Differential Activation of Insulin Receptor Isoforms by Insulin-Like Growth Factors Is Determined by the C Domain
http://www.100md.com
《内分泌学杂志》
School of Molecular and Biomedical Science (A.D., G.V.B., G.W.B., J.C.W., B.E.F.), The University of Adelaide, Adelaide 5005, Australia
Commonwealth Scientific and Industrial Research Organisation Division of Molecular and Health Technologies (A.D., G.V.B., L.J.C.), Adelaide 5000, Australia
Department of Pediatrics (J.M.C., A.L., C.T.R.), Oregon Health and Science University, Portland, Oregon 97239
Abstract
The actions of IGF-I and IGF-II are thought to be largely due to their activation of the IGF-I receptor. However, IGF-II can also bind with high affinity to, and effectively activate, an isoform of the insulin receptor (IR-A) that lacks a sequence at the carboxyl-terminal end of the extracellular subunit due to the alternative splicing of exon 11. This isoform is poorly activated by IGF-I. Here, we show that IGF-II, but not IGF-I, induces potent autophosphorylation of residues Y1158, Y1162, and Y1163 in the activation loop of the kinase domain and tyrosine 960 in the juxtamembrane region of both IR-A and IR-B (exon 11+) isoforms. We have also found, by using IGF chimeras, that the C domain of IGF-II completely accounts for the ability of IGF-II to stimulate IR autophosphorylation compared with IGF-I. We further show that the C domains are responsible for the differential abilities of IGF-II and IGF-I to activate phosphorylation of insulin receptor substrate-1 and Akt, as well as their ability to induce migration and cell survival via the IR-A. Finally, we show for the first time that IGF signaling through the IR-A can protect cells from butyrate-induced apoptosis. In summary, our studies define the structural determinants that allow potent IGF-II signaling and regulation of cellular functions through the IR-A and provide novel insights into IGF signaling via the IR.
Introduction
MOUSE KNOCKOUT STUDIES have revealed that the insulin receptor (IR) mediates the growth-promoting effects of IGF-II in early development (1, 2, 3). The alternatively spliced form of the IR that lacks residues encoded by exon 11 (IR-A) binds insulin and IGF-II with high affinity, but binds IGF-I poorly. Insulin and IGF-II, but not IGF-I, can potently induce IR-A autophosphorylation (4). Numerous studies have shown that IGF-II, but not IGF-I, can signal via the IR-A and induce cancer cell proliferation and migration (5, 6, 7, 8). An increase in the relative expression of the IR-A has been shown in several cancers, including those of the lung, colon (4), breast (4, 7), ovaries (9), thyroid (8), and smooth and striated muscle (5). Together, these studies suggest that the binding and activation of the IR-A by IGF-II is significant in development and can possibly contribute to certain pathologies (10).
Autophosphorylation of the IR occurs initially on tyrosines 1162, 1158, and 1163 in the activation loop of the kinase domain, allowing unobstructed access of ATP and substrate to the kinase active site (11, 12, 13, 14). The activated catalytic core then phosphorylates additional tyrosine residues such as 960, which provides a docking site for the adaptor proteins insulin receptor substrate-1 (IRS)-1 (15, 16), IRS-2 (17), and Shc (18) (reviewed in Refs.19 and 20). Activation of IRS-1, Shc, Erk1/2, and Akt by IGF-II, but not IGF-I, has been shown in both transfected (4, 21) and tumor cell lines (SKUT-1) (5) devoid of the IGF-I receptor (IGF-IR) and expressing >95% IR-A. To date, the structural determinants of IGF-II that allow the activation of IR-A signaling pathways are not known.
Mature IGF-I and IGF-II are comprised of four domains (B, C, A, and D) and share extensive sequence and structural similarity. We have previously demonstrated by domain swapping that the IGF-II C and D domains conferred high-affinity binding to the IR-A and IR-B, whereas the C and D domains of IGF-I did not (22). These studies, however, did not define the particular domain primarily responsible for determining the differential abilities of the IGFs to activate the IR or induce downstream signaling pathways that mediate the biological effects of the IGFs.
Here, we report for the first time that IGF-II and IGF-I differentially activate specific tyrosine residues on IR-A and IR-B. In addition, by using IGF chimeras (22), we show that the sole structural determinant for the differential ability of IGF-I and IGF-II to induce autophosphorylation of specific IR tyrosine residues and activate downstream signaling molecules is the C domain. We also demonstrate that substitution of the C domain of IGF-I with that of IGF-II results in a chimera whose ability to induce protection from butyrate-induced apoptosis and promote cell migration via the IR-A is similar to that of authentic IGF-II. Our studies may have significant ramifications for the design of cancer therapeutics currently directed against IGF-IR signaling.
Materials and Methods
Materials
Antibodies against the following proteins were obtained from the indicated sources: IRS-1, phospho-Erk1/2, Erk1/2, and Akt from Cell Signaling Technologies (Beverly, MA); phospho-Akt [pS473], phospho-IR/IGF-1R [pYpYpY1158/1162/1163], and phospho-IR [pY972)] from Biosource International (Camarillo, CA); and rat carboxyl-terminal IRS-1 from Upstate Biotechnology (Lake Placid, NY). Protease inhibitors, pepstatin, type 1 collagen, and sodium orthovanadate were from Sigma Chemical Co. (St. Louis, MO). Criterion 12% Tris-tricine gels were from Bio-Rad (Hercules, CA). Human insulin was from Novo Nordisk (Bagsvrd, Denmark). R– cells (mouse 3T3-like cells with a targeted ablation of the IGF-IR gene) (23) were a kind gift from Prof. R. Baserga (Thomas Jefferson University, Philadelphia, PA). The construction, expression, and purification of chimeras of IGF-I and IGF-II and R– cells expressing the human IR-A (R–IR-A) and IR-B (R–IR-B) cDNAs have been previously reported (22). 3T3-IR-A cells have been described previously (24).
IR-A and IR-B phosphorylation and activation of intracellular signaling molecules
3T3-IR-A cells were treated with 10 nM ligand for 2, 5, 10, and 60 min in time-course experiments, and R–IR-A or R–IR-B cells were treated with 10 nM ligand for 5 min. For whole-cell lysates, cells were lysed in 1x sodium dodecyl sulfate (SDS) sample buffer (25) without dithiothreitol or bromophenol blue and boiled immediately to inhibit protease and phosphatase action. Protein concentration was determined with a detergent-compatible protein assay kit (Bio-Rad). dithiothreitol was then added to 100 mM and bromophenol blue to 0.1%. For immunoprecipitations, cells were lysed in 50 mM Tris (pH 6.8), 1% (wt/vol) SDS, and 10% (vol/vol) glycerol or 150 mM NaCl, 10% (vol/vol) glycerol, 20 mM Tris (pH 8), 1 mM EDTA, 0.2% SDS, 1 tablet of complete protease inhibitors per 10 ml, 1 mM sodium orthovanadate, and 1 μg/ml pepstatin. Lysates were incubated on ice for 30 min, inverting occasionally. They were then microcentrifuged at 12,000 x g for 15 min and the supernatants transferred to new tubes. Protein concentration was determined as above. For immunoprecipitations, lysates (500 μg) were precleared with protein A-agarose beads for 30 min before incubation with anti-IRS-1 antibody overnight at 4 C. Protein A-agarose beads were added for 3 h at 4 C and immunoprecipitates were then washed in 3x lysis buffer, eluted in 2x SDS sample buffer, and subjected to SDS-PAGE. Immunoprecipitates or whole-cell lysates (20 μg) were subjected to reducing SDS-PAGE on Criterion gels. Blots were probed with relevant phospho-antibodies, or, for IRS-1 immunoprecipitates, blots were probed with the antiphosphotyrosine antibody PY20. In all cases, after probing with the phospho-specific antibody, the blot was stripped in 100 mM Tris-HCl (pH 6.8), 10% SDS, and 100 mM -mercaptoethanol for 30 min at 60 C and reprobed with a pan-specific antibody. Paired Student’s t tests were used for all statistical analyses. Significance was accepted at P < 0.05.
Cell viability assays
R–IR-A cells were serum-starved for 5 h at 37 C/5% CO2 before being treated with ligands in the presence of 0.1% BSA/5 mM sodium butyrate. After 48 h, ATP levels were measured as an indication of cell viability with a Cell-Titer Glo Cell Viability Luminescent Assay according to the instructions of the manufacturer (Promega, Madison, WI).
Migration assays
Migration assays were conducted as essentially as described (26) using a 96-well modified Boyden chamber (Neuro Probe, Inc., Gaithersburg, MD) and a 12-μm polycarbonate filter coated with type 1 collagen. Cells (200,000/well) were prelabeled with 2.2 μg/ml calcein (Molecular Probes, Eugene, OR) and migrated toward IGFs, IGF chimeras, or insulin for 5 h.
Results
Time course of IR-A tyrosine 960 phosphorylation
To examine in detail the activation of IR-A by insulin, IGF-II, and IGF-I, the time course of Y960 phosphorylation was examined. In this experiment, the effect of exchanging the C domains between IGF-I and IGF-II on the kinetics of receptor phosphorylation was investigated. As shown in Figure 1, insulin induced a 27-fold increase in Y960 phosphorylation after 5 min in 3T3-IR-A cells, which was maintained for 60 min. IGF-II increased Y960 phosphorylation up to 8-fold after 5 min, which slowly decreased to 4-fold over basal by 60 min. IGF-I was extremely poor at inducing Y960 phosphorylation at each time tested. Interestingly, over the entire time course, IGF-I CII stimulated Y960 phosphorylation to the same extent as IGF-II, with a maximum induction of 9-fold and a slow decline to 6-fold by 60 min. IGF-II CI was equally as poor as IGF-I at inducing phosphorylation of Y960. These results suggested that IGF-II was substantially more potent than IGF-I at inducing IR-A Y960 phosphorylation and that this was due to its C domain. Because maximal phosphorylation was observed 5 min after stimulation with all ligands, this time point was used for all subsequent experiments. Although the relative levels of Y960 phosphorylation induced by insulin, IGF-II, and IGF-I were as expected for the IR-A, the presence of endogenous mouse IGF-IR in the 3T3-IR-A cells could influence signaling. For this reason, R– cells expressing IR-A or IR-B (R–IR-A and R–IR-B) cells were used for all subsequent experiments. R– cells express approximately 5 x 103 endogenous IR per cell (6), and these are the IR-B isoform (data not shown). However, insulin, IGF-I, and IGF-II did not stimulate proliferation in R– cells alone (data not shown) (6). Both R–IR-A and R–IR-B cells were sorted by FACS analysis to generate cell lines that express equivalent levels of receptor (75,000 receptors/cell) (data not shown); this is an order of magnitude lower level than those used in previous studies (4, 21) and reflects a more physiological cell surface receptor density (27).
Induction of IR-A and IR-B tyrosine autophosphorylation
Because phosphorylation of tyrosine residues Y1158, Y1162, and Y1163 in the activation loop is the first detectable event after ligand binding to the IR, we examined the ability of insulin, IGF-II, IGF-I, and IGF chimeras to induce phosphorylation of these residues in the IR-A (Y1170, Y1174, and Y1175 in the IR-B; abbreviated as 3Y) (Fig. 2, A and C). Insulin caused a 22-fold increase in 3Y phosphorylation, whereas IGF-II was 4-fold less potent, and IGF-I only slightly activated 3Y phosphorylation over basal levels (IGF-II vs. IGF-I, P < 0.01). IGF-I chimeras containing the C and D domains of IGF-II (IGF-I CIIDII), or only the C domain (IGF-I CII), were equally as potent as IGF-II at stimulating 3Y phosphorylation in R–IR-A cells (IGF-II vs. IGF-I CII, P > 0.5). IGF-I DII was slightly better than IGF-I (P < 0.05). However, when measuring activation of other signaling molecules in subsequent analyses, IGF-I DII was equipotent to IGF-I (see below). IGF-II CIDI and IGF-II CI were not statistically significantly different from IGF-I in their ability to stimulate 3Y phosphorylation (IGF-I vs. IGF-II CI, P > 0.5), and IGF-II DI was slightly poorer than IGF-II at stimulating 3Y phosphorylation (P < 0.05).
Stimulation of IR-B by insulin caused a 45-fold increase in 3Y phosphorylation, greater than the fold-over-basal stimulation of IR-A (Fig. 2C). This result is supported by previous observations that insulin-activated IR-B had greater autophosphorylation activity compared with IR-A (28). IGF-II stimulated 3Y phosphorylation 8-fold lower relative to insulin and was able to induce only 1.9-fold higher 3Y phosphorylation relative to IGF-I. Exchanging the C or D domains, or both, between IGF-I and IGF-II had the same relative effect on 3Y phosphorylation on the IR-B as on the IR-A.
These results suggest that both insulin and IGF-II are potent activators of IR-A 3Y phosphorylation, whereas IGF-I is extremely poor, and that the potency of IGF-II in activating the IR-A is due to its C domain.
After activation of the IR kinase domain per se, phosphorylation of tyrosine 960 in the juxtamembrane domain of IR-A (Y972 in the IR-B) provides a docking site for binding of several adaptor molecules. We examined the ability of insulin, IGF-II, IGF-I, and IGF chimeras to induce phosphorylation of this site in both IR-A and IR-B (Fig. 2, B and D). IR-A Y960 phosphorylation was stimulated 10-fold over basal by insulin and 4-fold by IGF-II, whereas IGF-I stimulation of Y960 phosphorylation was only 1.6-fold over basal (IGF-II vs. IGF-I, P < 0.05). IGF-I CIIDII and IGF-I CII both stimulated Y960 phosphorylation of the IR-A to the same extent as IGF-II (IGF-II vs. IGF-I CII, P > 0.5), whereas IGF-II CIDI and IGF-II CI were both as poor as IGF-I (IGF-I vs. IGF-II CI, P > 0.5). Swapping the D domains alone had only a modest effect on the relative activation of Y960.
Insulin induced a 16-fold increase in IR-B Y972 phosphorylation over basal (Fig. 2D). This was slightly higher than that seen with IR-A (Fig. 2B). IGF-II only induced a 3-fold increase in IR-B Y972 phosphorylation, whereas IGF-I did not increase Y972 phosphorylation over basal by a statistically significant margin. IGF-I CII-stimulated IR-B Y972 phosphorylation 3.6-fold, whereas IGF-II CI did not activate this site.
Phosphorylation of IRS-1 in R–IR-A and R–IR-B cells
We next examined the ability of insulin, IGF-II, IGF-I, IGF-I CII, and IGF-II CI to stimulate IRS-1 phosphorylation in R–IR-A and R–IR-B cells (Fig. 3, A and B). Five minutes after stimulation of IR-A with insulin, IRS-1 was strongly phosphorylated. IGF-II stimulated a 4-fold lower level of IRS-1 phosphorylation relative to insulin, whereas IGF-I induced 3.5-fold less IRS-1 phosphorylation compared with IGF-II (IGF-II vs. IGF-I, P < 0.05). IGF-I CII stimulated IRS-1 phosphorylation to the same level as IGF-II (IGF-II vs. IGF-I CII, P > 0.5), whereas IGF-II CI was as ineffective as IGF-I at inducing IRS-1 phosphorylation in R–IR-A cells (IGF-I vs. IGF-II CI, P > 0.5). Interestingly, in R–IR-B cells, the ability of IGF-II to activate IRS-1 phosphorylation relative to insulin was similar to that in R–IR-A cells (Fig. 3B). Surprisingly, IGF-I was more potent at inducing IRS-1 phosphorylation in R–IR-B cells than in R–IR-A cells. The difference between IGF-II and IGF-I-induced phosphorylation of IRS-1 was only 1.4-fold in R–IR-B cells. This trend is in line with their relative binding affinities, where the difference in binding affinity between IGF-II and IGF-I for IR-B is smaller than the difference in their affinity for IR-A (22). IGF-I CII had a slightly increased ability relative to IGF-II to activate IRS-1 tyrosine phosphorylation in R–IR-B cells, whereas IGF-II CI induced IRS-1 phosphorylation to the same extent as IGF-I (Fig. 3B).
Activation of Akt/Protein Kinase B in R–IR-A and R–IR-B cells
As shown in Fig. 4A, insulin strongly activated Akt/protein kinase B (PKB) via the IR-A, whereas IGF-II and IGF-I were only 30 and 11% as potent as insulin. Interestingly, with R–IR-A cells, all chimeras containing the IGF-II C domain (IGF-I CIIDII, IGF-I CII, and IGF-II DI) stimulated Akt/PKB phosphorylation to the same level as IGF-II, whereas all chimeras containing the IGF-I C domain (IGF-II CIDI, IGF-II CI, and IGF-I DII) were as poor as IGF-I at stimulating Akt/PKB phosphorylation in R–IR-A cells. These results highlight the importance of the IGF-I and IGF-II C domains in determining the downstream signaling properties of the IGFs.
In R–IR-B cells, insulin also stimulated Akt/PKB phosphorylation, and IGF-II and IGF-I were 30 and 15% as potent as insulin, respectively (Fig. 4B). The IGF-I CIIDII and IGF-I CII chimeras were both more effective at inducing Akt/PKB phosphorylation than IGF-II, whereas IGF-I DII was equally as potent as IGF-II, unlike the case in R–IR-A cells. IGF-II CIDI and IGF-II CI stimulated Akt/PKB phosphorylation to the same level as IGF-I in R–IR-B cells.
Activation of Erk1/2 in R–IR-A and R–IR-B cells
Insulin was the only ligand that significantly increased Erk1/2 phosphorylation over basal in R–IR-A cells (Supplemental Fig. 1, published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). No ligand induced Erk1/2 phosphorylation in R–IR-B cells (data not shown).
R–IR-A cell viability and migration
Interestingly, despite the decreased ability of IGF-II to stimulate downstream signaling relative to insulin, IGF-II was as potent as insulin at stimulating R–IR-A cell survival (P > 0.5) (Fig. 5). As expected from receptor binding and activation experiments, IGF-II was more potent than IGF-I in stimulating cell survival in IR-A cells (P < 0.001). IGF-II CI was as potent as IGF-I in promoting R–IR-A cell survival (P > 0.5). Likewise, IGF-I CII was as potent as IGF-II in stimulating R–IR-A cell survival. Similarly, exchange of the C domains demonstrated that this domain alone was sufficient to account for the differential ability of the IGFs to stimulate chemotaxis via IR-A (Fig. 6). There was no significant difference between IGF-I and IGF-II CI or between IGF-II and IGF-I CII. These results suggest that the C domains of the IGFs account both for the differential recruitment and stimulation of downstream signaling molecules by IGF-I and IGF-II via the IR-A and their differential ability to promote cell survival and migration through the IR-A.
Discussion
This study reveals that the IGF C domain determines the difference in signaling potential of IGF-II and IGF-I via the IR-A and IR-B. In contrast to other receptor tyrosine kinases, the IR and the IGF-IR rely on the phosphorylation of docking proteins rather than autophosphorylation alone to enable the recruitment and activation of downstream signaling molecules (29, 30). One such docking protein, IRS-1, is the major adaptor phosphorylated by the IR, and phosphorylated tyrosines on IRS-1 can then provide binding sites for a number of SH2 domain-containing proteins, including Grb-2 (31), which links the IR to ras (32), and the p85 regulatory subunit of phosphatidylinositol 3-kinase (33), which links the IR to Akt/PKB (34). For that reason, we compared the ability of insulin, IGF-II, IGF-I, and IGF chimeras to induce IRS-1 phosphorylation. Our results suggest that the C domain not only mediates the differential ability of IGF-II and IGF-I to bind and induce autophosphorylation of specific tyrosines in the cytoplasmic domain of the IR-A but also accounts for the characteristic recruitment and stimulation of IRS-1 by the IR-A of each IGF.
We have shown that IGF-II, due to its C domain, can potently regulate the phosphorylation of Akt/PKB via both IR-A and IR-B. Interestingly, in both R–IR-A and R–IR-B cells, Erk1/2 phosphorylation was not increased over basal by any ligand other than insulin, which itself only induced a small 3-fold increase and only in R–IR-A cells. Previous studies of IGF activation of Erk via the IR-A have shown that IGF-II, but not IGF-I, can induce Erk1/2 activation above basal levels (4, 21). In those studies, the IR-A was overexpressed to an approximate level of 500,000 receptors per cell (4, 21), whereas the cells used here and in our previous studies (22) express only 75,000 receptors per cell. This could account for the differences in the ability of IGF-II to stimulate Erk1/2 phosphorylation via the IR-A in previous work and our studies. Interestingly, in R–IR-B cells, Erk1/2 was still not activated, even though IGF-II can induce both IR and IRS-1 phosphorylation in these cells.
We also assessed the ability of IGFs to influence two processes important to cancer progression, i.e. survival from apoptosis and cellular migration. We show here for the first time that IGF signaling through IR-A can protect cells from butyrate-induced apoptosis. Butyrate is a potent proapoptotic compound whose mechanism of action is not definitively determined, but which may reflect its ability to inhibit histone deacetylase activity (35). Previous studies have shown that IGF-II protected SKUT-1 cells (which express no IGF-IR and >95% IR-A) from staurosporine-induced apoptosis (5). This study, along with our results, suggests that IGF-II signaling through the IR-A can protect cells from apoptotic agents with different mechanisms of action. IGF-II was able to inhibit butyrate-induced apoptosis in R–IR-A cells to a significantly greater extent than IGF-I. This trend was also observed with migration. In both cell survival and migration, the exchange of the IGF C domains accounted for the differential ability of the IGFs to stimulate biological responses via the IR-A. Interestingly, despite IGF-II having a lower affinity for the IR-A relative to insulin and being substantially poorer at inducing phosphorylation of IRS-1 and Akt/PKB relative to insulin, IGF-II was as potent as insulin at protecting R–IR-A cells from butyrate-induced apoptosis. Both insulin and IGF-II were equipotent at protecting SKUT-1 cells from staurosporine-induced apoptosis (5). This highlights the importance of delineating exactly what signaling pathways are activated by either IGF-II or insulin via the IR-A. The ability of the IGF-II to mediate cell survival and migration via the IR-A suggests a possible mechanism whereby cells can remain viable in the presence of IGF-IR inhibitors. Previous studies have suggested that insulin-stimulated cell migration is due to activation of IGF-IR signaling (21). Here, we show that insulin can induce cell migration exclusively through the IR-A.
Our results provide novel insights into the mechanism of IGF action via the IR and, more generally, the distinction between ligand binding and receptor signaling. In our previous binding studies, swapping both the C and D domains was a requirement for complete exchange of the binding specificity for the IR-A, as well as the IR-B. However, here we show that substitution of the IGF-I C domain alone with that of IGF-II is sufficient to allow the chimera to activate the IR, IRS-1, and Akt/PKB to the same extent as IGF-II. It is possible that the C domain of IGF-II, although not accounting for the entire difference in the free energy of IR-A binding by IGF-II compared with IGF-I, can, nevertheless, induce a conformational change in the receptor similar to that induced by authentic IGF-II.
Inhibition of IGF-IR signaling is being investigated as a potential target for cancer therapy (36). Small-molecule inhibitors of the IGF-IR that do not inhibit the IR kinase have been shown in mouse models to be effective at reducing tumor formation and growth (37, 38). The presence of the IR-A on tumor cells may reduce the efficacy of IGF-IR-directed therapies by providing an avenue for cell survival and proliferation, especially because many tumor cells overexpress IGF-II (39, 40). Overcoming this potential problem by inhibiting the IR is also not advisable, because reducing IR signaling may affect glucose metabolism. Specifically inhibiting IGF-II action is an attractive strategy (41, 42) that would prevent its action via both the IGF-IR and IR-A but allow insulin to signal unaffected through the IR. Supporting the validity of this approach is the finding that a phage-displayed peptide, isolated by screening for binding to IGF-I, inhibits IGF-I binding to both the IGF-IR and IR, although the effect on insulin signaling via the IR in the presence of the peptide was not reported (43). In this report, we show that the IGF-II C domain alone, for which there is no analogous region in insulin, is critical for signaling, cell survival, and migration induced by IGF-II via the IR-A. The IGF-II C domain, therefore, provides a potential site for design of specific inhibitors of IGF-II, and possibly IGF-I, binding to the IR and IGF-IR. Thus, our results provide novel insights into the biological response of IGF ligand-receptor interactions and have ramifications for the design of therapeutic IGF-IR inhibitors.
Footnotes
This work was supported in part by a grant from the McCoy Foundation (to C.T.R.) and by a fellowship from the Novartis Foundation (to A.D.)
Present address for A.D.: Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
First Published Online October 20, 2005
Abbreviations: IR, Insulin receptor; IRS, insulin receptor substrate; PKB, protein kinase B; SDS, sodium dodecyl sulfate.
Accepted for publication October 13, 2005.
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Commonwealth Scientific and Industrial Research Organisation Division of Molecular and Health Technologies (A.D., G.V.B., L.J.C.), Adelaide 5000, Australia
Department of Pediatrics (J.M.C., A.L., C.T.R.), Oregon Health and Science University, Portland, Oregon 97239
Abstract
The actions of IGF-I and IGF-II are thought to be largely due to their activation of the IGF-I receptor. However, IGF-II can also bind with high affinity to, and effectively activate, an isoform of the insulin receptor (IR-A) that lacks a sequence at the carboxyl-terminal end of the extracellular subunit due to the alternative splicing of exon 11. This isoform is poorly activated by IGF-I. Here, we show that IGF-II, but not IGF-I, induces potent autophosphorylation of residues Y1158, Y1162, and Y1163 in the activation loop of the kinase domain and tyrosine 960 in the juxtamembrane region of both IR-A and IR-B (exon 11+) isoforms. We have also found, by using IGF chimeras, that the C domain of IGF-II completely accounts for the ability of IGF-II to stimulate IR autophosphorylation compared with IGF-I. We further show that the C domains are responsible for the differential abilities of IGF-II and IGF-I to activate phosphorylation of insulin receptor substrate-1 and Akt, as well as their ability to induce migration and cell survival via the IR-A. Finally, we show for the first time that IGF signaling through the IR-A can protect cells from butyrate-induced apoptosis. In summary, our studies define the structural determinants that allow potent IGF-II signaling and regulation of cellular functions through the IR-A and provide novel insights into IGF signaling via the IR.
Introduction
MOUSE KNOCKOUT STUDIES have revealed that the insulin receptor (IR) mediates the growth-promoting effects of IGF-II in early development (1, 2, 3). The alternatively spliced form of the IR that lacks residues encoded by exon 11 (IR-A) binds insulin and IGF-II with high affinity, but binds IGF-I poorly. Insulin and IGF-II, but not IGF-I, can potently induce IR-A autophosphorylation (4). Numerous studies have shown that IGF-II, but not IGF-I, can signal via the IR-A and induce cancer cell proliferation and migration (5, 6, 7, 8). An increase in the relative expression of the IR-A has been shown in several cancers, including those of the lung, colon (4), breast (4, 7), ovaries (9), thyroid (8), and smooth and striated muscle (5). Together, these studies suggest that the binding and activation of the IR-A by IGF-II is significant in development and can possibly contribute to certain pathologies (10).
Autophosphorylation of the IR occurs initially on tyrosines 1162, 1158, and 1163 in the activation loop of the kinase domain, allowing unobstructed access of ATP and substrate to the kinase active site (11, 12, 13, 14). The activated catalytic core then phosphorylates additional tyrosine residues such as 960, which provides a docking site for the adaptor proteins insulin receptor substrate-1 (IRS)-1 (15, 16), IRS-2 (17), and Shc (18) (reviewed in Refs.19 and 20). Activation of IRS-1, Shc, Erk1/2, and Akt by IGF-II, but not IGF-I, has been shown in both transfected (4, 21) and tumor cell lines (SKUT-1) (5) devoid of the IGF-I receptor (IGF-IR) and expressing >95% IR-A. To date, the structural determinants of IGF-II that allow the activation of IR-A signaling pathways are not known.
Mature IGF-I and IGF-II are comprised of four domains (B, C, A, and D) and share extensive sequence and structural similarity. We have previously demonstrated by domain swapping that the IGF-II C and D domains conferred high-affinity binding to the IR-A and IR-B, whereas the C and D domains of IGF-I did not (22). These studies, however, did not define the particular domain primarily responsible for determining the differential abilities of the IGFs to activate the IR or induce downstream signaling pathways that mediate the biological effects of the IGFs.
Here, we report for the first time that IGF-II and IGF-I differentially activate specific tyrosine residues on IR-A and IR-B. In addition, by using IGF chimeras (22), we show that the sole structural determinant for the differential ability of IGF-I and IGF-II to induce autophosphorylation of specific IR tyrosine residues and activate downstream signaling molecules is the C domain. We also demonstrate that substitution of the C domain of IGF-I with that of IGF-II results in a chimera whose ability to induce protection from butyrate-induced apoptosis and promote cell migration via the IR-A is similar to that of authentic IGF-II. Our studies may have significant ramifications for the design of cancer therapeutics currently directed against IGF-IR signaling.
Materials and Methods
Materials
Antibodies against the following proteins were obtained from the indicated sources: IRS-1, phospho-Erk1/2, Erk1/2, and Akt from Cell Signaling Technologies (Beverly, MA); phospho-Akt [pS473], phospho-IR/IGF-1R [pYpYpY1158/1162/1163], and phospho-IR [pY972)] from Biosource International (Camarillo, CA); and rat carboxyl-terminal IRS-1 from Upstate Biotechnology (Lake Placid, NY). Protease inhibitors, pepstatin, type 1 collagen, and sodium orthovanadate were from Sigma Chemical Co. (St. Louis, MO). Criterion 12% Tris-tricine gels were from Bio-Rad (Hercules, CA). Human insulin was from Novo Nordisk (Bagsvrd, Denmark). R– cells (mouse 3T3-like cells with a targeted ablation of the IGF-IR gene) (23) were a kind gift from Prof. R. Baserga (Thomas Jefferson University, Philadelphia, PA). The construction, expression, and purification of chimeras of IGF-I and IGF-II and R– cells expressing the human IR-A (R–IR-A) and IR-B (R–IR-B) cDNAs have been previously reported (22). 3T3-IR-A cells have been described previously (24).
IR-A and IR-B phosphorylation and activation of intracellular signaling molecules
3T3-IR-A cells were treated with 10 nM ligand for 2, 5, 10, and 60 min in time-course experiments, and R–IR-A or R–IR-B cells were treated with 10 nM ligand for 5 min. For whole-cell lysates, cells were lysed in 1x sodium dodecyl sulfate (SDS) sample buffer (25) without dithiothreitol or bromophenol blue and boiled immediately to inhibit protease and phosphatase action. Protein concentration was determined with a detergent-compatible protein assay kit (Bio-Rad). dithiothreitol was then added to 100 mM and bromophenol blue to 0.1%. For immunoprecipitations, cells were lysed in 50 mM Tris (pH 6.8), 1% (wt/vol) SDS, and 10% (vol/vol) glycerol or 150 mM NaCl, 10% (vol/vol) glycerol, 20 mM Tris (pH 8), 1 mM EDTA, 0.2% SDS, 1 tablet of complete protease inhibitors per 10 ml, 1 mM sodium orthovanadate, and 1 μg/ml pepstatin. Lysates were incubated on ice for 30 min, inverting occasionally. They were then microcentrifuged at 12,000 x g for 15 min and the supernatants transferred to new tubes. Protein concentration was determined as above. For immunoprecipitations, lysates (500 μg) were precleared with protein A-agarose beads for 30 min before incubation with anti-IRS-1 antibody overnight at 4 C. Protein A-agarose beads were added for 3 h at 4 C and immunoprecipitates were then washed in 3x lysis buffer, eluted in 2x SDS sample buffer, and subjected to SDS-PAGE. Immunoprecipitates or whole-cell lysates (20 μg) were subjected to reducing SDS-PAGE on Criterion gels. Blots were probed with relevant phospho-antibodies, or, for IRS-1 immunoprecipitates, blots were probed with the antiphosphotyrosine antibody PY20. In all cases, after probing with the phospho-specific antibody, the blot was stripped in 100 mM Tris-HCl (pH 6.8), 10% SDS, and 100 mM -mercaptoethanol for 30 min at 60 C and reprobed with a pan-specific antibody. Paired Student’s t tests were used for all statistical analyses. Significance was accepted at P < 0.05.
Cell viability assays
R–IR-A cells were serum-starved for 5 h at 37 C/5% CO2 before being treated with ligands in the presence of 0.1% BSA/5 mM sodium butyrate. After 48 h, ATP levels were measured as an indication of cell viability with a Cell-Titer Glo Cell Viability Luminescent Assay according to the instructions of the manufacturer (Promega, Madison, WI).
Migration assays
Migration assays were conducted as essentially as described (26) using a 96-well modified Boyden chamber (Neuro Probe, Inc., Gaithersburg, MD) and a 12-μm polycarbonate filter coated with type 1 collagen. Cells (200,000/well) were prelabeled with 2.2 μg/ml calcein (Molecular Probes, Eugene, OR) and migrated toward IGFs, IGF chimeras, or insulin for 5 h.
Results
Time course of IR-A tyrosine 960 phosphorylation
To examine in detail the activation of IR-A by insulin, IGF-II, and IGF-I, the time course of Y960 phosphorylation was examined. In this experiment, the effect of exchanging the C domains between IGF-I and IGF-II on the kinetics of receptor phosphorylation was investigated. As shown in Figure 1, insulin induced a 27-fold increase in Y960 phosphorylation after 5 min in 3T3-IR-A cells, which was maintained for 60 min. IGF-II increased Y960 phosphorylation up to 8-fold after 5 min, which slowly decreased to 4-fold over basal by 60 min. IGF-I was extremely poor at inducing Y960 phosphorylation at each time tested. Interestingly, over the entire time course, IGF-I CII stimulated Y960 phosphorylation to the same extent as IGF-II, with a maximum induction of 9-fold and a slow decline to 6-fold by 60 min. IGF-II CI was equally as poor as IGF-I at inducing phosphorylation of Y960. These results suggested that IGF-II was substantially more potent than IGF-I at inducing IR-A Y960 phosphorylation and that this was due to its C domain. Because maximal phosphorylation was observed 5 min after stimulation with all ligands, this time point was used for all subsequent experiments. Although the relative levels of Y960 phosphorylation induced by insulin, IGF-II, and IGF-I were as expected for the IR-A, the presence of endogenous mouse IGF-IR in the 3T3-IR-A cells could influence signaling. For this reason, R– cells expressing IR-A or IR-B (R–IR-A and R–IR-B) cells were used for all subsequent experiments. R– cells express approximately 5 x 103 endogenous IR per cell (6), and these are the IR-B isoform (data not shown). However, insulin, IGF-I, and IGF-II did not stimulate proliferation in R– cells alone (data not shown) (6). Both R–IR-A and R–IR-B cells were sorted by FACS analysis to generate cell lines that express equivalent levels of receptor (75,000 receptors/cell) (data not shown); this is an order of magnitude lower level than those used in previous studies (4, 21) and reflects a more physiological cell surface receptor density (27).
Induction of IR-A and IR-B tyrosine autophosphorylation
Because phosphorylation of tyrosine residues Y1158, Y1162, and Y1163 in the activation loop is the first detectable event after ligand binding to the IR, we examined the ability of insulin, IGF-II, IGF-I, and IGF chimeras to induce phosphorylation of these residues in the IR-A (Y1170, Y1174, and Y1175 in the IR-B; abbreviated as 3Y) (Fig. 2, A and C). Insulin caused a 22-fold increase in 3Y phosphorylation, whereas IGF-II was 4-fold less potent, and IGF-I only slightly activated 3Y phosphorylation over basal levels (IGF-II vs. IGF-I, P < 0.01). IGF-I chimeras containing the C and D domains of IGF-II (IGF-I CIIDII), or only the C domain (IGF-I CII), were equally as potent as IGF-II at stimulating 3Y phosphorylation in R–IR-A cells (IGF-II vs. IGF-I CII, P > 0.5). IGF-I DII was slightly better than IGF-I (P < 0.05). However, when measuring activation of other signaling molecules in subsequent analyses, IGF-I DII was equipotent to IGF-I (see below). IGF-II CIDI and IGF-II CI were not statistically significantly different from IGF-I in their ability to stimulate 3Y phosphorylation (IGF-I vs. IGF-II CI, P > 0.5), and IGF-II DI was slightly poorer than IGF-II at stimulating 3Y phosphorylation (P < 0.05).
Stimulation of IR-B by insulin caused a 45-fold increase in 3Y phosphorylation, greater than the fold-over-basal stimulation of IR-A (Fig. 2C). This result is supported by previous observations that insulin-activated IR-B had greater autophosphorylation activity compared with IR-A (28). IGF-II stimulated 3Y phosphorylation 8-fold lower relative to insulin and was able to induce only 1.9-fold higher 3Y phosphorylation relative to IGF-I. Exchanging the C or D domains, or both, between IGF-I and IGF-II had the same relative effect on 3Y phosphorylation on the IR-B as on the IR-A.
These results suggest that both insulin and IGF-II are potent activators of IR-A 3Y phosphorylation, whereas IGF-I is extremely poor, and that the potency of IGF-II in activating the IR-A is due to its C domain.
After activation of the IR kinase domain per se, phosphorylation of tyrosine 960 in the juxtamembrane domain of IR-A (Y972 in the IR-B) provides a docking site for binding of several adaptor molecules. We examined the ability of insulin, IGF-II, IGF-I, and IGF chimeras to induce phosphorylation of this site in both IR-A and IR-B (Fig. 2, B and D). IR-A Y960 phosphorylation was stimulated 10-fold over basal by insulin and 4-fold by IGF-II, whereas IGF-I stimulation of Y960 phosphorylation was only 1.6-fold over basal (IGF-II vs. IGF-I, P < 0.05). IGF-I CIIDII and IGF-I CII both stimulated Y960 phosphorylation of the IR-A to the same extent as IGF-II (IGF-II vs. IGF-I CII, P > 0.5), whereas IGF-II CIDI and IGF-II CI were both as poor as IGF-I (IGF-I vs. IGF-II CI, P > 0.5). Swapping the D domains alone had only a modest effect on the relative activation of Y960.
Insulin induced a 16-fold increase in IR-B Y972 phosphorylation over basal (Fig. 2D). This was slightly higher than that seen with IR-A (Fig. 2B). IGF-II only induced a 3-fold increase in IR-B Y972 phosphorylation, whereas IGF-I did not increase Y972 phosphorylation over basal by a statistically significant margin. IGF-I CII-stimulated IR-B Y972 phosphorylation 3.6-fold, whereas IGF-II CI did not activate this site.
Phosphorylation of IRS-1 in R–IR-A and R–IR-B cells
We next examined the ability of insulin, IGF-II, IGF-I, IGF-I CII, and IGF-II CI to stimulate IRS-1 phosphorylation in R–IR-A and R–IR-B cells (Fig. 3, A and B). Five minutes after stimulation of IR-A with insulin, IRS-1 was strongly phosphorylated. IGF-II stimulated a 4-fold lower level of IRS-1 phosphorylation relative to insulin, whereas IGF-I induced 3.5-fold less IRS-1 phosphorylation compared with IGF-II (IGF-II vs. IGF-I, P < 0.05). IGF-I CII stimulated IRS-1 phosphorylation to the same level as IGF-II (IGF-II vs. IGF-I CII, P > 0.5), whereas IGF-II CI was as ineffective as IGF-I at inducing IRS-1 phosphorylation in R–IR-A cells (IGF-I vs. IGF-II CI, P > 0.5). Interestingly, in R–IR-B cells, the ability of IGF-II to activate IRS-1 phosphorylation relative to insulin was similar to that in R–IR-A cells (Fig. 3B). Surprisingly, IGF-I was more potent at inducing IRS-1 phosphorylation in R–IR-B cells than in R–IR-A cells. The difference between IGF-II and IGF-I-induced phosphorylation of IRS-1 was only 1.4-fold in R–IR-B cells. This trend is in line with their relative binding affinities, where the difference in binding affinity between IGF-II and IGF-I for IR-B is smaller than the difference in their affinity for IR-A (22). IGF-I CII had a slightly increased ability relative to IGF-II to activate IRS-1 tyrosine phosphorylation in R–IR-B cells, whereas IGF-II CI induced IRS-1 phosphorylation to the same extent as IGF-I (Fig. 3B).
Activation of Akt/Protein Kinase B in R–IR-A and R–IR-B cells
As shown in Fig. 4A, insulin strongly activated Akt/protein kinase B (PKB) via the IR-A, whereas IGF-II and IGF-I were only 30 and 11% as potent as insulin. Interestingly, with R–IR-A cells, all chimeras containing the IGF-II C domain (IGF-I CIIDII, IGF-I CII, and IGF-II DI) stimulated Akt/PKB phosphorylation to the same level as IGF-II, whereas all chimeras containing the IGF-I C domain (IGF-II CIDI, IGF-II CI, and IGF-I DII) were as poor as IGF-I at stimulating Akt/PKB phosphorylation in R–IR-A cells. These results highlight the importance of the IGF-I and IGF-II C domains in determining the downstream signaling properties of the IGFs.
In R–IR-B cells, insulin also stimulated Akt/PKB phosphorylation, and IGF-II and IGF-I were 30 and 15% as potent as insulin, respectively (Fig. 4B). The IGF-I CIIDII and IGF-I CII chimeras were both more effective at inducing Akt/PKB phosphorylation than IGF-II, whereas IGF-I DII was equally as potent as IGF-II, unlike the case in R–IR-A cells. IGF-II CIDI and IGF-II CI stimulated Akt/PKB phosphorylation to the same level as IGF-I in R–IR-B cells.
Activation of Erk1/2 in R–IR-A and R–IR-B cells
Insulin was the only ligand that significantly increased Erk1/2 phosphorylation over basal in R–IR-A cells (Supplemental Fig. 1, published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). No ligand induced Erk1/2 phosphorylation in R–IR-B cells (data not shown).
R–IR-A cell viability and migration
Interestingly, despite the decreased ability of IGF-II to stimulate downstream signaling relative to insulin, IGF-II was as potent as insulin at stimulating R–IR-A cell survival (P > 0.5) (Fig. 5). As expected from receptor binding and activation experiments, IGF-II was more potent than IGF-I in stimulating cell survival in IR-A cells (P < 0.001). IGF-II CI was as potent as IGF-I in promoting R–IR-A cell survival (P > 0.5). Likewise, IGF-I CII was as potent as IGF-II in stimulating R–IR-A cell survival. Similarly, exchange of the C domains demonstrated that this domain alone was sufficient to account for the differential ability of the IGFs to stimulate chemotaxis via IR-A (Fig. 6). There was no significant difference between IGF-I and IGF-II CI or between IGF-II and IGF-I CII. These results suggest that the C domains of the IGFs account both for the differential recruitment and stimulation of downstream signaling molecules by IGF-I and IGF-II via the IR-A and their differential ability to promote cell survival and migration through the IR-A.
Discussion
This study reveals that the IGF C domain determines the difference in signaling potential of IGF-II and IGF-I via the IR-A and IR-B. In contrast to other receptor tyrosine kinases, the IR and the IGF-IR rely on the phosphorylation of docking proteins rather than autophosphorylation alone to enable the recruitment and activation of downstream signaling molecules (29, 30). One such docking protein, IRS-1, is the major adaptor phosphorylated by the IR, and phosphorylated tyrosines on IRS-1 can then provide binding sites for a number of SH2 domain-containing proteins, including Grb-2 (31), which links the IR to ras (32), and the p85 regulatory subunit of phosphatidylinositol 3-kinase (33), which links the IR to Akt/PKB (34). For that reason, we compared the ability of insulin, IGF-II, IGF-I, and IGF chimeras to induce IRS-1 phosphorylation. Our results suggest that the C domain not only mediates the differential ability of IGF-II and IGF-I to bind and induce autophosphorylation of specific tyrosines in the cytoplasmic domain of the IR-A but also accounts for the characteristic recruitment and stimulation of IRS-1 by the IR-A of each IGF.
We have shown that IGF-II, due to its C domain, can potently regulate the phosphorylation of Akt/PKB via both IR-A and IR-B. Interestingly, in both R–IR-A and R–IR-B cells, Erk1/2 phosphorylation was not increased over basal by any ligand other than insulin, which itself only induced a small 3-fold increase and only in R–IR-A cells. Previous studies of IGF activation of Erk via the IR-A have shown that IGF-II, but not IGF-I, can induce Erk1/2 activation above basal levels (4, 21). In those studies, the IR-A was overexpressed to an approximate level of 500,000 receptors per cell (4, 21), whereas the cells used here and in our previous studies (22) express only 75,000 receptors per cell. This could account for the differences in the ability of IGF-II to stimulate Erk1/2 phosphorylation via the IR-A in previous work and our studies. Interestingly, in R–IR-B cells, Erk1/2 was still not activated, even though IGF-II can induce both IR and IRS-1 phosphorylation in these cells.
We also assessed the ability of IGFs to influence two processes important to cancer progression, i.e. survival from apoptosis and cellular migration. We show here for the first time that IGF signaling through IR-A can protect cells from butyrate-induced apoptosis. Butyrate is a potent proapoptotic compound whose mechanism of action is not definitively determined, but which may reflect its ability to inhibit histone deacetylase activity (35). Previous studies have shown that IGF-II protected SKUT-1 cells (which express no IGF-IR and >95% IR-A) from staurosporine-induced apoptosis (5). This study, along with our results, suggests that IGF-II signaling through the IR-A can protect cells from apoptotic agents with different mechanisms of action. IGF-II was able to inhibit butyrate-induced apoptosis in R–IR-A cells to a significantly greater extent than IGF-I. This trend was also observed with migration. In both cell survival and migration, the exchange of the IGF C domains accounted for the differential ability of the IGFs to stimulate biological responses via the IR-A. Interestingly, despite IGF-II having a lower affinity for the IR-A relative to insulin and being substantially poorer at inducing phosphorylation of IRS-1 and Akt/PKB relative to insulin, IGF-II was as potent as insulin at protecting R–IR-A cells from butyrate-induced apoptosis. Both insulin and IGF-II were equipotent at protecting SKUT-1 cells from staurosporine-induced apoptosis (5). This highlights the importance of delineating exactly what signaling pathways are activated by either IGF-II or insulin via the IR-A. The ability of the IGF-II to mediate cell survival and migration via the IR-A suggests a possible mechanism whereby cells can remain viable in the presence of IGF-IR inhibitors. Previous studies have suggested that insulin-stimulated cell migration is due to activation of IGF-IR signaling (21). Here, we show that insulin can induce cell migration exclusively through the IR-A.
Our results provide novel insights into the mechanism of IGF action via the IR and, more generally, the distinction between ligand binding and receptor signaling. In our previous binding studies, swapping both the C and D domains was a requirement for complete exchange of the binding specificity for the IR-A, as well as the IR-B. However, here we show that substitution of the IGF-I C domain alone with that of IGF-II is sufficient to allow the chimera to activate the IR, IRS-1, and Akt/PKB to the same extent as IGF-II. It is possible that the C domain of IGF-II, although not accounting for the entire difference in the free energy of IR-A binding by IGF-II compared with IGF-I, can, nevertheless, induce a conformational change in the receptor similar to that induced by authentic IGF-II.
Inhibition of IGF-IR signaling is being investigated as a potential target for cancer therapy (36). Small-molecule inhibitors of the IGF-IR that do not inhibit the IR kinase have been shown in mouse models to be effective at reducing tumor formation and growth (37, 38). The presence of the IR-A on tumor cells may reduce the efficacy of IGF-IR-directed therapies by providing an avenue for cell survival and proliferation, especially because many tumor cells overexpress IGF-II (39, 40). Overcoming this potential problem by inhibiting the IR is also not advisable, because reducing IR signaling may affect glucose metabolism. Specifically inhibiting IGF-II action is an attractive strategy (41, 42) that would prevent its action via both the IGF-IR and IR-A but allow insulin to signal unaffected through the IR. Supporting the validity of this approach is the finding that a phage-displayed peptide, isolated by screening for binding to IGF-I, inhibits IGF-I binding to both the IGF-IR and IR, although the effect on insulin signaling via the IR in the presence of the peptide was not reported (43). In this report, we show that the IGF-II C domain alone, for which there is no analogous region in insulin, is critical for signaling, cell survival, and migration induced by IGF-II via the IR-A. The IGF-II C domain, therefore, provides a potential site for design of specific inhibitors of IGF-II, and possibly IGF-I, binding to the IR and IGF-IR. Thus, our results provide novel insights into the biological response of IGF ligand-receptor interactions and have ramifications for the design of therapeutic IGF-IR inhibitors.
Footnotes
This work was supported in part by a grant from the McCoy Foundation (to C.T.R.) and by a fellowship from the Novartis Foundation (to A.D.)
Present address for A.D.: Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
First Published Online October 20, 2005
Abbreviations: IR, Insulin receptor; IRS, insulin receptor substrate; PKB, protein kinase B; SDS, sodium dodecyl sulfate.
Accepted for publication October 13, 2005.
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