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Abstract
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 42, Issue of October 18, pp. 39684 –39695, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Insulin/Insulin-like Growth Factor I Hybrid Receptors Have Different Biological Characteristics Depending on the Insulin Receptor Isoform Involved* Received for publication, March 22, 2002, and in revised form, July 20, 2002 Published, JBC Papers in Press, July 22, 2002, DOI 10.1074/jbc.M202766200 Giuseppe Pandini‡§ , Francesco Frasca‡ , Rossana Mineo‡ , Laura Sciacca‡, Riccardo Vigneri‡§, and Antonino Belfiore**‡‡ From the ‡Istituto di Medicina Interna, Malattie Endocrine e del Metabolismo, University of Catania, Ospedale Garibaldi, 95123 Catania, Italy, the §Istituto Mediterraneo di Oncologia, 95100 Catania, Italy, and the **Dipartimento di Medicina Clinica e Sperimentale, University of Catanzaro, Policlinico Mater Domini, via T. Campanella 115, 88100 Catanzaro, Italy posed of two extracellular - and two transmembrane -sub- The insulin receptor (IR) and the insulin-like growth factor I receptor (IGF-IR) have a highly homologous units linked by disulfide bonds. Each -subunit, containing the structure, but different biological effects. Insulin and ligand-binding site, is 130 kDa, whereas each -subunit, con- IGF-I half-receptors can heterodimerize, leading to the taining the tyrosine kinase domain, is 95–97 kDa. These formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) receptors share 50% overall amino acid sequence homology that bind IGF-I with high affinity. As the IR exists in two and 84% homology in the tyrosine kinase domains. After ligand isoforms (IR-A and IR-B), we evaluated whether the as- binding, activated receptors recruit and phosphorylate docking sembly of the IGF-IR with either IR-A or IR-B moieties proteins, including the insulin receptor substrate-1 family pro- may differently affect Hybrid-R signaling and biological teins Gab1 and Shc (1–5), leading to the activation of many role. Three different models were studied: (a) 3T3-like intracellular mediators, including phosphatidylinositol 3-ki- mouse fibroblasts with a disrupted IGF-IR gene (R nase, Akt, and ERK1/2, involved in the regulation of cell me- cells) cotransfected with the human IGF-IR and with tabolism, proliferation, and survival. Although both the IR and either the IR-A or IR-B cDNA; (b) a panel of human cell IGF-IR similarly activate major signaling pathways, subtle lines variably expressing the two IR isoforms; and (c) differences exist in the recruitment of certain docking proteins HepG2 human hepatoblastoma cells predominantly ex- and intracellular mediators between the two receptors (6 –9). pressing either IR-A or IR-B, depending on their differ- These differences are the basis for the predominant metabolic entiation state. We found that Hybrid-Rs containing effect elicited by IR activation and the predominant mitogenic, IR-A (Hybrid-Rs ) bound to and were activated by IGF-I, IGF-II, and insulin. By binding to Hybrid-Rs , insulin transforming, and anti-apoptotic effect elicited by IGF-IR acti- activated the IGF-I half-receptor -subunit and the IGF- vation (10 –13). According to the classical view, insulin binds IR-specific substrate CrkII. In contrast, Hybrid-Rs with high affinity to the IR (100-fold higher than to the IGF- bound to and were activated with high affinity by IGF-I, IR), whereas both insulin-like growth factors (IGF-I and IGF- with low affinity by IGF-II, and insignificantly by insu- II) bind to the IGF-IR (with 100-fold higher affinity than to the lin. As a consequence, cell proliferation and migration IR). in response to both insulin and IGFs were more effec- Given the high degree of homology, the insulin and IGF-I tively stimulated in Hybrid-R -containing cells than in half-receptors (composed of one - and one -subunit) can het- Hybrid-R -containing cells. The relative abundance of erodimerize, leading to the formation of insulin/IGF-I hybrid IR isoforms therefore affects IGF system activation receptors (Hybrid-Rs) (14 –16). In many tissues, Hybrid-Rs are through Hybrid-Rs, with important consequences for the most represented receptor subtype (17). Hybrid-Rs may tissue-specific responses to both insulin and IGFs. also be overexpressed in a variety of human malignancies as a result of both IR and IGF-IR overexpression (18 –21). However, 1 the biological role of these Hybrid-Rs is still unclear. Func- The insulin receptor (IR) and the insulin-like growth factor tional studies have indicated that Hybrid-Rs behave more like (IGF) I receptor (IGF-IR) are tetrameric glycoproteins com- IGF-IRs than IRs because they bind to and are activated by IGF-I with an affinity similar to that of the typical IGF-IR. In * This work was supported in part by grants from the Associazione contrast, Hybrid-R activation in response to insulin occurs with Italiana per la Ricerca sul Cancro and Ministero dell’Universita ` e della much lower affinity (22, 23). Hybrid-Rs are therefore believed Ricerca Scientifica e Tecnologica (1999, 2001) (to A. B.). The costs of to provide additional binding sites to IGF-I and to increase cell publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” sensitivity to this growth factor (17–19). These studies have in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. not, however, taken into account the different IR isoform con- Recipient of a fellowship from the Fondazione Giuseppe Alazio per tribution to Hybrid-R formation and function. la Ricerca sul Cancro. The human IR exists in two isoforms (IR-A and IR-B), gen- Recipients of fellowships from the Fondazione Italiana per la Ricerca sul Cancro. ‡‡ To whom correspondence should be addressed. Tel.: 39-0961- 712423; Fax: 39-0957-158072; E-mail: [email protected]. Hybrid-R , insulin/insulin-like growth factor I hybrid receptor contain- The abbreviations used are: IR, insulin receptor; IGF, insulin-like ing the insulin receptor B isoform; BSA, bovine serum albumin; PMSF, growth factor; IGF-IR, insulin-like growth factor I receptor; ERK, ex- phenylmethylsulfonyl fluoride; BrdUrd, bromodeoxyuridine; ELISA, tracellular signal-regulated kinase; Hybrid-R, insulin/insulin-like enzyme-linked immunosorbent assay; GFP, green fluorescent protein; growth factor I hybrid receptor; Hybrid-R , insulin/insulin-like growth PBS, phosphate-buffered saline; RT, reverse transcription; X-gal, 5-bro- factor I hybrid receptor containing the insulin receptor A isoform; mo-4-chloro-3-indolyl--D-galactopyranoside; SH, Src homology. 39684 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. IR Isoforms Affect Insulin/IGF-I Hybrid Receptor Properties 39685 TABLE I erated by alternative splicing of the insulin receptor gene that Description of receptors and tranfected cells studied either excludes or includes 12 amino acid residues encoded by Description a small exon (exon 11) at the carboxyl terminus of the IR -subunit (see Table I). The relative abundance of IR isoforms Receptors is regulated by tissue-specific and unknown factors (24, 25). IR-A IR isoform lacking 12 amino acid residues encoded by exon 11 Recently, we found that IR-A (but not IR-B) binds IGF-II with IR-B IR isoform containing 12 amino acid residues high affinity and behaves as a second physiological receptor for encoded by exon 11 IGF-II in fetal and dedifferentiated (malignant) cells (26 –28). A Hybrid-R Receptor composed of one - and one -subunit of We therefore hypothesized that the relative abundance of the the IGF-IR and one - and one -subunit of IR-A Hybrid-R Receptor composed of one - and one -subunit of two isoforms may affect the functional properties of Hybrid-Rs the IGF-IR and one - and one -subunit of IR-B and modulate, in this way, the activation of the IGF system. Cells To investigate these issues, we used three different cellular R 3T3-like fetal fibroblasts derived from IGF-IR models. First, we used R fibroblasts, which are 3T3-like cells knockout mice derived from IGF-IR knockout mice. These cells also have low R IR-A R cells transfected with a construct encoding IR-A R IR-B R cells transfected with a construct encoding IR-B levels of endogenous IR. We cotransfected these cells with both R R cells transfected with the human IGF-IR gene the human IGF-IR gene and a construct encoding either IR-A R A R cells transfected with a construct encoding IR-A or IR-B to obtain cells expressing either Hybrid-Rs or Hybrid- A to obtain cells expressing the Hybrid-R Rs , respectively (see Table I). Second, we employed a panel of R B R cells transfected with a construct encoding for IR-B to obtain cells expressing the Hybrid-R human cell lines that express the two IR isoforms in variable amounts. Third, we used HepG2 hepatoblastoma cells that express predominantly either IR-A or IR-B depending on the K. Siddle, University of Cambridge, Cambridge, UK) (32, 33); a rabbit polyclonal antibody that recognizes the IR -subunit (Transduction culture conditions (29). Laboratories, Lexington, KY); and polyclonal antibody 29B4 (which We found that each of the IR isoforms is equally able to form A B recognizes the IR -subunit) (Santa Cruz Biotechnology Inc., Santa hybrids with the IGF-IR. Hybrid-Rs and Hybrid-Rs , how- Cruz, CA). ever, have different functional characteristics. Hybrid-Rs The following anti-IGF-IR antibodies were employed: monoclonal have a high affinity only for IGF-I. Hybrid-Rs have an even antibody IR-3 (which recognizes the IGF-IR -subunit and only poorly higher affinity for IGF-I and bind also IGF-II and insulin. recognizes the Hybrid-R) (Oncogene Research, Cambridge, MA) (34); monoclonal antibody 17-69 (which recognizes the -subunits of both the Insulin binding to Hybrid-Rs phosphorylates the IGF-IR IGF-IR and Hybrid-R) (Dr. K. Siddle) (35); and a chicken polyclonal -subunit and activates CrkII, an IGF-IR-specific substrate. antibody that recognizes the IGF-IR -subunit (Upstate Biotechnology, Accordingly, cell transfection with IR-A cDNA (but not with Inc., Lake Placid, NY). Anti-phospho-ERK1/2 and anti-phospho-Akt IR-B cDNA) markedly increases cell motility in response not antibodies were purchased from New England Biolabs (Beverly, MA); only to IGF-I, but also to insulin and IGF-II. anti-phosphotyrosine monoclonal antibody 4G10 was from Upstate Bio- These data therefore suggest that the relative abundance of technology, Inc.; and anti-BrdUrd antibody was from BD PharMingen (Erembodegem, Belgium). IR isoforms modulates the activation of the IGF system by regulating both binding and signaling characteristics of Hy- Cells brid-Rs. They also provide clues to the mechanism by which ARO cells were kindly provided by Dr. A. Pontecorvi (Regina Elena insulin may activate the IGF-IR phosphorylation cascade and Cancer Institute, Rome, Italy). A549, IM-9, HepG2, MDA-MB157, and biological effects in a tissue-specific manner. These findings PC-3 cells were obtained from American Type Culture Collection. R may have important implications for cell biological responses to mouse fibroblasts (3T3-like mouse cells derived from animals with a insulin, IGF-I, and IGF-II. targeted disruption of the IGF-IR gene, expressing 5 10 insulin receptors/cell) were kindly provided by Dr. R. Baserga (Kimmel Cancer EXPERIMENTAL PROCEDURES Center, Jefferson University, Philadelphia, PA) (Table I). HepG2 and Materials MDA-MB157 cells were routinely grown in minimum essential medium supplemented with 10% fetal bovine serum. A549, PC-3, IM-9 and ARO The pNTK2 expression vectors containing the cDNAs for the A cells were routinely grown in RPMI 1640 medium supplemented with (Ex11 ) and B (Ex11 ) isoforms of the human IR were kindly provided 10% fetal bovine serum. The R mouse fibroblasts were routinely grown by Dr. Axel Ullrich (Max Planck Institute of Biochemistry, Martinsried, in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal Germany). The pECE expression vector containing the cDNA encoding bovine serum. the human IGF-IR was a gift of Dr. R. Roth (Department of Molecular Pharmacology, Stanford University, Stanford, CA). The pCH110 ex- Transfection Experiments pression vector for -galactosidase was kindly provided by Dr. F. Tato ` (Universita ´ di Roma “La Sapienza,” Rome, Italy). The expression vector R cells were grown in 35-mm plates until 60 –70% confluent. They for pBOS-H2B-GFP was kindly provided by Dr. J. Y. Wang (University were first transfected with 2 g of pECE expression vector containing of California at San Diego, San Diego, CA). the cDNA encoding the IGF-IR (36) and cotransfected with 0.2 gof The following materials were purchased from the indicated manu- pSV2 plasmid encoding the hygromycin resistance gene by the Lipo- facturers: fetal calf serum, glutamine, LipofectAMINE, and DNase I fectAMINE method according to the manufacturer’s protocol. Cells from Invitrogen (Paisley, UK); RPMI 1640 medium, Dulbecco’s modified were then subjected to antibiotic selection in medium supplemented Eagle’s medium, minimum essential medium, Ham’s nutrient mixture with 400 g/ml hygromycin for 3 weeks. Stably transfected clones were F-12, bovine serum albumin (BSA; radioimmunoassay grade), bacitra- tested for receptor content by ELISA. Cell clones were further trans- cin, phenylmethylsulfonyl fluoride (PMSF), puromycin, bromodeoxyuri- fected with the pNTK2 expression vector containing the cDNA for dine (BrdUrd), and porcine insulin from Sigma; protein G-Sepharose either the A (Ex11 ) or B (Ex11 ) isoform of the human IR (37) and from Amersham Biosciences (Uppsala, Sweden); and I-labeled IGF-I cotransfected with the pPDV6 plasmid encoding the puromycin resist- (specific activity of 11.1 MBq/g) from PerkinElmer Life Sciences ance gene. Cells were subsequently subjected to antibiotic selection in (Zaventem, Belgium). IGF-I and IGF-II were obtained from Calbio- medium supplemented with 400 g/ml hygromycin and 2.4 g/ml pu- chem, and FuGENE 6 transfection reagent was obtained from Roche romycin for 3 weeks. Receptor content was evaluated in selected clones Molecular Biochemicals (Mannheim, Germany). by ELISA. Cell clones expressing similar amounts of either IR-A or A B The following anti-IR antibodies were employed: monoclonal antibod- IR-B, IGF-IR, and Hybrid-R (either the Hybrid-R or Hybrid-R ) were ies MA-10 and MA-20 (which recognize the IR -subunit, but only selected for subsequent studies. For migration studies, HepG2 cells poorly recognize the Hybrid-R) (Dr. I. D. Goldfine, University of Cali- were transiently transfected by the FuGENE 6 method according to the fornia at San Francisco, San Francisco, CA) (30, 31); monoclonal anti- manufacturer’s protocol. Briefly, 4 10 cells were seeded in six-well body CT-1 (which recognizes the IR -subunit) and monoclonal antibody plates and grown for 24 h in complete medium (minimum essential 83-7 (which recognizes the -subunits of both the IR and Hybrid-R) (Dr. medium with 10% fetal bovine serum). Thereafter, a transfection mix- 39686 IR Isoforms Affect Insulin/IGF-I Hybrid Receptor Properties ture containing 2 g of pNTK2-IR-A/IR-B 0.2 gof -galactosidase or Western Blotting—To confirm data obtained by ELISA, aliquots of histone H2B-GFP 12 l of FuGENE 6 in 100 l of minimal essential the same lysates were subjected to Western blot analysis. Cell lysates medium without serum or antibiotics was added to each well. Cells were were incubated at 4 °C under constant rotation for 2 h with 4 g of the grown in complete medium; and after 48 h, they were assayed for specific anti-receptor antibody and then for 2 h with protein G-Sepha- -galactosidase activity or scored under a fluorescence microscope for rose. Immunoprecipitates were eluted and subjected to SDS-PAGE and GFP expression. then immunoblotted (1 g/ml) as described below. IRs were immuno- precipitated with anti-IR antibody MA-20 and blotted with the rabbit Preparation of Cell Lysate anti-IR polyclonal antibody. IGF-IRs were immunoprecipitated with anti-IGF-IR antibody IR-3 and blotted with the chicken anti-IGF-IR Cells were grown until 80% confluent and serum-starved 24 h before polyclonal antibody. Hybrid-Rs were immunoprecipitated with anti-IR stimulation with the various ligands. For receptor and ERK/Akt acti- antibody 83-7 and blotted with the chicken anti-IGF-IR polyclonal vation, cells were stimulated with 10 nM insulin, IGF-I, or IGF-II for 10 antibody. Western blot specificity was evaluated by examining the min. For in vitro Crk phosphorylation, cells were stimulated with 50 nM interference of 200 ng of purified receptor of each subtype added to a cell insulin, IGF-I, or IGF-II for 5 min. After three washes with ice-cold lysate containing 200 ng of IR, IGF-IR, or Hybrid-R. PBS, cells were lysed in cold radioimmune precipitation assay buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% Nonidet P-40, 0.5% Hybrid-R Autophosphorylation Triton X-100, 0.25% sodium deoxycholate, 10 mM sodium pyrophos- phate, 1 mM NaF, 1 mM sodium orthovanadate, 2 mM PMSF, 10 g/ml Western Blotting—Cell lysates were incubated at 4 °C under con- aprotinin, 10 g/ml pepstatin, and 10 g/ml leupeptin. After being stant rotation for 1 h with protein G-Sepharose to eliminate antibody scraped, samples were rotated for 15 min at 4 °C. Insoluble material MA-10 bound to the IR. After centrifugation, the supernatant was was separated from the soluble extract by microcentrifugation at incubated at 4 °C under constant rotation for 2 h with 4 g of anti- 10,000 g for 10 min at 4 °C. Protein concentration was determined by Hybrid-R antibody 83-7 coated with protein G-Sepharose. Immunopre- the Bradford assay. cipitates were eluted and subjected to SDS-PAGE. The resolved pro- teins were transferred to nitrocellulose membranes, immunoblotted A B Ligand Binding Assay for the Hybrid-R or Hybrid-R with anti-phosphotyrosine monoclonal antibody 4G10, and revealed by A B an ECL method. The nitrocellulose membrane was then stripped with Either the Hybrid-R or Hybrid-R was captured by incubating cell Restore stripping buffer (Pierce) for 30 min at room temperature and lysates for 22 h in Maxisorp Break-Apart immunoplates (Nunc, Ros- subsequently reprobed with the chicken anti-IGF-IR polyclonal kilde, Denmark) precoated with 2 g/ml antibody 83-7. After washing, 125 antibody. the immunocaptured receptors were incubated with I-labeled IGF-I ELISA—As previously described (38), 100 l of the cell lysates pre- (10 pM in 50 mM HEPES-buffered saline (pH 7.6) containing 0.05% pared as described above were immunocaptured in Maxisorp plates Tween 20, 1% BSA, 2 mM sodium orthovanadate, 1 mg/ml bacitracin, coated with antibodies 83-7 (which recognizes both the IR and Hy- and1mM PMSF) in the presence or absence of increasing concentra- brid-R) and MA-20 (which recognizes the IR only) at a concentration of tions of various unlabeled ligands (insulin, IGF-I, and IGF-II). After 2 h 2 g/ml in 50 mM sodium bicarbonate (pH 9.0) overnight at 4 °C. After at room temperature, the plates were washed, and the radioactivity in washing, the captured phosphorylated proteins were incubated with each well was counted in a -counter. biotin-conjugated anti-phosphotyrosine antibody 4G10 (0.3 g/ml in 50 mM HEPES (pH 7.6), 150 mM NaCl, 0.05% Tween 20, 1% BSA, 2 mM IR, IGF-IR, and Hybrid-R Measurements sodium orthovanadate, 1 mg/ml bacitracin, and 1 mM PMSF) for2hat Cell lysates were prepared as described above and used for receptor 22 °C and then with peroxidase-conjugated streptavidin. The peroxi- measurement both by ELISA and Western blot analysis. dase activity was determined colorimetrically by adding 100 lof ELISA—The characteristics and specificity of these ELISAs have 3,3,5,5-tetramethylbenzidine (0.4 mg/ml in 0.1 M citrate/phosphate been previously described (18). Receptors were captured by incubating buffer (pH 5.0) with 0.4 l/ml 30% H O ). The reaction was stopped 2 2 lysates (0.5– 60 g/well) in Maxisorp immunoplates precoated with the by the addition of 1.0 M H PO , and the absorbance was measured at 3 4 specific monoclonal antibody (2 g/ml) indicated below. After washing, 450 nm. the immunocaptured receptors were incubated with the specific biotin- ylated monoclonal antibody indicated below (0.3 g/ml in 50 mM In Vitro CrkII Phosphorylation HEPES-buffered saline (pH 7.6) containing 0.05% Tween 20, 1% BSA, In vitro receptor tyrosine kinase activity for CrkII was measured as 2mM sodium orthovanadate, 1 mg/ml bacitracin, and 1 mM PMSF) and previously described (9) with modifications. 500 g of proteins were then with peroxidase-conjugated streptavidin. The peroxidase activity immunoprecipitated with either anti-IR monoclonal antibody MA-20 or was determined colorimetrically by adding 100 lof3,3,5,5-tetra- anti-Hybrid-R antibody 83-7 coupled to protein G-Sepharose. Pellets methylbenzidine (0.4 mg/ml in 0.1 M citrate/phosphate buffer (pH 5.0) were washed twice with radioimmune precipitation assay buffer and with 0.4 l/ml 30% H O ). The reaction was stopped by the addition of 2 2 twice with kinase buffer without ATP and resuspended in 100 lof 1.0 M H PO , and the absorbance was measured at 450 nm. 3 4 kinase buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 0.1% IRs were captured with anti-IR antibody MA-20 and detected with Triton X-100, 10 mM MgCl ,2mM MnCl , 0.05% BSA, 50 M ATP, and 2 2 biotinylated anti-IR antibody CT-1 (30, 33). IGF-IRs were captured 1 g of glutathione S-transferase-Crk (provided by Dr. Raymond Birge, with anti-IGF-IR antibody IR-3 and detected with biotinylated anti- Rockefeller University). Reaction mixtures were incubated at room body 17-69 (34, 35). Hybrid-Rs were captured with anti-IR antibody temperature for 20 min under continuous agitation. After rapid centrif- 83-7, which recognizes both the Hybrid-R and IR, and detected with ugation at 14,000 rpm, supernatants were collected, and 4 sample biotinylated anti-IGF-IR antibody 17-69 (32, 35). The receptor content buffer was added. Samples were boiled for 3 min; subjected to SDS- was evaluated by comparing each sample with a standard curve, as PAGE; and transferred to nitrocellulose membranes, which were blot- previously described (18). ted with anti-phosphotyrosine antibody 4G10. Membranes were The minimal detectable amount of hybrids was 0.125 ng/well (1.25 stripped and reprobed with anti-CrkII polyclonal antibody (Santa Cruz ng/ml). The assay was linear from 0.125 to 1.0 ng/well. There was no Biotechnology Inc.) where required. interference from either 1 ng/well purified IR (from human IR cDNA-transfected NIH/3T3 cells) or 1 ng/well purified IGF-IR (from ERK1/2 and Akt Phosphorylation in Response to Insulin, human IGF-IR cDNA-transfected Chinese hamster ovary cells). Multi- IGF-I, or IGF-II ple dilutions of cells and tissues containing either Hybrid-Rs or Hy- brid-Rs produced dose-response curves parallel to those obtained with After the addition of 5 sample buffer, samples were heated at the purified IR/IGF-IR hybrid standard (Ref. 18 and data not shown). 95–100 °C for 5 min and subjected to reducing SDS-PAGE on 10% Intra-assay coefficients of variation were 7% at 0.5 ng/tube and 8% polyacrylamide gel. After electrophoresis, the resolved proteins were at 1.0 ng/tube. Inter-assay coefficients of variation were 8 and 10%, transferred to nitrocellulose membranes and subjected to immunoblot respectively (18). analysis. For ERK1/2 activation studies, the blots were probed with the The ELISAs for the IR and IGF-IR had similar characteristics of phospho-specific ERK1/2 polyclonal antibody. For Akt phosphorylation sensitivity and specificity, as previously described (18). Purified IGF-IR studies, the blots were probed with anti-phospho-Akt polyclonal anti- or Hybrid-R (up 1 ng/well) did not interfere in the IR assay, and purified body. The nitrocellulose membranes were then stripped with stripping IR or Hybrid-R did not interfere in the IGF-IR assay. The minimal buffer for 30 min at room temperature and subsequently reprobed with detectable amounts were 0.05 ng/tube for the IR and 0.0625 ng/tube for either anti-ERK1/2 polyclonal antibody or anti-Akt polyclonal antibody. the IGF-IR. Intra-assay coefficients of variation were 8%, and inter- All immunoblots were revealed by the ECL method, autoradiographed, assay coefficients of variation were 10% for both assays (18). and subjected to densitometric analysis. IR Isoforms Affect Insulin/IGF-I Hybrid Receptor Properties 39687 TABLE II IR, IGF-IR, and Hybrid-R content in cell clones obtained from R fibroblasts transfected with the IGF-IR and with either the IR-A (clones A28, A25, and A48) or IR-B (clones B15, B22, and B3) cDNA Clones A25 and B22, with a similar receptor content, were selected for subsequent studies. Data represent mean S.E. of three different experiments. Receptor content Clones Hybrid-R IR IGF-IR Measured Predicted ng/100 g protein R A28 11.0 0.8 5.4 0.3 24.2 1.5 15.4 R A25 18.4 1.2 6.8 0.4 21.8 2.2 22.4 R A48 25.1 1.1 5.6 0.7 28.6 3.2 23.7 R B15 12.3 1.2 8.5 0.6 19.3 2.3 20.4 R B22 18.2 0.7 7.2 0.9 22.4 1.6 22.9 R B3 20.6 1.7 9.3 0.6 24.5 2.8 27.7 If the total concentrations of insulin and IGF-I half-receptors are I and G, respectively, and these half-receptors combine randomly, then it 2 2 would be predicted that the relative concentrations of IR/IGF-IR/Hybrid-R would be I :G :2IG. Thus, the measured content of Hybrid-Rs can be compared with the expected content on the basis of random assembly, since Hybrid-Rs 2 IR IGF-IR. IR Isoform RT-PCR mount (Biomeda). Coverslips were scored at 40 magnification under an Olympus microscope, and images were randomly acquired with an RT-PCR for IR isoforms was carried out as previously described (39) ORCA digital camera (Hamamatsu) and superimposed with ImagePro- using oligonucleotide primers spanning nucleotides 2230 –2251 (5- Plus software. Numbers were calculated as the percent of BrdUrd- AAC-CAG-AGT-GAG-TAT-GAG-GAT-3) and 2846 –2867 (accession incorporating cells among GFP-positive cells, and the increases induced M10051) (5-CCG-TTC-CAG-AGC-GAA-GTG-CTT-3) of the human IR. by growth factors were calculated as the percent over basal levels. PCR amplification was carried out for 30 cycles of 20 s at 96 °C, 30 s at 58 °C, and 1.5 min at 72 °C using a DNA thermal cycler (PerkinElmer RESULTS Life Sciences). After electrophoresis of the PCR products, the 600- and 636-bp DNA fragments representing the Ex11 and Ex11 IR isoforms IR-A and IR-B Moieties Can Form Hybrid-Rs were analyzed by scanning densitometry and compared with the stand- with the Same Efficiency ards. Standard preparation was carried out using mRNA from NIH/3T3 cells transfected with both IR isoform cDNAs mixed at various ratios Transfected R Cells—R cells, which do not express endog- and co-amplified by RT-PCR. To verify that the larger cDNA was really enous IGF-IR and have low levels of endogenous IR (which are IR-B, RT-PCR products were subjected to BanI digestion. Only cDNA not recognized by the anti-human IR antibodies used), were containing exon 11, the restriction site for the enzyme, was digested. first transfected with a plasmid containing the cDNA of the Migration Assays human IGF-IR and then with a plasmid containing either the Cell migration assays were performed as previously described (40, IR-A or IR-B cDNA. The stable transfectants obtained were 41) with minor modifications using modified Boyden chambers (6.5-mm evaluated for IR, IGF-IR, and Hybrid-R content, as described diameter, 10-m thickness, 8-m pores; Transwell, Costar Corp., Cam- under “Experimental Procedures.” In these cotransfected cell bridge, MA) containing polycarbonate membranes coated with 10 g/ml clones (IGF-IR IR-A or IGF-IR IR-B), Hybrid-R content collagen type IV. 36 h after transfection, HepG2 cells were serum- was in close accordance with the value predicted by the random starved for 12 h. Cells were then removed from the plates with Hanks’ balanced salt solution containing 5 mM EDTA, 25 mM HEPES (pH 7.2), assembly model, indicating that each of the two IR isoforms can and 0.01% trypsin; resuspended at 10 cells/ml; and added to the top of form Hybrid-Rs with the same efficiency (Table II). Western each migration chamber. Cells were allowed to migrate to the underside blot analyses, carried out as described under “Experimental of the top chamber for6hinthe presence or absence of 10 nM insulin, Procedures,” proved to be specific for each receptor measured IGF-I, or IGF-II, which had been added to the lower chamber. Filters (Fig. 1A) and confirmed ELISA data (Fig. 1B and Table II). containing migrated and non-migrated cells were incubated with X-gal (Promega) as substrate according to the manufacturer’s recommenda- Established Human Cell Lines—To study native Hybrid-R tions. Total cells stained with X-gal were scored using a 40 objective. functional characteristics in non-transfected cells, we studied a The non-migrated cells on the upper membrane surface were removed panel of established human cell lines (IM-9 lymphoblasts, ARO with a cotton swab, and the migrated cells attached to the bottom thyroid cancer cells, MDA-MB157 breast cancer cells, PC-3 surface of the membrane stained with X-gal were counted as described prostate cancer cells, A549 lung cancer cells, and HepG2 hepa- above. Cell migration was expressed as the percent of migrated cells over total cells. Each determination was performed in triplicate. toblastoma cells). In these cells, we measured the IR isoform relative abundance and the IR, IGF-IR, and Hybrid-R content. BrdUrd Incorporation With the exception of IM-9 cells, which expressed only IR-A, HepG2 cells were seeded onto coverslips in six-well plates in com- the remaining cell lines expressed both IR-A and IR-B. In these plete medium. 24 h later, they were transfected with empty vector-IR- cell lines, IR-A content ranged from 24 to 82% of the total IR A/IR-B histone H2B-GFP in triplicates as described above. 12 h later, content. All these cells also expressed IGF-IRs and Hybrid-Rs. the medium was replaced with Dulbecco’s modified Eagle’s medium and Hybrid-R content was in all cases in accordance with the ran- 0.1% BSA, and the cells were serum-starved for 24 h. Then, 10 nM insulin, IGF-I, or IGF-II was added, and the cells were further incu- dom assembly model (Table II), confirming data obtained in bated for 36 h. Cells were incubated with 10 M BrdUrd for 1 h, fixed in transfected cells. 3.7% paraformaldehyde in PBS for 15 min at room temperature, and We also evaluated Hybrid-R content in HepG2 hepatoblas- incubated with 50 nM NH Cl in PBS. Cells were then permeabilized toma cells before and after exposure to dexamethasone, which with 0.3% Triton X-100 in PBS; incubated with blocking solution con- causes cell differentiation and a change in the IR isoform rel- taining 10% normal goat serum in PBS for 45 min at room temperature; and exposed to a mixture containing anti-BrdUrd antibody (diluted ative abundance (29). In agreement with previous reports, IR-A 1:200 in PBS plus 10% normal goat serum), 20 mM MgCl , 0.5% Nonidet 2 decreased from 82 to 14% of the total cell IR content after P-40, and DNase I (1:500) for1hat room temperature. Coverslips were dexamethasone-induced differentiation (Fig. 2 and Table III). washed three times with PBS and incubated with Texas Red-conju- Undifferentiated HepG2 cells therefore predominantly ex- gated goat anti-mouse antibody (1:200) in PBS plus 10% normal goat pressed Hybrid-Rs , whereas differentiated HepG2 cells pre- serum for 45 min at room temperature. Cells were counterstained with Hoechst 33258, and coverslips were mounted onto glass slides with gel/ dominantly expressed Hybrid-Rs . 39688 IR Isoforms Affect Insulin/IGF-I Hybrid Receptor Properties cDNAs for IGF-IR, IR-A, or IR-B. Binding studies were carried out on immunopurified receptors from these cells by displacing 125 125 either I-labeled IGF-I or I-labeled insulin with increasing concentrations of unlabeled ligands (insulin, IGF-I, and IGF- II). As previously reported (26), the IGF-IR bound both IGFs (but not insulin) with high affinity, and both IR isoforms bound insulin with high affinity and IGF-I poorly. However, only IR-A bound IGF-II with high affinity. EC values are given in Table IV. Data consistent with those obtained in stable transfectants of R cells were also obtained in Hybrid-Rs immunopurified from HepG2 cells (Fig. 3). In undifferentiated HepG2 cells (which predominantly express IR-A and Hybrid-Rs ), IGF-I, IGF-II, or insulin displaced I-labeled IGF-I with an affinity in the physiological concentration range (EC 0.4, 0.6, and 4.5 nM, respectively). In contrast, in differentiated HepG2 cells (which predominantly express IR-B and Hybrid-Rs ), the EC values were 1.8 for IGF-I, 4.0 for IGF-II, and 20 nM for insulin (Fig. 3). The binding characteristics of Hybrid-Rs were also studied in FIG.1. A, specificity of Western blot analysis. To cell lysates from transfected fibroblast cell clones containing the IR (upper panel), the a variety of established human cell lines (Table II). In Hy- IGF-IR (middle panel), or the Hybrid-R (lower panel) were added 200 ng brid-Rs immunopurified from IM-9 cells (which express only of purified IR (lane 2), Hybrid-R (lane 3), or IGF-IR (lane 4). In meas- IR-A and Hybrid-Rs ) or from PC-3, MDA-MB157, and ARO urements of each receptor, no interference by the other two related cells (all which predominantly express Hybrid-Rs ), both IGFs receptors was observed. B, expression of the IR, IGF-IR, and Hybrid-R in stably transfected R cell clones. R cells were transfected either and insulin efficiently displaced I-labeled IGF-I. EC values with IGF-IR and IR-A cDNAs (clones R A28, R A25, and R A48) or ranged 0.2 to 0.6 nM for IGF-I, 0.3 to 0.7 nM for IGF-II, and 1.8 with IGF-IR and IR-B cDNAs (clones R B15, R B22, and R B3). Re- to 3.2 nM for insulin. In contrast, in A549 cells (which predom- ceptors were immunoprecipitated (IP) and detected by Western blot inantly express IR-B (76%) and Hybrid-Rs ), the EC values analysis as described under “Experimental Procedures.” Clones R A25 50 were 1.5 nM for IGF-I, 10 nM for IGF-II, and 100 nM for and R B22 had a similar receptor content and were selected for func- tional studies. IB, immunoblot. insulin. Receptor autophosphorylation was evaluated in intact cells A B expressing either only Hybrid-Rs or Hybrid-Rs after expo- sure to either insulin or IGFs in the presence of a molar excess of the IR-blocking antibody MA-10, which does not recognizes Hybrid-Rs, as evaluated by immunoprecipitation experiments (data not shown). This procedure was used to avoid the inter- ference of IRs. Cells were then solubilized, and receptors were immunopurified with antibody 83-7 (which recognizes the IR and Hybrid-R, but not the IGF-IR). Autophosphorylation/acti- FIG.2. Time course of IR isoform expression in HepG2 cells vation of immunopurified Hybrid-Rs was measured by Western during differentiation. HepG2 cells were cultured in the absence blotting. As shown in Fig. 4A, IGF-I, IGF-II, and insulin were (time 0) or in the presence of dexamethasone for the indicated times, and IR isoform expression was measured by RT-PCR. Numbers on the all able to efficiently activate Hybrid-Rs , whereas only IGF-I bottom indicate the relative abundance of IR isoform expression (%) was able to efficiently activate Hybrid-Rs . Both IGF-II and calculated from densitometric analysis. The results are representative B insulin were much less effective in Hybrid-Rs than in Hybrid- of three separate experiments. M, MARKER 600 bp. Rs . Similar results were obtained in parallel experiments in A B which Hybrid-R autophosphorylation was quantitated by Hybrid-Rs and Hybrid-Rs Have Different Binding and ELISA (Fig. 4B). These autophosphorylation data are therefore Activation Properties with Regard to Insulin and IGFs in close accordance with results from binding studies and sug- To study the different binding characteristics of Hybrid-Rs gest that Hybrid-Rs may be regarded as additional receptors and Hybrid-Rs , we used two double-transfected cell clones for IGF-I, IGF-II, and also insulin, whereas, in contrast, Hy- (R A25 and R B22) expressing similar amounts of either Hy- brid-Rs should be regarded as selective receptors for IGF-I. A B brid-Rs or Hybrid-Rs (Fig. 1 and Table II). Cells were solu- bilized, and Hybrid-Rs were immunopurified with monoclonal A B Hybrid-Rs (but Not Hybrid-Rs ) Shift Insulin antibody 83-7, which does not recognize the IGF-IR. I-La- to IGF-IR Signaling beled IGF-I was then allowed to bind to immunocaptured re- ceptors in the absence or presence of increasing concentrations Because insulin bound to the Hybrid-R with an affinity of various unlabeled ligands (insulin, IGF-I, and IGF-II). within the physiological range, we evaluated the ability of A A The displacement curves indicate that Hybrid-Rs bound insulin to activate the IGF-IR -subunit of the Hybrid-R . For IGF-I with high affinity, 8-fold higher compared with Hybrid- this purpose, either R A25 or R B22 cell clones were stimu- B A Rs (Fig. 3). Moreover, Hybrid-Rs also bound insulin and lated with insulin, IGF-I, or IGF-II and then solubilized as IGF-II with an affinity 30-fold higher than that of Hybrid- described under “Experimental Procedures.” Samples were im- B B Rs . In contrast, Hybrid-Rs bound only IGF-I with high affin- munoprecipitated with anti-phosphotyrosine antibody 4G10, ity (Fig. 3). Half-maximal inhibition of I-labeled IGF-I subjected to SDS-PAGE, and blotted with anti-IGF-IR anti- A B A (EC ) by the three ligands in both Hybrid-Rs and Hybrid-Rs body. In R A25 cells, which express only Hybrid-Rs , insulin is given in Table IV. recruited the IGF-IR to the tyrosine phosphorylation cascade To compare the ligand affinity of Hybrid-Rs with that of with a potency similar to that of IGF-II, albeit lower than that homodimeric receptors, R cells were stably transfected with of IGF-I (Fig. 5). By contrast, in R B22 cells, which express IR Isoforms Affect Insulin/IGF-I Hybrid Receptor Properties 39689 TABLE III IR-A relative abundance and IR, IGF-IR, and Hybrid-R content in a panel of human malignant cells and in human hepatoma HepG2 cells before and after differentiation Hybrid-R a a Cells IR-A IR IGF-IR Measured Predicted IM-9 100 12.0 0.6 26.0 4.4 30.0 4.2 35.3 ARO 80 8.0 0.9 10.8 1.0 25.5 3.4 18.6 MDA-MB157 70 11.0 0.87 2.0 0.5 12.0 2.2 9.4 PC-3 68 8.5 2.2 0.9 0.1 9.5 3.1 5.5 A549 24 0.18 0.03 17.0 4.2 2.5 0.1 3.5 HepG2 Undifferentiated 82 3.4 0.8 2.1 0.4 6.5 0.4 5.3 Differentiated 14 8.2 0.6 3.2 0.9 10.6 0.2 10.2 Receptor content expressed as ng/100 g of protein. 125 B A FIG.3. Competition inhibition curves of I-labeled IGF-I binding to immunopurified Hybrid-Rs or Hybrid-Rs . Immunopurified A B 125 Hybrid-Rs or Hybrid-Rs were incubated with I-labeled IGF-I (10 pM) in the absence or presence of increasing concentrations of insulin, IGF-I, or IGF-II as described under “Experimental Procedures.” The data represent means S.E. of three separate experiments run in triplicate. Hybrid-Rs were immunopurified either from R cells transfected with both IR-A and IGF-IR (clone R A25) or from undifferentiated HepG2 cells. Hybrid-Rs were immunopurified either from R cells transfected with both IR-B and IGF-IR (clone R B22) or from differentiated HepG2 cells. only Hybrid-Rs , IGF-IR recruitment by insulin was very weak -subunits) with a higher potency than in R B22 cells. and much lower than that induced by IGF-I or IGF-II (Fig. 5). We then evaluated whether insulin, via the Hybrid-R ,is Reblotting with anti-phosphotyrosine antibody 4G10 showed able to activate IGF-IR-specific intracellular mediators like the that, in R A25 cells, IGF-II stimulated the tyrosine phospho- small adapter protein CrkII, which is phosphorylated by the rylation of the 97-kDa band (containing both the IR and IGF-IR IGF-IR, but not by the IR (9, 42, 43). To this purpose, either 39690 IR Isoforms Affect Insulin/IGF-I Hybrid Receptor Properties TABLE IV tion and migration) to either insulin or IGFs. To avoid possible Binding affinity of insulin, IGF-I, and IGF-II for immunopurified proliferation and migration differences due to the differentia- A B receptors (Hybrid-R , Hybrid-R , IGF-IR, IR-A, and IR-B from tion state, undifferentiated HepG2 cells were forced to overex- transfected R cells A B press either Hybrid-Rs or Hybrid-Rs by transient IR-A or EC of unlabeled ligand IR-B cDNA transfection. Control cells were obtained by trans- R cells Insulin IGF-I IGF-II fection of an empty vector. Transfection efficiency, evaluated by nM histone H2B-GFP and -galactosidase, ranged from 15 to 20% Hybrid-R 3.7 0.9 0.3 0.2 0.6 0.1 (Fig. 9A). Hybrid-R 100 2.5 0.5 15.0 0.9 Cell proliferation was measured by scoring BrdUrd-labeled IGF-IR 30.0 0.2 0.3 0.6 1.0 nuclei in GFP-positive cells. Both IR-A and IR-B transfection IR-A 0.2 0.2 30.0 0.9 0.4 enhanced cell proliferation in response to insulin as compared IR-B 0.3 0.4 30.0 11.0 5.0 with empty vector transfection. By contrast, only IR-A trans- fection significantly enhanced cell proliferation in response to R A25 or R B22 cell clones were stimulated with insulin or both IGFs. IR-B transfection only slightly enhanced prolifera- IGF-I, and immunopurified receptors were incubated with tion in response to IGF-I and was totally ineffective for IGF- CrkII and ATP in kinase buffer as described under “Experi- II-stimulated cell proliferation (Fig. 9B). mental Procedures.” When IRs were immunopurified (with an- We also measured cell migration by scoring -galactosidase- tibody MA-20), no CrkII phosphorylation was observed (Fig. 6), positive cells that migrated to the lower side of Transwells (Fig. confirming that CrkII is not a substrate of the IR. In contrast, 10A). IR-A transfection significantly enhanced cell migration in when Hybrid-Rs were immunopurified (with antibody 83-7), A B response to all three ligands as compared with empty vector Hybrid-Rs (but not Hybrid-Rs ) were able to phosphorylate transfection. In contrast, IR-B transfection only slightly en- CrkII in response to insulin (Fig. 6), a difference that may be hanced cell migration in response to IGF-I, but not in response explained by the high affinity of insulin for Hybrid-Rs . Both A B to insulin or IGF-II (Fig. 10B). Hybrid-Rs and Hybrid-Rs were able to phosphorylate CrkII Taken together, these data suggest that the relative abun- in response to IGF-I. Taken together, these data suggest that dance of IR isoforms differentially regulates two major biolog- insulin may activate IGF-IR-specific intracellular pathways by ical effects (such as cell proliferation and migration) in re- interacting with Hybrid-Rs . sponse to both insulin and IGFs. IR-A overexpression and A B Hybrid-R and Hybrid-R Post-receptor Signaling subsequent Hybrid-R formation markedly enhance cell biolog- Double-transfected R A25 and R B22 cell clones were used ical responses to both IGFs, whereas IR-B overexpression does to study the ligand ability to activate the post-receptor signal- not. In addition, whereas cell proliferation in response to insu- ing pathways in intact cells expressing similar amounts of the lin is activated via both IR-A and IR-B, only IR-A increases cell three receptor subtypes (IGF-IR, IR, and Hybrid-R), but differ- migration in response to insulin, an effect most likely mediated ent isoforms. Parallel experiments were also carried out in cells by the activation of the IGF signaling pathway, via insulin containing only IR-A (R IR-A cells), IR-B (R IR-B cells), or binding to the Hybrid-R . IGF-IR (R cells). Cells were exposed to each ligand (10 nM) for DISCUSSION 10 min, and phosphorylation of the intracellular substrates The main finding of our study is that the differential expres- ERK1/2 kinase (p42/p44 mitogen-activated protein kinase) and Akt was subsequently measured by Western blotting. sion of the two isoforms of the human IR constitutes a molec- ular switch for the preferential activation of either the IR or Both substrates ERK1/2 and Akt had similar activation pat- terns in response to the different ligands. Insulin was the most IGF-I pathway. This is determined by both binding and signal- ing specificities of the two Hybrid-R types that are formed. In potent stimulating factor in both double-transfected cell clones, as expected by the presence of elevated IR levels (Fig. 7). IGF-II particular, predominant IR-A expression in cells coexpressing the IGF-IR leads to increased formation of Hybrid-Rs , which was approximately as potent as IGF-I in R A25 cells (Fig. 7) because of its high affinity for both IR-A and Hybrid-Rs , up-regulates the IGF system by two different mechanisms: (a) binding and activation with high affinity by both IGF-I and whereas it was less potent than IGF-I in R B22 cells (Fig. 7), in accordance with data obtained from the anti-phosphotyrosine IGF-II (which do not occur with the Hybrid-R ) and (b) activa- tion of the IGF-IR pathway also after insulin binding. antibody blot in Fig. 5. These data confirm that IR-A predom- inance enhances the cell sensitivity to IGF-II (which can bind In contrast, predominant IR-B expression leads to high bind- ing specificity whereby insulin activates only its own receptor to IGF-IRs, IR-A, and Hybrid-Rs ). Similar results were ob- tained in HepG2 cells: undifferentiated cells (mostly expressing and post-receptor signaling. Moreover, IR-B will sequestrate A B part of the IGF-IR moieties to form Hybrid-Rs , which have a Hybrid-Rs ) behaved similarly to R A25 cells, whereas differ- entiated cells (mostly expressing Hybrid-Rs ) behaved simi- reduced affinity for IGF-I and especially for IGF-II. This com- bined effect will result in reduced IGF system activity. larly to R B22 cells (data not shown). In cell clones containing only IR-A, both insulin and IGF-II Although IR isoforms and insulin/IGF-I hybrid receptors have been extensively studied (18, 19, 22–25, 29), their biolog- stimulated Akt and ERK1/2 phosphorylation to a similar ex- tent (Fig. 8). In contrast, in cell clones containing only IR-B, ical role was unclear. Hybrid-Rs are present in cells and tissues coexpressing both IRs and IGF-IRs and are often the most insulin (but not IGFs) was able to stimulate Akt and ERK1/2 phosphorylation. In R cells (which express only the IGF-IR), abundant receptor subtype (14, 16, 17). Functional studies have consistently shown that Hybrid-Rs the two IGFs were roughly equally potent in stimulating Akt and ERK1/2 phosphorylation, whereas insulin was not very behave similarly to homotypic IGF-IRs rather than to homo- typic IRs (14 –19, 22, 23). Using immunopurified receptors, effective (Fig. 8). Soos et al. (22) have shown that Hybrid-Rs bind IGF-I with Biological Effects of Either Insulin or IGFs high affinity, similar to typical IGF-IRs, whereas they bind in Cells Predominantly Expressing Either Hybrid-Rs insulin with much lower affinity (20-fold lower compared or Hybrid-Rs with IRs). Moreover, insulin does not effectively displace Hy- We evaluated whether the presence of Hybrid-Rs or Hybrid- brid-R-bound IGF-I, possibly because IGF-I interaction with Rs may affect cell biological responses (such as cell prolifera- the -subunit of the IGF-IR allosterically inhibits insulin bind- IR Isoforms Affect Insulin/IGF-I Hybrid Receptor Properties 39691 A B FIG.4. Autophosphorylation of Hybrid-Rs and Hybrid-Rs in response to insulin, IGF-I, and IGF-II. Cultured cells containing either A B Hybrid-Rs (clone R A25) or Hybrid-Rs (clone R B22) were exposed to insulin, IGF-I, or IGF-II (10 nM) in the presence of the IR-blocking antibody MA-10. Cells were then solubilized, and Hybrid-Rs were immunopurified with antibody 83-7. A, Western blot analysis. Upper panel, anti- phosphotyrosine (PY) antibody immunoblot (I.B.). Numbers on the bottom indicate means S.D. of the densitometric reading of three independent experiments (arbitrary units). Lower panel, reblotting with anti-IGF-IR antibody. A representative experiment is shown. B, ELISA. Receptor autophosphorylation in response to ligands was measured by ELISA as described under “Experimental Procedures.” The data represent means S.E. of three separate experiments. ing (23). According to these observations, Hybrid-Rs are auto- has been suggested to reduce the availability of typical IRs, phosphorylated more efficiently after binding IGF-I compared thus contributing to insulin resistance in diabetes (44 – 46); with insulin (22). however, these data are controversial. Interestingly, certain As Hybrid-Rs are believed to result from random assembly of human cancers (namely thyroid and breast cancers) (18 –21, insulin and IGF-I half-receptors (17), their cell content is di- 28, 47) have been shown to overexpress IRs and, as a conse- rectly related to the expression level of the two receptors. quence, to express very high levels of Hybrid-Rs. In these Therefore, in cells expressing high IR levels, Hybrid-R content models, Hybrid-Rs were able to mediate cancer cell growth in may exceed typical IR and IGF-IR content (18, 19). This will response to IGF-I, suggesting that they may provide a selective shift the major ligand binding from insulin to IGFs and may growth advantage to malignant cells (18, 19). have relevant biological consequences in both metabolic disor- No previous study has addressed the functional characteris- ders and cancer. For instance, increased Hybrid-R formation tics of the Hybrid-R with relation to the IR isoform involved. 39692 IR Isoforms Affect Insulin/IGF-I Hybrid Receptor Properties FIG.5. Involvement of the IGF-IR moiety in Hybrid-R and Hybrid-R activation. R A25 and R B22 cells were treated with the indicated ligands (10 nM), and tyrosine-phosphorylated proteins were immunoprecipitated as indicated under “Experimental Procedures.” Upper panel, immunoblot (I.B.) with anti-IGF-IR antibody. A represent- ative experiment is shown. Middle panel, densitometric data represent- ing means S.E. of three separate experiments. Lower panel, reblot- ting with anti-phosphotyrosine (PY) antibody. FIG.7. ERK1/2 and Akt activation by insulin, IGF-I, and IGF-II in transfected R fibroblasts expressing either Hybrid-Rs or Hybrid-Rs . Serum-starved R A25 and R B22 cells were exposed to 10 nM insulin, IGF-I, or IGF-II. A, ERK1/2 activation. Upper panel, anti-phospho-ERK antibody blot showing results representative of three separate experiments; middle panel, reblotting with anti-ERK antibody; lower panel, densitometric reading (phospho-ERK/total ERK) representing means S.E. of three separate experiments. B, Akt acti- vation. Upper panel, anti-phospho-Akt antibody blot showing results representative of three separate experiments; middle panel, reblotting with anti-Akt antibody; lower panel, densitometric reading (phos- pho-Akt/total Akt) representing means S.E. of three separate FIG.6. In vitro tyrosine kinase activity of the IR and Hybrid-R experiments. for CrkII. R A25 and R B22 cells were stimulated in vivo with the indicated ligands. The tyrosine kinase activity of immunoprecipitated (I.P.) receptors for CrkII was determined in vitro as indicated under A B “Experimental Procedures.” Upper panel, anti-phosphotyrosine (PY) only either the Hybrid-R or Hybrid-R . HepG2 cells provide a blot; lower panel, anti-Crk antibody reblotting. A representative exper- natural model expressing up to 80% IR-A of the total IR content iment is shown. H-R, Hybrid-R. under basal conditions (undifferentiated state), but only 15% after differentiation with dexamethasone. In these models, we found that the two IR isoforms have a similar ability to form Although the precise role of the two IR isoforms is not entirely hybrids with the IGF-IR because Hybrid-R content, measured clear, this issue has become relevant following recent evidence by a specific ELISA, was very close to the value predicted that the relative abundance of IR isoforms is tightly regulated according to the random assembly model on the basis of the cell by tissue-specific factors, stage of development, and cell differ- content of IRs and IGF-IRs. entiation (24, 25, 29). IR-A is the predominant isoform in fetal We first studied ligand binding and observed that the two tissues; binds IGF-II with high affinity (26); and mediates fetal Hybrid-R types bind ligands with different affinity. Immuno- growth in response to IGF-II, as also suggested by genetic purified Hybrid-Rs have a high affinity for IGF-I (ED 2.5 studies carried out in transgenic mice (48, 49). Moreover, when nM IGF-I), bind IGF-II with 6-fold lower affinity, and do not cells transform and become malignant, dedifferentiation is of- appreciably bind insulin. Accordingly, Hybrid-Rs are acti- ten associated with an increased IR-A relative abundance, pro- vated by IGF-I and to a lesser extent by IGF-II and are not by viding a selective growth advantage to malignant cells via an insulin. In contrast, immunopurified Hybrid-Rs have a higher autocrine or paracrine loop with locally produced IGF-II (27, affinity for IGF-I (ED 0.3 nM IGF-I) compared with Hybrid- 28). IR-B is the predominant IR isoform in normal adult tissues Rs and bind IGF-II with a similar affinity (ED 0.6 nM that are major target tissues for the metabolic effects of insulin IGF-II) and insulin with a lower affinity (ED 3.7 nM insu- (adipose tissue, liver, and muscle) (24, 25). lin), but still in the physiological range. In agreement with In this study, we have demonstrated that each IR isoform binding data, Hybrid-Rs can be activated by both IGFs and affects Hybrid-R biology by using three different models: trans- also by insulin. fected R mouse fibroblasts, undifferentiated and differenti- We then studied post-receptor signaling and, more specifi- ated HepG2 human hepatoblastoma cells, and a panel of hu- cally, whether insulin can induce IGF-IR -subunit phospho- man cell lines with different relative abundance of the two IR rylation in intact cells expressing Hybrid-Rs. As expected from isoforms. R mouse fibroblasts were transfected to coexpress the binding data, exposure to insulin caused IGF-IR -subunit the IGF-IR and either IR-A or IR-B to obtain cells containing phosphorylation in cells expressing Hybrid-Rs , but not in cells IR Isoforms Affect Insulin/IGF-I Hybrid Receptor Properties 39693 FIG.8. ERK1/2 and Akt activation by insulin, IGF-I, and IGF-II in trans- fected R fibroblasts expressing only IR-A(R IR-A) or IR-B(R IR-B and in R fibroblasts expressing only IGF- IRs (R ). Serum-starved cells were ex- posed to 10 nM insulin, IGF-I, or IGF-II. A, ERK1/2 activation. Upper panel, anti- phospho-ERK antibody blot showing re- sults representative of three separate ex- periments; middle panel, reblotting with anti-ERK antibody; lower panel, densito- metric reading (phospho-ERK/total ERK) representing means S.E. of three sepa- rate experiments. B, Akt activation. Up- per panel, anti-phospho-Akt antibody blot showing results representative of three separate experiments; middle panel, re- blotting with anti-Akt antibody; lower panel, densitometric reading (phospho- Akt/total Akt) representing means S.E. of three separate experiments. FIG.9. Proliferation in HepG2 cells transfected with either IR-A or IR-B cDNA in response to insulin, IGF-I, or IGF-II. A, proliferation was measured by scoring BrdUrd (BrdU) incorporation in GFP-positive cells under an immunofluo- rescence microscope. B, bars indicate cell proliferation over basal levels in response to insulin, IGF-I, or IGF-II in cells trans- fected with an empty vector (E.V.) or IR-A or IR-B cDNA. Values are means S.D. of three experiments performed in tripli- cate and were calculated as described un- der “Experimental Procedures.” expressing Hybrid-Rs . Although the -subunits of the IR and (6 –13) are the consequence of the different activation of intra- IGF-IR share 80% homology, differences exist in the recruit- cellular mediators. CrkII is an adapter protein consisting pri- ment of intracellular mediators and the biological effects elic- marily of SH2 and SH3 domains; is a specific substrate of the ited by the two receptors: more pronounced metabolic effects IGF-IR (9, 42, 43); and mediates certain protein-protein inter- follow activation of the IR, whereas more pronounced mito- actions involved in signaling pathways that lead to cytoskeletal genic, anti-apoptotic, and transforming effects follow activation rearrangement, cell growth, differentiation, apoptosis, and of the IGF-IR (1–13). These differences in biological effects transformation (41). We found here that CrkII is also a sub- 39694 IR Isoforms Affect Insulin/IGF-I Hybrid Receptor Properties This study indicates for the first time that regulation of IR isoform expression has important implications in both insulin and IGF signaling. In cells predominantly expressing IR-A (and coexpressing the IGF-IR), the IGF-IR intracellular cas- cade may be activated in response to insulin and IGFs via Hybrid-R activation. In contrast, in cells predominantly ex- pressing IR-B (as most differentiated cells do), insulin will activate only the typical IR signaling pathway, whereas the response to IGFs will mainly occur via typical IGF-IRs because Hybrid-Rs have a reduced affinity for IGFs and because insu- lin, at physiological concentrations, will not bind. A better understanding of the molecular mechanisms regulating the alternative splicing process of the IR gene will therefore pro- vide important information for the regulation of cell metabo- lism and proliferation and other biological functions. Acknowledgments—We thank Drs. I. D. Goldfine and K. Siddle for kindly providing anti-IR and anti-IGF-IR antibodies. We warmly thank Dr. R. Baserga for helpful discussion and critical reading of the manuscript. REFERENCES 1. Avruch, J. 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Journal
Journal of Biological Chemistry – Unpaywall
Published: Oct 1, 2002