DD, Lin Q, et al. Advanced rat mammary cancers are growth hormone dependent. Endocrinology. 2007;148:4536–4544 [DOI] [PubMed] [Google Scholar]
7. Swanson SM, Unterman TG. The growth hormone-deficient Spontaneous Dwarf rat is resistant to chemically induced mammary carcinogenesis. Carcinogenesis. 2002;23:977–982 [DOI] [PubMed] [Google Scholar]
8. Wang Z, Luque RM, Kineman RD, et al. Disruption of growth hormone signaling retards prostate carcinogenesis in the probasin/TAg rat. Endocrinology. 2008;149:1366–1376 [DOI] [PMC free article] [PubMed] [Google Scholar]
9. Wang Z, Prins GS, Coschigano KT, et al. Disruption of growth hormone signaling retards early stages of prostate carcinogenesis in the C3(1)/T antigen mouse. Endocrinology. 2005;146:5188–5196 [DOI] [PubMed] [Google Scholar]
10. Brooks AJ, Waters MJ. The growth hormone receptor: mechanism of activation and clinical implications. Nat Rev Endocrinol. 2010;6:515–525 [DOI] [PubMed] [Google Scholar]
11. Frank SJ, Messina JL. Growth hormone receptor. In: Oppenheim JJ, Feldman M, eds. Cytokine Reference On-Line. London: Academic Press, Harcourt; 2002:1–21 [Google Scholar]
12. Herrington J, Carter-Su C. Signaling pathways activated by the growth hormone receptor. Trends Endocrinol Metab. 2001;12:252–257 [DOI] [PubMed] [Google Scholar]
13. Davey HW, Xie T, McLachlan MJ, Wilkins RJ, Waxman DJ, Grattan DR. STAT5b is required for GH-induced liver IGF-I gene expression. Endocrinology. 2001;142:3836–3841 [DOI] [PubMed] [Google Scholar]
14. Woelfle J, Billiard J, Rotwein P. Acute control of insulin-like growth factor-I gene transcription by growth hormone through Stat5b. J Biol Chem. 2003;278:22696–22702 [DOI] [PubMed] [Google Scholar]
15. Ullrich A, Gray A, Tam AW, et al. Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J. 1986;5:2503–2512 [DOI] [PMC free article] [PubMed] [Google Scholar]
16. LeRoith D. Insulin-like growth factor I receptor signaling—overlapping or redundant pathways? Endocrinology. 2000;141:1287–1288 [DOI] [PubMed] [Google Scholar]
17. Nakae J, Kido Y, Accili D. Distinct and overlapping functions of insulin and IGF-I receptors. Endocr Rev. 2001;22:818–835 [DOI] [PubMed] [Google Scholar]
18. Salmon WD, Daughaday WH. A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. J Lab Clin Med. 1957;49:825–836 [PubMed] [Google Scholar]
19. Daughaday WH. Growth hormone axis overview—somatomedin hypothesis. Pediatr Nephrol. 2000;14:537–540 [DOI] [PubMed] [Google Scholar]
20. Le Roith D, Bondy C, Yakar S, Liu JL, Butler A. The somatomedin hypothesis: 2001. Endocr Rev. 2001;22:53–74 [DOI] [PubMed] [Google Scholar]
21. Fan Y, Menon RK, Cohen P, et al. Liver-specific deletion of the growth hormone receptor reveals essential role of growth hormone signaling in hepatic lipid metabolism. J Biol Chem. 2009;284:19937–19944 [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Lupu F, Terwilliger JD, Lee K, Segre GV, Efstratiadis A. Roles of growth hormone and insulin-like growth factor 1 in mouse postnatal growth. Dev Biol. 2001;229:141–162 [DOI] [PubMed] [Google Scholar]
23. Frank SJ. Growth hormone, insulin-like growth factor I, and growth: local knowledge. Endocrinology. 2007;148:1486–1488 [DOI] [PubMed] [Google Scholar]
24. Huang Y, Kim S-O, Yang N, Jiang J, Frank SJ. Physical and functional interaction of GH and IGF-1 signaling elements. Mol Endocrinol. 2004;18:1471–1485 [DOI] [PubMed] [Google Scholar]
25. Ma F, Wei Z, Shi C, et al. Signaling cross talk between growth hormone (GH) and insulin-like growth factor-I (IGF-I) in pancreatic islet beta-cells. Mol Endocrinol. 2011;25:2119–2133 [DOI] [PMC free article] [PubMed] [Google Scholar]
26. Gan Y, Zhang Y, Digirolamo DJ, et al. Deletion of IGF-I receptor (IGF-IR) in primary osteoblasts reduces GH-induced STAT5 signaling. Mol Endocrinol. 2010;24:644–656 [DOI] [PMC free article] [PubMed] [Google Scholar]
27. Gan Y, Zhang Y, Buckels A, et al. IGF-1R modulation of acute GH-induced STAT5 signaling: role of protein tyrosine phosphatase activity. Mol Endocrinol. 2013;27(11):1969–1979 [DOI] [PMC free article] [PubMed] [Google Scholar]
28. DiGirolamo DJ, Mukherjee A, Fulzele K, et al. Mode of growth hormone action in osteoblasts. J Biol Chem. 2007;282:31666–31674 [DOI] [PubMed] [Google Scholar]
29. Fulzele K, DiGirolamo DJ, Liu Z, Xu J, Messina JL, Clemens TL. Disruption of the insulin-like growth factor type 1 receptor in osteoblasts enhances insulin signaling and action. J Biol Chem. 2007;282:25649–25658 [DOI] [PubMed] [Google Scholar]
30. Xu J, Zhang Y, Berry PA, et al. Growth hormone signaling in human T47D breast cancer cells: potential role for a growth hormone receptor-prolactin receptor complex. Mol Endocrinol. 2011;25:597–610 [DOI] [PMC free article] [PubMed] [Google Scholar]
31. Wang X, Yang N, Deng L, et al. Interruption of growth hormone signaling via SHC and ERK in 3T3–F442A preadipocytes upon knockdown of insulin receptor substrate-1. Mol Endocrinol. 2009;23:486–496 [DOI] [PMC free article] [PubMed] [Google Scholar]
32. Lee CT, Park KH, Adachi Y, et al. Recombinant adenoviruses expressing dominant negative insulin-like growth factor-I receptor demonstrate antitumor effects on lung cancer. Cancer Gene Ther. 2003;10:57–63 [DOI] [PubMed] [Google Scholar]
33. Frank SJ, Wang X, He K, et al. In vivo imaging of hepatic growth hormone signaling. Mol Endocrinol. 2006;20:2819–2830 [DOI] [PubMed] [Google Scholar]
34. McKern NM, Lawrence MC, Streltsov VA, et al. Structure of the insulin receptor ectodomain reveals a folded-over conformation. Nature. 2006;443:218–221 [DOI] [PubMed] [Google Scholar]
35. Lou M, Garrett TP, McKern NM, et al. The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity. Proc Natl Acad Sci USA. 2006;103:12429–12434 [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Garrett TP, McKern NM, Lou M, et al. Crystal structure of the first three domains of the type-1 insulin-like growth factor receptor. Nature. 1998;394:395–399 [DOI] [PubMed] [Google Scholar]
37. Lawrence MC, McKern NM, Ward CW. Insulin receptor structure and its implications for the IGF-1 receptor. Curr Opin Struct Biol. 2007;17:699–705 [DOI] [PubMed] [Google Scholar]
38. Ward CW, Lawrence MC, Streltsov VA, Adams TE, McKern NM. The insulin and EGF receptor structures: new insights into ligand-induced receptor activation. Trends Biochem Sci. 2007;32:129–137 [DOI] [PubMed] [Google Scholar]
39. Kristensen C, Wiberg FC, Andersen AS. Specificity of insulin and insulin-like growth factor I receptors investigated using chimeric mini-receptors. Role of C-terminal of receptor α subunit. J Biol Chem. 1999;274:37351–37356 [DOI] [PubMed] [Google Scholar]
40. Schumacher R, Soos MA, Schlessinger J, Brandenburg D, Siddle K, Ullrich A. Signaling-competent receptor chimeras allow mapping of major insulin receptor binding domain determinants. J Biol Chem. 1993;268:1087–1094 [PubMed] [Google Scholar]
41. Menting JG, Whittaker J, Margetts MB, et al. How insulin engages its primary binding site on the insulin receptor. Nature. 2013;493:241–245 [DOI] [PMC free article] [PubMed] [Google Scholar]
42. Hubbard SR. Structural biology: insulin meets its receptor. Nature. 2013;493:171–172 [DOI] [PubMed] [Google Scholar]
43. Kaplan SA, Cohen P. The somatomedin hypothesis 2007: 50 years later. J Clin Endocrinol Metab. 2007;92:4529–4535 [DOI] [PubMed] [Google Scholar]
44. Wu Y, Sun H, Basta-Pljakic J, et al. Serum IGF-1 is insufficient to restore skeletal size in the total absence of the growth hormone receptor. J Bone Miner Res. 2013;28:1575–1586 [DOI] [PMC free article] [PubMed] [Google Scholar]
45. Ashcom G, Gurland G, Schwartz J. Growth hormone synergizes with serum growth factors in inducing c-fos transcription in 3T3–F442A cells. Endocrinology. 1992;131:1915–1921 [DOI] [PubMed] [Google Scholar]
46. Edmondson SR, Russo VC, McFarlane AC, Wraight CJ, Werther GA. Interactions between growth hormone, insulin-like growth factor I, and basic fibroblast growth factor in melanocyte growth. J Clin Endocrinol Metab. 1999;84:1638–1644 [DOI] [PubMed] [Google Scholar]

[B1] 1. Moller N, Jorgensen JO. Effects of growth hormone on glucose, lipid, and protein metabolism in human subjects. Endocr Rev. 2009;30:152–177 [DOI] [PubMed] [Google Scholar]

[B2] 2. Waters MJ, Hoang HN, Fairlie DP, Pelekanos RA, Brown RJ. New insights into growth hormone action. J Mol Endocrinol. 2006;36:1–7 [DOI] [PubMed] [Google Scholar]

[B3] 3. Hernandez LM, Lee PD, Camacho-Hubner C. Isolated growth hormone deficiency. Pituitary. 2007;10:351–357 [DOI] [PubMed] [Google Scholar]

[B4] 4. David A, Hwa V, Metherell LA, et al. Evidence for a continuum of genetic, phenotypic, and biochemical abnormalities in children with growth hormone insensitivity. Endocr Rev. 2011;32:472–497 [DOI] [PubMed] [Google Scholar]

[B5] 5. Duncan E, Wass JA. Investigation protocol: acromegaly and its investigation. Clin Endocrinol (Oxf). 1999;50:285–293 [DOI] [PubMed] [Google Scholar]

[B6] 6. Shen Q, Lantvit DD, Lin Q, et al. Advanced rat mammary cancers are growth hormone dependent. Endocrinology. 2007;148:4536–4544 [DOI] [PubMed] [Google Scholar]

[B7] 7. Swanson SM, Unterman TG. The growth hormone-deficient Spontaneous Dwarf rat is resistant to chemically induced mammary carcinogenesis. Carcinogenesis. 2002;23:977–982 [DOI] [PubMed] [Google Scholar]

[B8] 8. Wang Z, Luque RM, Kineman RD, et al. Disruption of growth hormone signaling retards prostate carcinogenesis in the probasin/TAg rat. Endocrinology. 2008;149:1366–1376 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9. Wang Z, Prins GS, Coschigano KT, et al. Disruption of growth hormone signaling retards early stages of prostate carcinogenesis in the C3(1)/T antigen mouse. Endocrinology. 2005;146:5188–5196 [DOI] [PubMed] [Google Scholar]

[B10] 10. Brooks AJ, Waters MJ. The growth hormone receptor: mechanism of activation and clinical implications. Nat Rev Endocrinol. 2010;6:515–525 [DOI] [PubMed] [Google Scholar]

[B11] 11. Frank SJ, Messina JL. Growth hormone receptor. In: Oppenheim JJ, Feldman M, eds. Cytokine Reference On-Line. London: Academic Press, Harcourt; 2002:1–21 [Google Scholar]

[B12] 12. Herrington J, Carter-Su C. Signaling pathways activated by the growth hormone receptor. Trends Endocrinol Metab. 2001;12:252–257 [DOI] [PubMed] [Google Scholar]

[B13] 13. Davey HW, Xie T, McLachlan MJ, Wilkins RJ, Waxman DJ, Grattan DR. STAT5b is required for GH-induced liver IGF-I gene expression. Endocrinology. 2001;142:3836–3841 [DOI] [PubMed] [Google Scholar]

[B14] 14. Woelfle J, Billiard J, Rotwein P. Acute control of insulin-like growth factor-I gene transcription by growth hormone through Stat5b. J Biol Chem. 2003;278:22696–22702 [DOI] [PubMed] [Google Scholar]

[B15] 15. Ullrich A, Gray A, Tam AW, et al. Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J. 1986;5:2503–2512 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16. LeRoith D. Insulin-like growth factor I receptor signaling—overlapping or redundant pathways? Endocrinology. 2000;141:1287–1288 [DOI] [PubMed] [Google Scholar]

[B17] 17. Nakae J, Kido Y, Accili D. Distinct and overlapping functions of insulin and IGF-I receptors. Endocr Rev. 2001;22:818–835 [DOI] [PubMed] [Google Scholar]

[B18] 18. Salmon WD, Daughaday WH. A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. J Lab Clin Med. 1957;49:825–836 [PubMed] [Google Scholar]

[B19] 19. Daughaday WH. Growth hormone axis overview—somatomedin hypothesis. Pediatr Nephrol. 2000;14:537–540 [DOI] [PubMed] [Google Scholar]

[B20] 20. Le Roith D, Bondy C, Yakar S, Liu JL, Butler A. The somatomedin hypothesis: 2001. Endocr Rev. 2001;22:53–74 [DOI] [PubMed] [Google Scholar]

[B21] 21. Fan Y, Menon RK, Cohen P, et al. Liver-specific deletion of the growth hormone receptor reveals essential role of growth hormone signaling in hepatic lipid metabolism. J Biol Chem. 2009;284:19937–19944 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22. Lupu F, Terwilliger JD, Lee K, Segre GV, Efstratiadis A. Roles of growth hormone and insulin-like growth factor 1 in mouse postnatal growth. Dev Biol. 2001;229:141–162 [DOI] [PubMed] [Google Scholar]

[B23] 23. Frank SJ. Growth hormone, insulin-like growth factor I, and growth: local knowledge. Endocrinology. 2007;148:1486–1488 [DOI] [PubMed] [Google Scholar]

[B24] 24. Huang Y, Kim S-O, Yang N, Jiang J, Frank SJ. Physical and functional interaction of GH and IGF-1 signaling elements. Mol Endocrinol. 2004;18:1471–1485 [DOI] [PubMed] [Google Scholar]

[B25] 25. Ma F, Wei Z, Shi C, et al. Signaling cross talk between growth hormone (GH) and insulin-like growth factor-I (IGF-I) in pancreatic islet beta-cells. Mol Endocrinol. 2011;25:2119–2133 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B26] 26. Gan Y, Zhang Y, Digirolamo DJ, et al. Deletion of IGF-I receptor (IGF-IR) in primary osteoblasts reduces GH-induced STAT5 signaling. Mol Endocrinol. 2010;24:644–656 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27. Gan Y, Zhang Y, Buckels A, et al. IGF-1R modulation of acute GH-induced STAT5 signaling: role of protein tyrosine phosphatase activity. Mol Endocrinol. 2013;27(11):1969–1979 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B28] 28. DiGirolamo DJ, Mukherjee A, Fulzele K, et al. Mode of growth hormone action in osteoblasts. J Biol Chem. 2007;282:31666–31674 [DOI] [PubMed] [Google Scholar]

[B29] 29. Fulzele K, DiGirolamo DJ, Liu Z, Xu J, Messina JL, Clemens TL. Disruption of the insulin-like growth factor type 1 receptor in osteoblasts enhances insulin signaling and action. J Biol Chem. 2007;282:25649–25658 [DOI] [PubMed] [Google Scholar]

[B30] 30. Xu J, Zhang Y, Berry PA, et al. Growth hormone signaling in human T47D breast cancer cells: potential role for a growth hormone receptor-prolactin receptor complex. Mol Endocrinol. 2011;25:597–610 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B31] 31. Wang X, Yang N, Deng L, et al. Interruption of growth hormone signaling via SHC and ERK in 3T3–F442A preadipocytes upon knockdown of insulin receptor substrate-1. Mol Endocrinol. 2009;23:486–496 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B32] 32. Lee CT, Park KH, Adachi Y, et al. Recombinant adenoviruses expressing dominant negative insulin-like growth factor-I receptor demonstrate antitumor effects on lung cancer. Cancer Gene Ther. 2003;10:57–63 [DOI] [PubMed] [Google Scholar]

[B33] 33. Frank SJ, Wang X, He K, et al. In vivo imaging of hepatic growth hormone signaling. Mol Endocrinol. 2006;20:2819–2830 [DOI] [PubMed] [Google Scholar]

[B34] 34. McKern NM, Lawrence MC, Streltsov VA, et al. Structure of the insulin receptor ectodomain reveals a folded-over conformation. Nature. 2006;443:218–221 [DOI] [PubMed] [Google Scholar]

[B35] 35. Lou M, Garrett TP, McKern NM, et al. The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity. Proc Natl Acad Sci USA. 2006;103:12429–12434 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36. Garrett TP, McKern NM, Lou M, et al. Crystal structure of the first three domains of the type-1 insulin-like growth factor receptor. Nature. 1998;394:395–399 [DOI] [PubMed] [Google Scholar]

[B37] 37. Lawrence MC, McKern NM, Ward CW. Insulin receptor structure and its implications for the IGF-1 receptor. Curr Opin Struct Biol. 2007;17:699–705 [DOI] [PubMed] [Google Scholar]

[B38] 38. Ward CW, Lawrence MC, Streltsov VA, Adams TE, McKern NM. The insulin and EGF receptor structures: new insights into ligand-induced receptor activation. Trends Biochem Sci. 2007;32:129–137 [DOI] [PubMed] [Google Scholar]

[B39] 39. Kristensen C, Wiberg FC, Andersen AS. Specificity of insulin and insulin-like growth factor I receptors investigated using chimeric mini-receptors. Role of C-terminal of receptor α subunit. J Biol Chem. 1999;274:37351–37356 [DOI] [PubMed] [Google Scholar]

[B40] 40. Schumacher R, Soos MA, Schlessinger J, Brandenburg D, Siddle K, Ullrich A. Signaling-competent receptor chimeras allow mapping of major insulin receptor binding domain determinants. J Biol Chem. 1993;268:1087–1094 [PubMed] [Google Scholar]

[B41] 41. Menting JG, Whittaker J, Margetts MB, et al. How insulin engages its primary binding site on the insulin receptor. Nature. 2013;493:241–245 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B42] 42. Hubbard SR. Structural biology: insulin meets its receptor. Nature. 2013;493:171–172 [DOI] [PubMed] [Google Scholar]

[B43] 43. Kaplan SA, Cohen P. The somatomedin hypothesis 2007: 50 years later. J Clin Endocrinol Metab. 2007;92:4529–4535 [DOI] [PubMed] [Google Scholar]

[B44] 44. Wu Y, Sun H, Basta-Pljakic J, et al. Serum IGF-1 is insufficient to restore skeletal size in the total absence of the growth hormone receptor. J Bone Miner Res. 2013;28:1575–1586 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B45] 45. Ashcom G, Gurland G, Schwartz J. Growth hormone synergizes with serum growth factors in inducing c-fos transcription in 3T3–F442A cells. Endocrinology. 1992;131:1915–1921 [DOI] [PubMed] [Google Scholar]

[B46] 46. Edmondson SR, Russo VC, McFarlane AC, Wraight CJ, Werther GA. Interactions between growth hormone, insulin-like growth factor I, and basic fibroblast growth factor in melanocyte growth. J Clin Endocrinol Metab. 1999;84:1638–1644 [DOI] [PubMed] [Google Scholar]

PERMALINK

Functional Collaboration of Insulin-Like Growth Factor-1 Receptor (IGF-1R), but Not Insulin Receptor (IR), With Acute GH Signaling in Mouse Calvarial Cells

Yujun Gan

Andrew J Paterson

Yue Zhang

Jing Jiang

Stuart J Frank

Abstract