Báo cáo Y học: Characteristics of binding of insulin-like growth factor (IGF)-I and IGF-II analogues to the type 1 IGF receptor determined by BIAcore analysis pptx

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Báo cáo Y học: Characteristics of binding of insulin-like growth factor (IGF)-I and IGF-II analogues to the type 1 IGF receptor determined by BIAcore analysis pptx

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Characteristics of binding of insulin-like growth factor (IGF)-I and IGF-II analogues to the type 1 IGF receptor determined by BIAcore analysis Correlation of binding af®nity with ability to prevent apoptosis Briony E. Forbes 1, *, Perry J. Hart®eld 2, *, Kerrie A. McNeil 1 , Kathy H. Surinya 1, ², Steven J. Milner 3 , Leah J. Cosgrove 4 and John C. Wallace 1 1 Department of Molecular Biosciences, Adelaide University, SA Australia; 2 School of Biomedical Sciences, Faculty of Medical and Health Sciences, University of Newcastle, Callaghan, NSW, Australia; 3 GroPep Ltd, Thebarton, SA, Australia; 4 CSIRO Health Sciences and Nutrition, Adelaide, SA, Australia Insulin-like growth factor ( IGF) binding to the type 1 IGF receptor (IGF1R) e licits mitogenic eects, promotion of dierentiation and protection f rom apoptosis. This study has systematically measured IGF1R binding anities of IGF-I, IGF-II and 14 IGF analogues to a recombinant high-anity form of the IGF1R using BIAcore technology. The analogues assessed could be divided into two groups: (a) those d esigned t o i nvestigate b inding of I GF-binding protein, which exhibited IGF1R-binding anities similar to those of IGF-I or IGF-II; (b) those generated to probe IGF1R i nteractions with greatly redu ced IGF1R-binding anities. The relative binding anities of IGF-I analogues and IGF-I for the IGF1R d etermined by B IAcore analysis agreed closely with existing data from receptor-binding assays using cells or tissue membranes, demonstrating that BIAcore technology is a powerful tool for measuring anities of IGFs for IGF1R. In parallel studies, IGF1R- binding anities were related to ability to protect against serum withdrawal-induced apoptosis in three dierent assays including Hoechst 33258 staining, cell survival, and DNA fragmentation assays using the rat pheochromo- cytoma cel l line, PC12. In this model s ystem, IGF-I a nd IGF-II at low nanomolar concentrations are able t o p revent apoptosis completely. We conclude that ability to protect against apoptosis is directly related t o ability t o bind t he IGF1R. Keywords: apoptosis; BIAcore analysis; insulin-like growth factor (IGF); type 1 IGF receptor. Insulin-like growth factors-I and -II ( IGF-I and IGF-II) are small peptides, which are able to promote cell proliferation, differentiation a nd survival resulting predominantly from interactions with the type 1 IGF receptor (IGF1R). Prevention of apoptosis by an activated IGF1R plays a major role in the survival of many cell types, including neurons and haemopoietic cells after interleukin-3 with- drawal (reviewed by B aserga et al. [1]). Signi®cantly, t he ability of IGFs to protect against apoptosis has been implicated in potentiation of aberrant growth in disease situations such as cancer, where abnormally high levels of circulating IGFs are evident [2]. The IGF1R is a ubiquitously expressed transmembrane homodimeric tyrosine kinase receptor [3,4]. It consists of two extracellular ligand-binding a domains and t wo transmem- brane b domains. The tyrosine kinase domain i s l ocated within the cytoplasmic region and is responsible for s ignal- ling events initiated by ligand activation via the extracellular domain. Upon receptor a utop hosphorylation, two m ajor downstream pathways are activated, namely the Ras/Raf/ mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways. In s ome situations, a third pathway involving 14.3.3 protein modulation of Raf activation is also involved in IGF1R signalling [5,6]. Anti-apoptotic activity of the IGF1R requires activation of at least two of these pathways [5], with the phosphatidylinositol 3-kinase/Akt pathway being perhaps the most important [1,7]. The IGF1R and insulin receptor (IR) are structurally related [3], as are the IGFs and insulin [8±10]. I t is not surprising therefore that IGF-I binds with high af®nity to the IGF1R and also binds the IR, but with 100-fold lower af®nity. IGF-I also binds the structurally unrelated type 2 IGF receptor (IGF2R) with low a f®nity [11]. IGF-II binds IGF2R and an isoform of the IR with h igh af®nity and also binds the IGF1R, albeit with  twofold to threefold lower af®nity th an IGF-I [11]. In ad dition, both IGFs bind t o six Correspondence to B. E. Forbes, Department of Molecular Biosciences, Adelaide University, SA 5005, Australia. Fax: + 61 8 8303 4348, Tel.: + 61 8 8303 5581, E-mail: briony.forbes@adelaide.edu.au Abbreviations: IGF, insulin-like growth factor; IGF1R, type 1 I GF receptor; rhIGF1R, recombinant human high-anity IGF1R; IR, insulin receptor; IGF2R, type 2 IGF recept or; IGFBP, IGF-binding protein; NGF, nerve growth factor; HBS, Hepes-buered saline; DMEM, Dulbecco's modi®ed Eagle's medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium. *Note: these authors contributed equally to this work. Present address: Department of Clinical Biochemistry, Addenbrooke's Hospital, University of Cambridge, UK. (Received 3 August 2001, revised 7 December 2001, accepted 11 December 2001) Eur. J. Biochem. 269, 961±968 (2002) Ó FEBS 2002 high-af®nity binding proteins (IGFBPs) with  10-fold higher af®nity than their binding to IGF receptors. IGFBPs thereby in¯uence t he availability o f IGF to bind to IGF receptors [1 2]. Over the past 14 years, a large number of IGF analogues have been designed (initially by comparing IGF-I and insulin sequences) a nd recombinantly expressed as tools to investigate which residues are important for interactions with IGF1R, IGF2R and the IGFBP family. Initially, key residues involved in IGF1R binding were identi®ed in the IGF-I B-domain (Tyr24) [13,14] and C-domain ( Tyr31) [14], whereas residues important for b inding of IGF2R (Phe49, Arg50, Ser51 [15]) and IGFBP (Glu3, Thr4, Gln15, Phe16 [16]) were located in the A and B domains, respectively. Further mutations have led to the general consensus that the B and C domains contain residues most involved in IGF1R binding, t he A domain represents the IGF2R-binding site, while the B domain and the ® rst A domain helix contain the major determinants for IGFBP binding. In this study we show, using IGF-I, IGF-II and 14 IGF analogues, that potency in an tiapoptotic activity is directly related to ability to b ind to the IGF1R. Previously we have expressed a recombinant f orm o f the human IGF1R by fusing the cDNA encoding the receptor ectodomain (resi- dues 1±944) to the cDNA of the mouse Fc domain o f immunoglobulin (encoding residues of the CH2 and CH3 domains) [17]. Expression in mammalian cells y ielded a homodimer, with the Fc domain replacing the transmem- brane domain of the high-af®nity IGF receptor. We determined that the af®nity of the r ecombinant receptor for IGF was comparable to that of native type 1 IGF receptors as measured using conventional ELISA-based and whole cell binding studies. With this valuable tool we have now assessed IGF±receptor interaction s using the BIAcore. With parallel studies, we h ave determined the a bilities of IGF and IGF analogue to prevent serum withdrawal- induced apoptosis in the rat pheochromocytoma PC12 cell line, a w ell-established model of neuronal apoptosis [18±20]. This study clearly i ndicates that the binding af®nity of IGF analogues to IGF1R correlates directly with their ability to prevent apoptosis. MATERIALS AND METHODS Materials IGF-I, IGF-II and IGF analogues (Table 1 ) were either obtained from GroPep Pty. L td. (Adelaide, South Austra- lia) or p rovided by S . J. Milner as a member o f the CRC for Tissue Growth and Repair (includes L ong IGF-I a nd Gly3- IGF-I). Nerve growth factor (NGF) was acquired from Alomone (Jerusalem, Israel). BIAcore C M5 sensor chips, amine coupling kits and surfactant A were from BIAcore Inc. (Melbourne, A ustralia). Hepes-buffered s aline (HBS) for BIAcore analysis contained 10 m M Hepes, 150 m M NaCl, 3.4 m M EDTA, pH 7.4, and 0.005% surfactant A. Hoechst 33258 was from Calbiochem. Preparation of recombinant high-af®nity type 1 IGF receptor (rhIGF1R) rhIGF1R was expressed in BHK-21 neonatal hamster kidney ®broblasts from a cDNA clone encoding the ectodomain of t he IGF1R fused to the mouse Fc domain of immunoglobulin (K. H. Surinya, B. E. Forbes, F. Occhidoro, K. A. McNeil, J. C. Wallace & L. J. Cosgrove, unpublished r esults). rhIGF1R was puri®ed from cell culture medium using an a nti-mouse IgG af®nity column and was dialysed against HBS before use i n further experiments. Table 1. Kinetic constants obtained from B IAcore analysis of binding o f IGF-I, IGF-II and mutant IGF analogue to rhIGF1R. Data wer e analy sed using BIAevaluation software 3.0 and ®tted to a Langmuir 1 : 1 binding model as outlined in Materials and methods. T he dissociation constant (K d ) was determined from th e calculation of k d /k a ,wherek a is t he association rate and k d is the dissociation rate. Relative K d is equal to K d of IGF-I/K d of IGF analogu e. Dashes ind icate d ata inap propriate f or assessing association and dissociation rates. N o d etectable bind ing (ND) was seen with Ala31-IGF-I (800 n M ), Leu60-IGF-I (800 n M ), Leu27-IGF-II (1 l M ), or des-(1±6,10)-Leu27-IGF-II (1 l M ). k a ´ 10 5 (1/ M ás) k d ´ 10 )3 (1/s) K d ´ 10 )9 ( M ) Relative K d IGF-I and analogues IGF-I 2.70 1.16 4.29 1.00 Long 2.52 1.07 4.25 1.01 Arg3 3.60 0.88 2.44 1.76 Gly3 3.65 1.90 5.20 0.83 des-(1±3) 2.89 0.94 3.25 1.32 Leu24 ± ± 88.6 0.05 des-(2,3)-Leu24 ± ± 161.0 0.03 Ala31 ± ± 53.4 0.08 des-(2,3)-Ala31 ± ± 48.2 0.09 Leu60 ± ± 945.0 0.005 Ala31Leu60 ± ± ND ND IGF-II and analogues IGF-II 1.00 2.35 23.50 1.000 Des-(1±6) 0.51 2.90 56.86 0.41 Arg6 0.97 2.25 23.20 1.01 Leu27 ± ± ND ND Des-(1±6,10)-Leu27 ± ± ND ND 962 B. E. Forbes et al.(Eur. J. Biochem. 269) Ó FEBS 2002 BIAcore analysis BIAcore sensor chip preparation. Coupling of rhIGF1R to CM5 BIAsensor chip via amine group linkage was achieved using standard coupling procedures [21]. Brie¯y, CM5 sensor chips were activated by injecting 35 lL N-ethyl- N¢-[(dimethylamino)propyl]carbodiimide/ N-hydroxysucci- nimide a t 5 lLámin )1 . Subsequently, rhIGF1R was coupled to the CM5 se nsor chip by injecting 3 5 lLrhIGF1R(4lg) in 10 m M sodium acetate, pH 4.5, at 5 lLámin )1 . Unreacted groups were inactivated w ith 35 lL1 M ethanolamine/HCl, pH 8.5. A sensor surface with 8±10 000 response units of coupled rhIGF1R would routinely result in a response of  100 response units with 200 n M IGF-I. Generation of kinetic binding data. Kinetic studies with a range of analyte concentrations were determined at a ¯ow rate of 30 lLámin )1 to minimize mass transfer effects, and by allowing 300 s for association and 900 s for dissociation. In the case of IGF-I and related analogues, the concentra- tions used were 12.5, 25, 50, 100, and 200 n M ,andfor IGF-II and r elated analogues c oncentrations of 25, 50, 100, 200, and 400 n M were utilized. The rhIGF1R-coated biosensor surface was regenerated with 0.3 M sodium citrate/0.4 M NaCl, pH 4.5. Kinetic data were analysed with BIAevaluation 3.0 software. For each binding curve, the response obtained u sing control surfaces (n o protein coupled) was subtracted. Both IGF-I and IGF-II binding ®tted a 1 : 1 Langmuir binding model using global ®tting. This model describes a simple reversible interaction of two molecules in a 1 : 1 complex. Goodness of ® t measured as a v 2 value w as not greater than 5 for rhIGF1R b inding. All binding experiments were repeated at least in duplicate and biosensor chips coupled at different times yielded surfaces with identical binding af®nities. T he binding af®nities of IGF-I and IGF-II to rhIGF1R (K d  4.45 and 2 3 n M , respectively) were comparable to the binding af®nities (K d  3.5 a nd 20 n M , respectively) repor ted i n t he study by Jansson et al. [ 22], who used a homodimer of the IGF1R ectodomain fused to the IgG-binding Z domain in BIAcore experiments. Apoptosis assays Cell culture. The rat PC12 pheochromocytoma cell line was generously provided by R. Rush (Flinders Medical Centre, Ad elaide, South Australia). Stock cultures were maintained in Dulbecco's modi®ed Eagle's medium (DMEM; high glucose) supplemented with 10% horse serum, 5% fetal bovine serum, 100 UámL )1 penicillin and 100 lgámL )1 streptomycin. PC12 cells were detached by trituration, resuspended in complete DMEM, and plated on poly( L -ornithine)-coated plastic culture dishes for 18±24 h before further treatments. Determination of levels of apoptosis by ¯uorescence microscopy Levels of apoptosis were quantitated after labelling the cells with the nuclear stain Hoechst 33258 and visualization by ¯uorescence microscopy as described previously [23]. Brie¯y, PC12 cells were plated in six-well plastic culture dishes (4 ´ 10 4 cells per well), washed in seru m-free DMEM and resuspended in DMEM in either the presence or absence (control) of IGF-I, IGF-II or IGF a nalogues at various concentrations. Treatments were for 24 h and the cells were subsequently stained with Hoechst 33258 (5 lgámL )1 ). Nuclei that were condensed or fragmented were scored as apoptotic. Cell survival assays. PC12 cells were plated in 96-well tissue culture plates (1 ´ 10 4 cells per well) in serum -free DMEM either in the absence or the presence of IGF-I, I GF- II or IGF analogues at the concentrations indicated. Cell survival was determined after 24 h using a commercial [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium (MTT)-based] assay (Cell Titer, 96Ò, Aqueous One Solution Assay; Promega). Absorbances were measured at 490 nm using a microtitre plate reader (Titertek Multiskan MCC). DNA fragmentation ELISA. PC12 ce lls (1 ´ 10 5 )were treated for 24 h in serum-free DMEM e ither in the absence or the presence of IGF-I, IGF-II, or IGF analogues at the concentrations indicated, scraped from the dish, centrifuged (400 g, 10 min), washed once, lysed (30 min, 4 °C). Cyto- plasmic f ractions were prepared from lysates by centrifuga- tion (20 000 g, 1 0 min). The cell death detection ELISA (Roche Molecular Biochemicals), which quantitatively determines levels of cytoplasmic histon e-associated D NA fragments generated during apoptotic DNA fragmentation, was performed according to the manufacturer's i nstruc- tions, and absorbances were measured at 405 nm. RESULTS BIAcore analysis of rhIGF1R binding by IGF analogues High af®nity binding of IGF-I, IGF-II and their analogues to rhIGF1R was measured by BIAcore analysis (Figs 1 and 2), and relative binding af®nities were determined (Table 1). Arg3-IGF-I, des-(1±3)-IGF-I, Gly3-IGF-I and Long IGF-I (with an N-terminal fusion partner consisting of 13 additional residues of porcine growth hormone [24]) exhibited rhIGF1R binding af®nities similar to IGF-I (no more than 1.5-fold difference in K d , see Fig. 1A±E and Table 1). These analogues were d esigned speci®cally to disrupt IGFBP binding and were therefore expected to maintain rhIGF1R-binding af®nities similar to IGF-I. Long Gly3-IGF-I and Long Arg3-IGF-I also had similar rhIGF1R-binding af®nities to IGF-I (data not shown). The a nalogues Leu24-IGF-I, des-(2,3)-Leu24-IGF-I, Ala31-IGF-I, des-(2,3)-Ala31-IGF-I and Leu60IGF-I were made to probe interactions with the IGF1R. These analogues bound with much lower af®nities to the rhIGF1R than IGF-I (Fig. 1F±H and Table 1). The association and dissociation rates of these analogues were too rapid to be ac curately measured, a nd therefore their s teady-state a f®nities were determined. T here was a 200-fold difference in af®nity between Leu60-IGF-I and IGF-I for binding to the rhIGF1R. In addition, the effect of the double substitution of Ala31Leu60-IGF-I resulted in no binding being detected, which s uggests its af®nity for t he rhIGF1R i s below the d etection limit of the BIAcore ( 10 )5 M ). This analogue has previously been shown to have a very low af®nity for IGF1R on Ó FEBS 2002 IGF analogue binding to IGF1R and cell survival (Eur. J. Biochem. 269) 963 human placental membranes ( >500-fold less than IGF-I [14]). There w as an approximately f ourfold difference in the af®nities of IGF-I (4.45 n M ) and IGF-II (17.8 n M )forthe rhIGF1R (Figs 1A and 2A, Table 1 ). Substitu tion of Glu at position 6 to A rg (Arg6-IGF-II) or deletion of the ®rst six amino acids of IGF-II (des-(1±6)-IGF-II), which are equivalent analogues to Arg3-IGF-I and des-(1±3)-IGF-I, had minimal effect on rhIGF1R-binding af®nity compared with native IGF-II (Fig. 2B,C and Table 1). However, replacement of Tyr27 of IGF-II with Leu results in comprehensive loss of af®nity for IGF1R [25]. The af®ni- ties of Leu27-IGF-II (equivalent analogue to Leu24-IGF-I) and des-(1±6,10)-Leu27-IGF-II we re too low to be detected in this study using BIAcore analysis (Table 1). Prevention of PC12 cell apoptosis by IGF analogues The effects of IGF analogues on the prevention of PC12 cell apoptosis were determined using three separate assays: ¯uorescent staining (Hoechst 33258) of nuclei, MTT-based cell survival assays and an apoptotic DNA fragmentation ELISA. PC12 cells cultured in complete serum are fully viable and actively proliferate. Deprivation of serum (24 h) induces 47.5% apoptosis (Fig. 3 C), and serum deprivation- induced apoptosis is completely prevented by NGF (100 ngámL )1 ,Fig.3C).IGF-I(1n M ) essentially prevented apoptosis induced by serum deprivation (Fig. 3A) and reduced the levels of apoptosis to an equivalent degree as NGF (100 ngámL )1 , < 5% apoptotic cells). IGF-II also strongly prevented apoptosis, reducing the levels of apop- tosisto8%with1n M IGF-II and completely preventing 20 100 180 50 450 850 A Response (RU) 20 100 180 50 450 850 B 20 100 180 50 450 850 20 100 180 50 450 850 D Time (s) C Time (s) 20 100 180 50 450 850 E 10 70 130 50 450 850 F 10 30 50 50 450 850 G 10 30 50 50 450 850 H 20 100 180 50 450 850 A Response (RU) 20 100 180 50 450 850 B 20 100 180 50 450 850 20 100 180 50 450 850 20 100 180 50 450 850 D Time (s) C Time (s) 20 100 180 50 450 850 E 10 70 130 50 450 850 10 70 130 50 450 850 F 10 30 50 50 450 850 G 10 30 50 50 450 850 H Fig. 1. BIAcore analysis of IGF-I and IGF-I analogue binding to high- anity recombinant IGF1R. Binding of IGF-I (A), des-(1±3)-IGF-I (B), Arg3-IGF -I (C), Gly3-IGF-I (D), Lon g IGF-I (E), Ala31-IGF-I (F), Leu 24-IGF-I (G) and L eu60-IGF-I (H) to rhIGF1R was mea- sured by using concentrations of 12.5, 25, 50, 100 and 200 n M .Asso- ciation w as measured for 300 s (starting after 100 s) and dissociation was m easured for 900 s in the presen ce o f HBS alon e. Receptor binding is expressed in response units (RU). These results are r epre- sentative of at l east du plicate experiments performed on dierent sensor chip surfaces. Binding of des-(2,3)-Leu24 and des-(2,3)-Ala31- IGF-I is not shown but kinetic analysis i s summarized in Table 1. Time (s) 0 40 80 0 400 800 1200 Response (RU) 0 40 80 120 400 800 12000 A 0 40 80 0 400 800 1200 B C Time (s) 0 40 80 0 400 800 1200 Response (RU) 0 40 80 120 400 800 12000 A 0 40 80 0 400 800 1200 B C Fig. 2. BIAcore analysis of IGF-II and IGF-II analogue binding to high- anity recombinant IGF1R. Bin ding of IGF-II (A), d es-(1±6)-IGF-II (B) and Arg6-IGF-II (C) to rhIGF1R measured by BIAcore analysis using concentrations of 25, 50, 100, 200 and 400 n M . Association was measured for 300 s (starting after 100 s) and dissociation was mea- sured for 900 s in the presence of HBS alone. Receptor binding is expressed in response units (RU). These results are representative of at le ast duplicate experiments performed on dierent sen sor chip surfaces . 964 B. E. Forbes et al.(Eur. J. Biochem. 269) Ó FEBS 2002 apoptosis at 10 n M IGF-II(Fig. 3C).LongIGF-I,des-(1±3)- IGF-I, Arg3-IGF-I and Gly3-IGF-I e ssentially prevented serum deprivation-induced PC12 cell a poptosis at concen- trations of 1 n M (Fig. 3A). In addition, the combination of the presence of the 13-amino acid N-terminal fusion p artner with charge reversal/neutralization (Long Arg3 and Long Gly3) did not alter the ability of the analogues to prevent apoptosis (data not shown). The second group of analogues were designed to probe for the IGF1R-binding site. Leu24-IGF-I and Ala31-IGF-I were not able to prevent apoptosis at concentrations of 1n M , where levels of apoptosis between 25 and 30% were measured, but results indicated that these analogues were able to fully protect against apoptosis at concentrations of 10 n M (Fig. 3B). The truncated forms, des-(2,3)-Leu24- IGF-I and des-(2,3)-Ala31-IGF-I, behaved in a similar manner to their full-length counterparts (Fig. 3). Interest- ingly, Leu60-IGF-I only prevented apoptosis at 100 n M , and high levels of apoptosis were measured at concentra- tions of 1 n M and 10 n M . Ala31Leu60-IGF-I failed to prevent serum deprivation-induced apoptosis at 1 n M and 10 n M and only reduced the levels o f apoptosis by < 50% at 100 n M (Fig. 3B). The IGF-II analogues Arg6-IGF-II a nd des-(1±6)-IGF-II were essentially as effective as native IGF-II in preventing serum deprivation-induced apoptosis with complete protec- tion at 10 n M (Fig. 3C). In contrast, Leu27-IGF-II only provided full protection at 100 n M . Interestingly, we cannot detect binding of Leu27-IGF-II to the I GF1R (Table 1). It is possible that there is a very w eak a f®nity for the IGF1R that results in a poor ability t o protect against apoptosis although binding was not detected using BIAcore or conventional cell-based assays. Des-(1±6,10)-Leu27-IGF-II was unable t o signi®cantly protect at all the concentrations tested and only reduced the levels of apoptosis to  30% at 100 n M (Fig. 3C). D es-(1±6,10)-Leu27-IGF-II does not bind IGFBPs but binds the IGF2R with equal af®nity to Leu27 (our unpublished data). We can conclud e that deletion of Gly at position 10 must result in an even poorer interaction with t he IGF1R leading to the inability of des- (1±6,10)-Leu27-IGF-II to protect PC12 cells from serum starvation-induced apoptosis. The present results con®rmed previous investigations [18,19] showing that PC12 cell apoptosis (induced by 24 h serum-free conditions) was completely prevented and that cell viability was fully maintained by IGF-I and IGF-II at concentrations of 1 n M and 10 n M , respectively (Figs 3 a nd 4). Indeed, I GF-I wa s a ble t o promote survival a t levels > 100%, indicating that IGF-I not only promotes cell survival but also stimulates a d egree o f cell proliferation even in serum-free conditions. Long I GF-I, des-(1±3)-IGF-I, Arg3-IGF-I and Gly3-IGF-I (all at 1 n M )promotedPC12 cellsurvivaltothesamedegreeasnativeIGF-I(Fig. 4A).In contrast, Leu24-IGF-I, d es-(2,3)-Leu24-IGF-I, Leu60-IGF-I and Ala31Leu60-IGF-I (all at 1 n M ) failed to prevent serum deprivation-induced cell death, and Ala31-IGF-I and des- (2,3)-Ala31-IGF-I e xhibited a s mall survival-promoting effect (Fig. 4C). IGF-II (10 n M ) completely prevented serum deprivation-induced cell death (Fig. 4E) and Arg6- IGF-II and des(1±6)-IGF-II were essentially equipotent to IGF-II. Leu27-IGF-II and d es-(1±6,10)-Leu27-IGF-II did not exhibit any survival-promoting effects at 10 n M (Fig. 4E). Finally, l evels o f PC12 cell a pop tosis w ere quantitated using a DNA fragmentation ELISA. I n the absence of serum, high levels of DNA fragmentation were detected, and IGF-I (1 n M ) prevented apoptotic DNA fragmentation (Fig. 4B). Long IGF-I, des-(1±3)-IGF-I, Arg3-IGF-I and Gly3-IGF-I ( all at 1 n M ) similarly prevented DNA fragmentation (Fig. 4B), whereas Ala31-IGF-I and des- (2,3)-Ala31-IGF-I slightly reduced the levels of DNA fragmentation (Fig. 4D). Leu24-IGF-I, des-(2,3)-Leu24- IGF-I, Leu60-IGF-I and Ala31Leu60-IGF-I (all at 1 n M ) had no effect and failed to reduce the levels of DNA fragmentation induced by serum deprivation (Fig. 4D). IGF-II (10 n M ) prevented DNA fragmentation and Arg6- IGF-II and des(1±6)-IGF-II showed comparable protective Fig. 3. Concentration-dependent eects of IGF-I, IGF-II and mutant IGF analogues on the prevention of serum deprivation-induced PC12 cell apoptosis measured by ¯uorescence microscopy analysis of nuclear morphology. PC12 cells were incubated in serum-free (SF) me dium for 24 h in either the presence or absence of IGF-I, IGF-II or mutant IGF analogues at the indicated c oncentrations. (A) E ects of IGF-I ana- logues that bind IGF-binding proteins with low ani ty; (B) eects of IGF-I analogues with substitutions that alter binding to IGF1R; (C) eects of IGF-II analogues t hat either b ind with low anity to IGF-binding pro teins [ Arg6 and d es-(1±6)] a nd/or bin d with low anity to IGF1R [Leu27 and d es-(1±6,10)-Leu27]. In addition, th e levels of apoptosis induced by serum deprivation (24 h) an d its pre- vention by NGF (100 ngámL )1 ) are shown in (C). PC12 cells were stained with Hoechst 33258 (5 lgámL )1 ) and le vels of apoptosis were quantitatively determined by ¯uorescence microscopy a s described in Materials and methods. The number of apoptotic cells is expressed as a percentage of all cells counted (% apoptosis), and data are presented as means  SEM from at least four independent experiments. Ó FEBS 2002 IGF analogue binding to IGF1R and cell survival (Eur. J. Biochem. 269) 965 effects, but Leu27-IGF-II and des-(1±6,10)-Leu27-IGF-II were completely unable to p revent apoptotic DNA frag- mentation (Fig. 4F). DISCUSSION We have made a direct comparison of rhIGF1R binding by IGF-I, IG F-II and 1 4 analogues using BIAcore analysis. IGF1R binding by all of these analogues has not previously been compared in a single binding assay. Analogues included in this study were originally designed to analyse e ither IGF1R [14,22] or IGFBP binding [24,26,27], a nd mutations consist of amino-acid substitutions (e.g. Tyr24 to Leu24 IGF-I), charge reversals (Glu3 to Arg3 I GF-I) or a mino-acid deletions (e.g. des-(1±3)-IGF-I). We have con®rmed that Tyr24, Tyr31 and Tyr60 o f IGF-I play a s igni®cant role in t he interaction with IGF1R, whereas the ®rst three amino acids of IGF-I, and in p articular Glu3, ar e not critical residues for binding to IGF1R. Corresponding residues of IGF-II (namely Tyr27 and residues 1±6 or Glu6) h ave similar importance in the interaction between IGF-II and IGF1R. When comparing the receptor's binding af®nities for the IGF analogues with its af®nity for IGF-I or IGF-II, respectively, our analyses generally con®rm t he r esults of conventional r eceptor binding assays. For example , there was a reported 1.36-fo ld difference in IGF1R binding between des-(1±3)-IGF-I and IGF-I as measured in L6 myoblast competition binding assays [24,26,28] and a 1.48- fold higher af®nity for r hIGF1R determined by BIAcore analysis in this study. Similarities were also seen between our present ®ndings and reported IGF1R-binding af®nities for Gly3-IGF-I, Arg3-IGF-I [24] and the ÔLongÕ forms of these analogues [26]. In addition, human placental membrane IGF1Rs had an 18-fold lower af®nity for Leu24-IGF-I than native IGF-I [14], and a similar reduction (20-fold) in rhIGF1R-binding af®nity was measured in our experi- ments. Therefore, this study demonstrates that BIAcore analysis is an excellent technique for the assessment of relative binding af®nities of IGF analogues and IGF-I for the IGF1R. The IGF1R-binding af®nities measured in the present study are comparable to the af®nities reported by J ansson et al . [22] using an immobilized recombinant IGF1R IgG- binding Z domain f usion protein in BIAcore experiments. The af®nities are, however,  10-fold lower than af®nities calculated from binding to soluble IGF1R preparations [29]. Coupling via amine g roups (predominantly through the N-terminus of the F c domain under these conditions) may be limiting interactions between rhIGF1R and IGF or hindering the ¯exibility o f r hIGF1R. Differences in relative binding af®nities between our BIAcore analyses a nd cell- based or membrane-based receptor-binding assays for Ala31-IGF-I and Leu60-IGF [14] may also re¯ect the Fig. 4. Comparative eects of IGF-I, I GF-II and mutant IGF analogues on PC12 measured by MTT-based cell survival assay and apoptotic DNA fragmentation. PC12 cells were incubated in serum-free medium for 24 h in either the presence or absence of IGF-I (1 n M ), IGF-II (10 n M ), IGF-I analogues (1 n M )orIGF-IIanalogues(10n M ). The eects of IGF-I analogues that b ind IGF-bindin g proteins with low anity (A, B), IGF a nalogues with substitution s that alter b inding to IGF1R (C, D) and IGF-II analogues (E, F) were determine d on cell survival (A, C, E) an d apoptotic DNA fragmentation (B, D, F). I GF-I (1 n M ) and IGF-II (10 n M ) complet ely prevented serum d ep rivation - induced PC12 cell death and DNA fragmentation. Survival assays (MTT-based assay) and DNA fragmentation ELISAs were performed as described in Materials and methods, and the d ata are presented as means  SD from at least three independent experiments. Fig. 5. Correlation b etween bind ing anities of IGF-I anal ogues t o recombinant human IGF1R and prevention of apoptosis by IGF-I ana- logues. The dissociation constants (K d ) for binding of IGF-I and IGF-I analogues to rhIGF1R were plotted against levels of apoptosis (%) determined by ¯u orescence m icroscopy (Ho echst 33258 staining) in the presence of 1 n M IGF-I a nd IGF-I analogues. Symbol representations are I GF-I (d), des-(1±3) (s), Arg3 (j), Gly3 (h), des-(2,3)-Ala31(m), Long (n), Leu24 (.), Ala31(,), Leu60 (r), des-(2,3)-Leu24 (e). 966 B. E. Forbes et al.(Eur. J. Biochem. 269) Ó FEBS 2002 differences in the assay systems used (i.e. immobilized rhIGF1R vs. cell membrane-bound receptor), with the greatest effect being on interactions involving residue 60 of IGF-I. We are currently introducing a biotinylated spacer arm at the C-terminus of rhIGF1R, which will allow a guaranteed homogeneously coupled chip and may perhaps improve ¯exibility and access to the receptor. Having established the re lative binding af®nities of IGF-I, IGF-II and the 14 analogues for rhIGF1R, we related these to their abilities to prevent apoptosis. IGFs have powerful antiapoptotic effects and promote survival in a d iverse array of cell types throu gh activation o f IGF1R signalling (for reviews, see [1] and [ 7]). This study investigated the survival- promoting effects of IGF analogues on serum deprivation- induced apoptosis in the rat PC12 cell line, which has been used extensively as a model system to study the mechanism of neuronal cell s urvival and apoptosis [18±20,23]. I GF-I and IGF-II c ompletely prevented serum deprivation- induced PC12 cell apoptosis at con centrations of 1 n M and 1 0 n M , r espectively, which are similar levels t o those reported previously [18±20]. Importantly, PC12 cells have a limited capacity to produce and secrete IGFBPs, and existing evidence indicates that PC12 cells only synthesize very low levels of IGFBP-6 [30]. This fact further suggests that PC12 cells constitute a useful cell model for investiga- ting direct biological actions of IGFs through IGF1R signalling, as IGFBPs will not regulate or negatively i mpact IGF±IGF1R interactions. We have made a direct correla- tion between r eceptor binding af®nity (K d ) and ability to prevent apoptosis for IGF-I and the IGF-I analogues (Fig. 5). Essentially, the ability of IGF-I o r IGF-I analogues to prevent apoptosis correlated directly with their IGF1R- binding af®nities, and a strong correlation coef®cient (r 2  0.97) was obtained for this relationship (Fig. 5). Thus, those IGF-I mutants designed to investigate IGFBP binding [Long IGF-I, des-(1±3)-IGF-I, Arg3-IGF-I, Gly3- IGF-I, Arg6-IGF-II and des(1±6)IGF-II] have basically equal abilities to prevent apoptosis. Conversely, IGF-I analogues with d isrupted IGF1R binding have a corre- sponding loss in their ability to p revent apoptosis. Despite the signi®cant role of IGFs in antiapoptotic actions, assays measuring apoptosis have not traditionally been used to analyse biological activity of analogues. However, comparisons of receptor binding and biological activities, such as protein and DNA synthesis and protein degradation, have been made in the past and they generally support our conclusions. For example, analogues designed to probe for interactions between IGFs and I GFBPs [des-(1±3)-IGF-I, Arg3-IGF-I a nd Long Arg3-IGF-I, des-(1±6)-IGF-II, Arg6-IGF-II] were analysed in L6 myo- blast receptor-binding assays and inhibition of protein breakdown assays [26,27]. In these studies the ability to inhibit protein breakdown was directly related to receptor- binding af®nity. A lso, IGF analogues with perturbed receptor interactions (Leu24-IGF-I, Ala31-IGF-I, Leu60- IGF-I and combinations of these) exhibited r educed ability to stimulate DNA synthesis in L7 murine ®broblasts compared with IGF-I [14]. Interestingly, strong correlations between insulin or insulin mutant binding to the insulin receptor and metabolic potencies (glucose transport and lipogenesis) have also been made [31]. However, mitogenic potency of insulin analogues does not correlate as tightly with overall receptor af®nity but rather with the dissociation rate or occupancy time of receptor [31,32]. In the present study, the analysis of association and dissociation rates of the IGF analogues did not reveal distinct mechanisms of IGF1R activation l eading to different b iological activities (for example apoptosis vs. protein degradation). The mechanism of signalling resulting from IGF1R activation by the analogues tested above was not investi- gated. However, both the Akt (via I RS-1) and the MAP kinase (via Shc) pathways are involved in IGF-induced antiapoptotic signalling via the IGF1R in PC12 cells [20]. It would be interesting in the future to determine whether analogues differ in their abilities to activate these pathways. In summary, we have clearly demonstrated a direct correlation b etween the IGF1R-binding af®nity of IGF and IGF analogues and the prevention of ap optosis mediated through IGF1R signalling, suggesting that IGF1R-binding af®nity is the primary determinant when assessing the antiapoptotic potential of IGF analogues. ACKNOWLEDGEMENTS This work was supported by an Australian government Cooperative Research Centre grant. We thank Mr A dam Denley and Ms. Filomena Occhiodoro for technical assistance. In addition, we gratefully acknowledge Mr Geo Francis (GroPep Ltd) for his critical comments. REFERENCES 1. Baserga, R., R esnico, M., D'Ambrosio, C. & Valentinis, B. (1997) The role of the IGF-I receptor in apoptosis. Vitam. Horm. 53, 65±98. 2. Chan, J.M., Stampfer, M.J., Giovanucci, E., Gann, P.H., Ma, J., Wilkinson, P., Hennekens, C.H. & Pollak, M. 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