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

Characteristics of binding of insulin-like growth factor IGF-I and IGF-II analogues to the type 1 IGF receptor determined by BlAcore analysis Correlation of binding affinity with abili

Characteristics of binding of insulin-like growth factor (IGF)-I

and IGF-II analogues to the type 1 IGF receptor determined

by BlAcore analysis

Correlation of binding affinity with ability to prevent apoptosis

Briony E Forbes"*, Perry J Hartfield”*, Kerrie A McNeil’, Kathy H Surinya''t, Steven J Milner’,

Leah J Cosgrove’ and John C Wallace’

"Department of Molecular Biosciences, Adelaide University, SA Australia; 7School of Biomedical Sciences, Faculty of Medical and Health Sciences, University of Newcastle, Callaghan, NSW, Australia; *GroPep Ltd, Thebarton, SA, Australia;

4CSIRO Health Sciences and Nutrition, Adelaide, SA, Australia

Insulin-like growth factor (IGF) binding to the type | IGF

receptor (IGFIR) elicits mitogenic effects, promotion of

differentiation and protection from apoptosis This study

has systematically measured IGFIR binding affinities of

IGF-I, IGF-I and 14 IGF analogues to a recombinant

high-affinity form of the IGFIR using BIAcore technology

The analogues assessed could be divided into two groups:

(a) those designed to investigate binding of IGF-binding

protein, which exhibited IGF1R-binding affinities similar to

those of IGF-I or IGF-II; (b) those generated to probe

IGFIR interactions with greatly reduced IGF1R-binding

affinities The relative binding affinities of IGF-I analogues

and IGF-I for the IGF1IR determined by BIAcore analysis

agreed closely with existing data from receptor-binding

assays using cells or tissue membranes, demonstrating that

BlAcore technology is a powerful tool for measuring affinities of IGFs for IGFIR In parallel studies, IGF1R- binding affinities were related to ability to protect against serum withdrawal-induced apoptosis in three different assays including Hoechst 33258 staining, cell survival, and DNA fragmentation assays using the rat pheochromo- cytoma cell line, PC12 In this model system, IGF-I and

IGF-II at low nanomolar concentrations are able to prevent

apoptosis completely We conclude that ability to protect against apoptosis is directly related to ability to bind the IGFIR

Keywords: apoptosis; BIAcore analysis; insulin-like growth factor (IGF); type 1 IGF receptor

Insulin-like growth factors-I and -II (IGF-I and IGF-ID are

small peptides, which are able to promote cell proliferation,

differentiation and survival resulting predominantly from

interactions with the type 1 IGF receptor (IGFIR)

Prevention of apoptosis by an activated IGFIR plays a

major role in the survival of many cell types, including

neurons and haemopoietic cells after interleukin-3 with-

drawal (reviewed by Baserga ef al [1]) Significantly, the

ability of IGFs to protect against apoptosis has been

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; IGFIR, type | IGF

receptor; rhIGFIR, recombinant human high-affinity IGF1R;

IR, insulin receptor; IGF2R, type 2 IGF receptor; IGFBP,

IGF-binding protein; NGF, nerve growth factor; HBS,

Hepes-buffered saline; DMEM, Dulbecco’s modified 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)

implicated in potentiation of aberrant growth in disease situations such as cancer, where abnormally high levels of

circulating IGFs are evident [2]

The IGFIR is a ubiquitously expressed transmembrane homodimeric tyrosine kinase receptor [3,4] It consists of two extracellular ligand-binding « domains and two transmem- brane 8 domains The tyrosine kinase domain is located within the cytoplasmic region and is responsible for signal- ling events initiated by ligand activation via the extracellular domain Upon receptor autophosphorylation, two major downstream pathways are activated, namely the Ras/Raf/ mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways In some situations, a third pathway involving 14.3.3 protein modulation of Raf activation is also involved in IGFIR signalling [5,6] Anti-apoptotic activity

of the IGFIR 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 IGFIR and insulin receptor (IR) are structurally related [3], as are the IGFs and insulin [8-10] It is not surprising therefore that IGF-I binds with high affinity to

the IGFIR and also binds the IR, but with 100-fold lower

affinity IGF-I also binds the structurally unrelated type 2 IGF receptor IGF2R) with low affinity [11] IGF-II binds IGF2R and an isoform of the IR with high affinity and also binds the IGFIR, albeit with ~ twofold to threefold lower affinity than IGF-I [11] In addition, both IGFs bind to six

high-affinity binding proteins ((GFBPs) with ~ 10-fold

higher affinity than their binding to IGF receptors IGFBPs

thereby influence the availability of IGF to bind to IGF

receptors [12]

Over the past 14 years, a large number of IGF analogues

have been designed (initially by comparing IGF-I and

insulin sequences) and recombinantly expressed as tools to

investigate which residues are important for interactions

with IGFIR, IGF2R and the IGFBP family Initially, key

residues involved in IGFIR binding were identified in the

IGF-I B-domain (Tyr24) [13,14] and C-domain (Tyr31) [14],

whereas residues important for binding of IGF2R (Phe49,

Arg50, Ser51 [15]) and IGFBP (Glu3, Thr4, Gln15, Phel6

[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 IGFIR

binding, the A domain represents the IGF2R-binding site,

while the B domain and the first A domain helix contain the

major determinants for IGFBP binding

In this study we show, using IGF-I, IGF-I and 14 IGF

analogues, that potency in antiapoptotic activity is directly

related to ability to bind to the IGFIR Previously we have

expressed a recombinant form of the human IGFIR by

fusing the cDNA encoding the receptor ectodomain (resi-

dues 1-944) to the cDNA of the mouse Fe domain of

immunoglobulin (encoding residues of the CH2 and CH3

domains) [17] Expression in mammalian cells yielded a

homodimer, with the Fc domain replacing the transmem-

brane domain of the high-affinity IGF receptor We

determined that the affinity of the recombinant 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 interactions using the BLAcore

With parallel studies, we have determined the abilities of

IGF and IGF analogue to prevent serum withdrawal- induced apoptosis in the rat pheochromocytoma PC12 cell

line, a well-established model of neuronal apoptosis [18-20]

This study clearly indicates that the binding affinity of IGF analogues to IGFIR 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 Ltd (Adelaide, South Austra- lia) or provided by S J Milner as a member of the CRC for Tissue Growth and Repair (includes Long IGF-I and Gly3- IGF-I) Nerve growth factor (NGF) was acquired from Alomone (Jerusalem, Israel) BIAcore CMS5 sensor chips, amine coupling kits and surfactant A were from BIAcore Inc (Melbourne, Australia) Hepes-buffered saline (HBS) for BlAcore analysis contained 10 mm Hepes, 150 mm

NaCl, 3.4 mm EDTA, pH 7.4, and 0.005% surfactant A

Hoechst 33258 was from Calbiochem

Preparation of recombinant high-affinity type 1 IGF receptor (rhIGF1R)

rhIGFIR was expressed in BHK-21 neonatal hamster kidney fibroblasts from a cDNA clone encoding the ectodomain of the IGFIR 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 results) rh[GFIR was purified from cell culture medium using an anti-mouse IgG affinity column and was dialysed against HBS before use in further

experiments

Table 1 Kinetic constants obtained from BI Acore analysis of binding of IGF-I, IGF-II and mutant IGF analogue to rh[GF1R Data were analysed using BIAevaluation software 3.0 and fitted to a Langmuir | : | binding model as outlined in Materials and methods The dissociation constant (Xa) was determined from the calculation of kg/k,, where k, is the association rate and kg is the dissociation rate Relative Kg is equal to Kg of IGF-I/Kg of IGF analogue Dashes indicate data inappropriate for assessing association and dissociation rates No detectable binding (ND) was seen with Ala31-IGF-I (800 nm), Leu60-IGF-I (800 nm), Leu27-IGF-II (1 um), or des-(1-6,10)-Leu27-IGF-II (1 pm)

IGF-I and analogues

IGF-II and analogues

BlAcore analysis

BlAcore sensor chip preparation Coupling of rhIGFIR

to CM5 BlAsensor chip via amine group linkage was

achieved using standard coupling procedures [21] Briefly,

CM5S sensor chips were activated by injecting 35 uL N-ethyl-

N’-[(dimethylamino)propyl|carbodiimide/N-hydroxysucci-

nimide at 5 L-min™' Subsequently, rhnIGF1R was coupled

to the CMS sensor chip by injecting 35 uL rhIGFIR (4 ng)

in 10 mM sodium acetate, pH 4.5, at 5 uLmin ` Unreacted

øroups were Inactivated with 3Š HL 1 m ethanolamine/HCl,

pH 8.5 A sensor surface with 8§—I0 000 response units of

coupled rhIGFIR would routinely result in a response of

~~ 100 response units with 200 nm IGF-I

Generation of kinetic binding data Kinetic studies with a

range of analyte concentrations were determined at a flow

rate of 30 wL-min™! 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 nm, and for

IGF-II and related analogues concentrations of 25, 50, 100,

200, and 400 nm were utilized The rhIGF1R-coated

biosensor surface was regenerated with 0.3m 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 using control surfaces (no protein

coupled) was subtracted Both IGF-I and IGF-II binding

fitted a 1 : 1 Langmuir binding model using global fitting

This model describes a simple reversible interaction of two

molecules ina 1 : | complex Goodness of fit measured as a

y°- value was not greater than 5 for rhnIGFIR binding All

binding experiments were repeated at least in duplicate and

biosensor chips coupled at different times yielded surfaces

with identical binding affinities The binding affinities of

IGF-I and IGF-II to rhIGFIR (Kg = 4.45 and 23 nm,

respectively) were comparable to the binding affinities

(Kg = 3.5 and 20 nM, respectively) reported in the study

by Jansson et al [22], who used a homodimer of the IGFIR

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, Adelaide, South Australia) Stock cultures were

maintained in Dulbecco’s modified Eagle’s medium

(DMEM; high glucose) supplemented with 10% horse

serum, 5% fetal bovine serum, 100 UL"! penicillin and

100 p:gmL7! 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 fluorescence microscopy

Levels of apoptosis were quantitated after labelling the cells

with the nuclear stain Hoechst 33258 and visualization by

fluorescence microscopy as described previously [23] Briefly,

PC12 cells were plated in six-well plastic culture dishes

(4 x 10° cells per well), washed in serum-free DMEM and

resuspended in DMEM in either the presence or absence (control) of IGF-I, IGF-II or IGF analogues at various concentrations Treatments were for 24 h and the cells were subsequently stained with Hoechst 33258 (5 ngmL””) Nuclei that were condensed or fragmented were scored as

apoptotic

Cell survival assays PC12 cells were plated in 96-well tissue culture plates (1 x 10* cells per well) in serum-free DMEM either in the absence or the presence of IGF-I, IGF-

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, 96®, Aqueous One Solution Assay; Promega) Absorbances were measured at 490 nm using a microtitre plate reader (Titertek Multiskan MCC)

DNA fragmentation ELISA PC12 cells (1 x 10°) were treated for 24 h in serum-free DMEM either 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 fractions were prepared from lysates by centrifuga- tion (20 000 g, 10 min) The cell death detection ELISA (Roche Molecular Biochemicals), which quantitatively determines levels of cytoplasmic histone-associated DNA fragments generated during apoptotic DNA fragmentation, was performed according to the manufacturer’s instruc-

tions, and absorbances were measured at 405 nm

RESULTS BlAcore analysis of rhIGF1R binding by IGF analogues High affinity binding of IGF-I, IGF-II and their analogues

to rhIGFIR was measured by BlAcore analysis (Figs | and 2), and relative binding affinities 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 rhIGFIR binding affinities similar to IGF-I (no more than 1.5-fold difference in Ky, see Fig 1A-E and Table 1) These analogues were designed specifically to disrupt IGFBP binding and were therefore expected to maintain rhIGFIR-binding affinities similar to IGF-I Long Gly3-IGF-I and Long Arg3-IGF-I also had similar rhIGF1R-binding affinities to IGF-I (data not shown) The analogues 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 IGFIR These analogues bound with much lower affinities to the rhIGFIR than IGF-I (Fig IF-H and Table 1) The association and dissociation rates of these analogues were too rapid to be accurately measured, and therefore their steady-state affinities were determined There was a 200-fold difference in affinity between Leu60-IGF-I and IGF-I for binding to the rhIGFIR In addition, the effect of the double substitution of Ala31lLeu60-IGF-I resulted in no binding being detected, which suggests its affinity for the rhIGFIR is below the detection limit

of the BIAcore ( 107° m) This analogue has previously been shown to have a very low affinity for IGFIR on

A

180

100

50 450 850

fc

—

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Fig 1 BlAcore analysis of IGF-I and IGF-I analogue binding to high-

affinity recombinant IGFIR Binding of IGF-I (A), des-(1-3)-IGF-I

(B), Arg3-IGF-I (C), Gly3-IGF-I (D), Long IGF-I (E), Ala31-IGF-I

(F), Leu24-IGF-I (G) and Leu60-IGF-I (H) to rhIGFIR was mea-

sured by using concentrations of 12.5, 25, 50, 100 and 200 nm Asso-

ciation was measured for 300 s (starting after 100 s) and dissociation

was measured for 900s in the presence of HBS alone Receptor

binding is expressed in response units (RU) These results are repre-

sentative of at least duplicate experiments performed on different

sensor chip surfaces Binding of des-(2,3)-Leu24 and des-(2,3)-Ala31-

IGF-I is not shown but kinetic analysis is summarized in Table 1

human placental membranes (> 500-fold less than IGF-I

[14])

There was an approximately fourfold difference in the

affinities of IGF-I (4.45 nm) and IGF-II (17.8 nm) for the

rhIGFIR (Figs 1A and 2A, Table 1) Substitution of Glu

at position 6 to Arg (Arg6-IGF-II) or deletion of the first

six amino acids of IGF-II (des-(1-6)-IGF-ID, which are

equivalent analogues to Arg3-IGF-I and des-(1—3)-IGF-I,

had minimal effect on rhIGF1R-binding affinity 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 affinity for IGF1IR [25] The affini-

ties of Leu27-IGF-II (equivalent analogue to Leu24-IGF-I)

and des-(1—6,10)-Leu27-IGF-II were 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:

fluorescent staining (Hoechst 33258) of nuclei, MTT-based

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Fig 2 BlAcore analysis of IGF-II and IGF-II analogue binding to high- affinity recombinant IGF1IR Binding of IGF-II (A), des-(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 nm Association was measured for 300 s (starting after 100 s) and dissociation was mea- sured for 900s in the presence of HBS alone Receptor binding

is expressed in response units (RU) These results are representative

of at least duplicate experiments performed on different sensor chip surfaces

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 3C), and serum deprivation- induced apoptosis is completely prevented by NGF (100 ngmL™', Fig 3C) IGF-I (1 nm) essentially prevented apoptosis induced by serum deprivation (Fig 3A) and reduced the levels of apoptosis to an equivalent degree as NGF (100 ngmL””, < 5% apoptotic cells) IGF-II also strongly prevented apoptosis, reducing the levels of apop- tosis to 8% with | nm IGF-II and completely preventing

g 20 Ứ2 Des(1-3)

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0.1 1.0 10 100 sué/

[IGF/IGF analog] / nM

Fig 3 Concentration-dependent effects of IGF-I, IGF-II and mutant

IGF analogues on the prevention of serum deprivation-induced PC12 cell

apoptosis measured by fluorescence microscopy analysis of nuclear

morphology PC12 cells were incubated in serum-free (SF) medium for

24 hin either the presence or absence of IGF-I, IGF-II or mutant IGF

analogues at the indicated concentrations (A) Effects of IGF-I ana-

logues that bind IGF-binding proteins with low affinity; (B) effects of

IGF-I analogues with substitutions that alter binding to IGFIR;

(C) effects of IGF-II analogues that either bind with low affinity to

IGF-binding proteins [Arg6 and des-(1-6)] and/or bind with low

affinity to IGFIR [Leu27 and des-(1-6,10)-Leu27] In addition, the

levels of apoptosis induced by serum deprivation (24 h) and its pre-

vention by NGF (100 ngmL~') are shown in (C) PC12 cells were

stained with Hoechst 33258 (5 ugmL“') and levels of apoptosis were

quantitatively determined by fluorescence microscopy as 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

apoptosis at 10 nm IGF-II (Fig 3C) Long IGF-I, des-(1—3)-

IGF-I, Arg3-IGF-I and Gly3-IGF-I essentially prevented

serum deprivation-induced PC12 cell apoptosis at concen-

trations of | nm (Fig 3A) In addition, the combination of

the presence of the 13-amino acid N-terminal fusion partner

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

1 nm, 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 nm (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 nM, and high levels of apoptosis were measured at concentra- tions of | nm and 10 nm Ala31Leu60-IGF-I failed to prevent serum deprivation-induced apoptosis at 1 nm and

10 nm and only reduced the levels of apoptosis by < 50% at

100 nm (Fig 3B)

The IGF-II analogues Arg6-IGF-II and 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 nm (Fig 3C) In contrast, Leu27-IGF-II only provided full protection at 100 nm Interestingly, we cannot detect binding of Leu27-IGF-II to the IGFIR (Table 1)

It is possible that there is a very weak affinity for the IGFIR that results in a poor ability to protect against apoptosis although binding was not detected using BlIAcore or conventional cell-based assays Des-(1—6,10)-Leu27-IGF-II was unable to significantly protect at all the concentrations tested and only reduced the levels of apoptosis to + 30% at

100 nm (Fig 3C) Des-(1—6,10)-Leu27-IGF-II does not

bind IGFBPs but binds the IGF2R with equal affinity to Leu27 (our unpublished data) We can conclude that deletion of Gly at position 10 must result in an even poorer interaction with the IGFIR leading to the inability of des-

(1-6,10)-Leu27-IGF-II to protect PC12 cells from serum

starvation-induced apoptosis

The present results confirmed 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 | nm and 10 nM, respectively (Figs 3 and 4) Indeed, IGF-I was able to promote survival at levels

> 100%, indicating that IGF-I not only promotes cell survival but also stimulates a degree of cell proliferation even in serum-free conditions Long IGF-I, des-(1—3)-IGF-I, Arg3-IGF-I and Gly3-IGF-I (all at 1 nm) promoted PC12 cell survival to the same degree as native IGF-I (Fig 4A) In

contrast, Leu24-IGF-I, des-(2,3)-Leu24-IGF-I, Leu60-IGF-I

and Ala31Leu60-IGF-I (all at 1 nm) failed to prevent serum deprivation-induced cell death, and Ala31-IGF-I and des- (2,3)-Ala31-IGF-I exhibited a small survival-promoting effect (Fig 4C) IGF-II (10 nm) completely prevented serum deprivation-induced cell death (Fig 4E) and Arg6- IGF-II and des(1-6)-IGF-I were essentially equipotent to IGF-II Leu27-IGF-II and des-(1—6,10)-Leu27-IGF-II_ did not exhibit any survival-promoting effects at 10 nm (Fig 4E)

Finally, levels of PC12 cell apoptosis were quantitated using a DNA fragmentation ELISA In the absence of serum, high levels of DNA fragmentation were detected, and IGF-I (1 nm) prevented apoptotic DNA fragmentation (Fig 4B) Long IGF-I, des-(1—-3)-IGF-I, Arg3-IGF-I and Gly3-IGF-I (all at 1 nm) 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 nm) had no effect and failed to reduce the levels of DNA fragmentation induced by serum deprivation (Fig 4D) IGF-II (10 nm) prevented DNA fragmentation and Arg6- IGF-II and des(1—-6)-IGF-II showed comparable protective

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Fig 4 Comparative effects of IGF-I, IGF-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 24h in either the presence or absence of IGF-I (1 nm), IGF-II

(10 nm), IGF-I analogues (1 nm) or IGF-I analogues (10 nm) The

effects of IGF-I analogues that bind IGF-binding proteins with low

affinity (A, B), IGF analogues with substitutions that alter binding to

IGFIR (C, D) and IGF-II analogues (E, F) were determined on cell

survival (A, C, E) and apoptotic DNA fragmentation (B, D, F) IGF-I

(1 nm) and IGF-II (10 nm) completely prevented serum deprivation-

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 data are presented as

means + SD from at least three independent experiments

effects, but Leu27-IGF-II and des-(1—6,10)-Leu27-IGF-II

were completely unable to prevent apoptotic DNA frag-

mentation (Fig 4F)

DISCUSSION

We have made a direct comparison of rhIGF1R binding by

IGF-I, IGF-II and 14 analogues using BIAcore analysis

IGFIR 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 either

IGFIR [14,22] or IGFBP binding [24,26,27], and mutations

consist of amino-acid substitutions (e.g Tyr24 to Leu24

IGF-I), charge reversals (Glu3 to Arg3 IGF-I) or amino-acid

(M) ° oO

0 10 20 30 40 50

Apoptosis (% at 1 nM)

Fig 5 Correlation between binding affinities of IGF-I analogues to recombinant human IGF1R and prevention of apoptosis by IGF-I ana- logues The dissociation constants (K,) for binding of IGF-I and IGF-I analogues to rhIGFIR were plotted against levels of apoptosis (%) determined by fluorescence microscopy (Hoechst 33258 staining) in the presence of | nm IGF-I and IGF-I analogues Symbol representations

are IGF-I (@), des-(1-3) (©), Arg3 (HD, Gly3 (C1), des-(2,3)-Ala31(A), Long (A), Leu24 (W), Ala31(V), Leu60 (@), des-(2,3)-Leu24 (©)

deletions (e.g des-(1—3)-IGF-I) We have confirmed that Tyr24, Tyr31 and Tyr60 of IGF-I play a significant role in the

interaction with IGF1R, whereas the first three amino acids

of IGF-I, and in particular Glu3, are not critical residues for

binding to IGFIR Corresponding residues of IGF-II (namely Tyr27 and residues 1-6 or Glu6) have similar importance in the interaction between IGF-II and IGFIR When comparing the receptor’s binding affinities for the IGF analogues with its affinity for IGF-I or IGF-II, respectively, our analyses generally confirm the results of conventional receptor binding assays For example, there was a reported 1.36-fold difference in IGFIR_ 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 affinity for rhIGFIR determined by BIAcore analysis in this study Similarities were also seen between our present findings and reported IGF1R-binding affinities for Gly3-IGF-I, Arg3-IGF-I [24] and the ‘Long’ forms of these

analogues [26] In addition, human placental membrane

IGF IRs had an 18-fold lower affinity for Leu24-IGF-I than native IGF-I [14], and a similar reduction (20-fold) in rhIGF1R-binding affinity was measured in our experi- ments Therefore, this study demonstrates that BIAcore analysis is an excellent technique for the assessment of relative binding affinities of IGF analogues and IGF-I for the IGFIR

The IGF1R-binding affinities measured in the present study are comparable to the affinities reported by Jansson

et al [22] using an immobilized recombinant IGFIR IgG- binding Z domain fusion protein in BIAcore experiments The affinities are, however, ~ 10-fold lower than affinities calculated from binding to soluble IGF1R preparations [29] Coupling via amine groups (predominantly through the N-terminus of the Fc domain under these conditions) may

be limiting interactions between rhIGFIR and IGF or hindering the flexibility of rhIGF1IR Differences in relative binding affinities between our BIAcore analyses and cell- based or membrane-based receptor-binding assays for Ala31-IGF-I and Leu60-IGF [14] may also reflect the

differences in the assay systems used (i.e immobilized

rhIGFIR_ 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 rhIGFIR, which will allow a

guaranteed homogeneously coupled chip and may perhaps

improve flexibility and access to the receptor

Having established the relative binding affinities of IGF-I,

IGF-II and the 14 analogues for rhIGFIR, we related these

to their abilities to prevent apoptosis IGFs have powerful

antiapoptotic effects and promote survival in a diverse array

of cell types through activation of IGFIR 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 survival and apoptosis [18—20,23] IGF-I

and IGF-II completely prevented serum deprivation-

induced PC12 cell apoptosis at concentrations of 1 nm

and 10 nM, respectively, which are similar levels to 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 IGFIR

signalling, as IGFBPs will not regulate or negatively impact

IGF-IGFI1R interactions We have made a direct correla-

tion between receptor binding affinity (Kg) and ability to

prevent apoptosis for IGF-I and the IGF-I analogues

(Fig 5) Essentially, the ability of IGF-I or IGF-I analogues

to prevent apoptosis correlated directly with their IGF1R-

binding affinities, and a strong correlation coefficient

(r°> = 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 disrupted IGFIR binding have a corre-

sponding loss in their ability to prevent apoptosis

Despite the significant 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 IGFBPs

[des-(1—3)-IGF-I, Arg3-IGF-I and 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 affinity Also, IGF analogues with perturbed

receptor interactions (Leu24-IGF-I, Ala31-IGF-I, Leu60-

IGF-I and combinations of these) exhibited reduced ability

to stimulate DNA synthesis in L7 murine fibroblasts

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 affinity 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 IGFIR activation leading to different biological activities (for example apoptosis vs protein degradation)

The mechanism of signalling resulting from IGFIR activation by the analogues tested above was not investi- gated However, both the Akt (via IRS-1) and the MAP kinase (via Shc) pathways are involved in IGF-induced

antiapoptotic signalling via the IGFIR 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 between the IGF1R-binding affinity of IGF and IGF analogues and the prevention of apoptosis mediated through IGFIR signalling, suggesting that IGF1R-binding affinity 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 Adam Denley and Ms Filomena Occhiodoro for technical assistance In addition, we gratefully acknowledge Mr Geoff Francis (GroPep Ltd) for his critical comments

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