Cell membrane association of insulin-like growth factor binding protein-2 (IGFBP-2) in the rat brain olfactory bulb

Progress in Growth Factor Research, Vol. 6. Nos. 2~,, pp. 329-336, 1995 Copyright © 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain. 0955-2235/95 $29.00 + .00

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CELL MEMBRANE ASSOCIATION OF INSULINLIKE GROWTH FACTOR BINDING PROTEIN-2 (IGFBP-2) IN THE RAT BRAIN OLFACTORY BULB V. C. Russo,* L. A. Bach t and G. A. Werther** *Centre for Hormone Research, Royal Children's Hospital, Parkville, Victoria 3052, Australia *Universityof Melbourne, Department of Medicine, Austin Hospital, Heidelberg, Victoria 3084, Australia

Identification o f sites o f expression o f lGF, IGF receptors and 1GFBPs in the olfactory bulb o f the rat brain suggested the presence o f a paracrine IGF system. Since cell association o f lGFBPs has been suggested as an important factor in their modulation o f IGF action, we investigated whether IGFBPs are cell associated in olfactory bulb (OB). This was supported by des(1-3)IGF-I only partially competing for [z25I]IGFI binding to rat OB membrane, suggesting the presence o f a cell associated IGFBP. Affinity cross-linking o f [I:51]IGF-I to rat OB membrane demonstrated a 39-kDa complex which was reduced by IGF-I and IGF-1I, but not by des(1-3jlGF-I or insulin. Western ligand blotting o f solubilised membrane showed a 38-kDa IGFBP which was immunoprecipitated by anti-lGFBP-2 antiserum but not by anti-IGFBP-5 antiserum. We conclude that in the rat IGFBP-2 is associated with membranes from OB. Whether the cell membrane association is due to integrin binding via its RGD sequence or glycosaminoglycan binding is currently under investigation. Cell associated IGFBP-2 may modulate IGF action in the neonatal rat OB.

Keywords: IGF-I, IGFBP-2, rat brain, olfactory bulb, cell membrane. INTRODUCTION The insulin-like growth factors are peptides which regulate growth and differentiation. They are synthesised in most tissues and may act locally in a endocrine, autocrine or paracrine manner through specific receptors [1, 2]. Cellular responses to I G F are modulated by insulin-like growth factor binding proteins (IGFBPs). Such modulation is the result of I G F - I G F B P interactions which occur in the pericellular and/or extracellular space, possibly involving extracellular matrix proteins [2]. Recent studies have demonstrated that some of the IGFBPs may be associated with the cell surface or extracellular matrix (ECM) [3-7] and, more recently, a

*Correspondenceto: G. A. Werther. Acknowledgements--This project was supported by grants from the National Health and Medical

Research Council of Australia, Royal Children's Hospital Research Foundation and Serono, Australia. 329

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specific membrane receptor has been identified for one of them (IGFBP-3) [8]. These findings support the hypothesis that IGFBPs are involved not only in stabilising and regulating levels of diffusible IGFs, but also in targeting of IGFs to their receptors [2]. Although sites of expression of mRNA for IGFs and IGFBPs have been precisely located in the rat brain [9-14], very little is known about IGF-IGFBP-ECM interaction occurring in the central nervous system (CNS). We therefore explored the presence of IGFBPs associated with cell membranes in the rat brain olfactory bulb (OB). MATERIALS AND METHODS Reagents

Recombinant human insulin-like growth factor-I and -II (IGF-I, IGF-II) were generous gifts from Dr A. Skottner (KabiPharmacia, Peptide Hormones, Sweden). Des(1-3)IGF-I was a gift from Dr C. Williams (University of Auckland, New Zealand). Insulin was purchased from Novo Nordisk Pharmaceuticals Pty Ltd (North Rocks, NSW, Australia). Anti-IGFBP-2 antiserum was kindly provided by Dr M. Rechler (NIH, Bethesda, MD, U.S.A.) and the anti-IGFBP-5 antiserum was a gift from Dr D. R. Clemmons (Chapel Hill, NC, U.S.A.). [125I]-IGF-I and 14C protein molecular weight markers were bought from Amersham (North Ryde, NSW, Australia). Disuccinimidyl suberate (DSS) was obtained from Pierce (Rockford, IL, U.S.A.). Chemical reagents (Analar grade) were purchased from BDH-Merk Pty Ltd (Kilsyth, Victoria, Australia). Phenyl-methyl-sulfonyl-fluoride (PMSF) and Titron X-100 were purchased from Boehringer (Mannheim, Germany). Nitrocellulose membranes were obtained from Schleicher and Schuell (Dassel, Germany). Kodak X-Omat AR films were from Eastman Kodak Co. (New York, U.S.A.). Brain Tissue Samples

One-day postnatal Sprague-Dawley rats were killed by decapitation and OBs were obtained as previously described [15]. All procedures were approved by the Royal Children's Hospital Animal Experimentation Ethics Committee. Membrane Preparation

Olfactory bulb membranes were obtained by a modification of a previously described method [16]. Olfactory bulbs were resuspended in ice cold homogenate buffer (10 mM) Tris-HCl, 2 mM PMSF, 1 TIU/ml of Aprotinin) and mechanically disaggregated. The tissue suspension was centrifuged in an Eppendorf 5415 C centrifuge at 800 r.p.m. × 5 min at 4°C and the resulting supernatant was centrifuged at 15,000 r.p.m. × 60 min at 4°C. This pellet (MF) was resuspended in ice-cold 10 mM Tris-HCL pH 7.4, 0.1% BSA. Aliquots of cell membrane suspension were adjusted to a total protein concentration of 50 ~g/80 ~1.

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[125I]-IGF-I Binding to Membranes Cell membranes from OB were incubated in a final volume of 100 /11 with [125I]IGF-I in the presence or absence of 1 #g/ml IGF-I, IGF-II, des(1-3)IGF-I or 10/zg/ml of insulin for 2 h at 37°C with periodic vortexing. Binding was quenched by addition of 400/zl of ice-cold 10 mM Tris-HC1 pH 7.4, 0.1% BSA and samples were centrifuged at 15,000 r.p.m, for 20 min at 4°C. Pellets were washed twice in 400/~1 of the above buffer. The radioactivity incorporated in the resulting pellet was measured by scintillation counting (data not shown).

[125I]-IGF-I Cross-linking OB membranes were incubated as described above and binding was stopped by incubating samples on ice for 10 min. Eleven microlitres of 10 mM disuccinimidyl suberate (DSS) (final concentration 1 mM) were added to each sample and incubated for 15 min on ice. The cross-linking reaction was quenched by addition of 45 /11 of 100 mM Tris pH 7.4-10 mM EDTA. Solubilised samples were subjected to 12% SDS-PAGE, under reducing conditions where specified. Gels were dried and affinity labelled IGF receptors and IGFBPs were visualised by autoradiography.

Western Ligand Blot ( WLB) Analysis Olfactory bulb membranes were solubilised in non-reducing sample buffer and electrophoresed according to the method of Laemmli [17]. Adult rat serum (5/zl) and ~4C protein MW markers were run in parallel lanes as necessary. Separated proteins were transferred to nitrocellulose filters and WLB of the transferred protein was carried out according to the method of Hossenlopp et al. [18] using [125I]IGF-I.

Immunoprecipitation Immunoprecipitation of IGFBPs was performed as we have described previously [14]. RESULTS

Binding and Affinity Cross-linking of [125I]IGF-I to OB Membrane Binding studies on crude OB membranes (50 /.tg total protein) showed that [125I]IGF-I was displaced by 1 /.tg/ml of cold IGF-I and IGF°II (~70% of the total binding) but was only partially abolished by des(1-3)IGF-I at the same concentration (~60% of the total binding). These findings suggested the presence of non-IGF receptor binding sites for IGF-I (data not shown). Affinity cross-linking of [~25I]IGF-I to OB membrane revealed a 39-kDa band consistent with IGF-I complexed to a 30-kDa IGFBP (Fig. 1, lane TMF). This band was ablated by excess IGF-I (1/.tg/ml) (Fig. 1, lane NMF). In order to further characterise the binding of [~25I]IGF-I to OB membrane, OB membranes were incubated with [125I]IGF-I in the presence or absence of 1/.tg/ml of

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TCF

NCF

TMF

NMF

CI

MW

67 kDa

kDa

30 kDa FIGURE 1. Autoradiographof 12% SDS-PAGE of [I~I]IGF-Icross-linking to OB membranes (MF) and cytosolic fraction (CF). Lanes are as follows: TCF, cross-linking of [lzsIIIGF-Ito OB CF; NCF, crosslinking of []~I]IGF-I to OB CF in presence of cold IGF-I (1/~,/ml); TMF, cross-linkingof pzsI]IGF-I to OB MF; NMF, cross-linking of [12sII1GF-Ito OB MF in presence of cold IGF-I (1/Jg/ml);CI, cross-linking of []zsIIIGF-Ito itself as control; MW, 14C methylated molecular weight standards. Exposure was for 5 days.

IGF-I, IGF-II, des(1-3)IGF-I or insulin at an excess concentration of 10 pg/ml. This was followed by affinity cross-linking and gel electrophoresis of the solubilised and reduced samples. Autoradiography (Fig. 2, lane D) clearly shows that des(1-3)IGF-I was equipotent to IGF-I and IGF-II in displacing [125I]IGF-I binding from the receptors (bands respectively at ~100 kDa and ~200 kDa) (lanes I, II) but did not displace the 38-kDa band. Insulin at 10 pg/ml partially compared with [125I]IGF-I binding to type I receptor, as expected, but showed negligible competition with []25I]IGF-I binding to the 38-kDa I G F B P - I G F complex (lane In). W L B o f Solubilised OB Membrane

Autoradiographs of the WLB showed a single band at about 32 kDa (Fig. 3, lane MF), the size of which is consistent with either IGFBP-2 or IGFBP-5. Both of these are known to be expressed in this brain region [12, 14, 19], but the band was immunoprecipitated by an antibody to IGFBP-2 but not by an antibody to IGFBP5, suggesting that the 32-kDa band was IGFBP-2 (data not shown). DISCUSSION We previously suggested the presence of an I G F paracrine system in the rat brain OB, based on our observation of IGF-I receptors located adjacent to sites of IGF-

Cell Associated IGFBP-2 in the Brain

MW

333

D

In

II

I

TB

20

6

38

FIGURE 2. Autoradiograph of 12% SDS-PAGE of [J25IIIGF-I cross-linking to OB membranes. Lanes are as follows: MW, 14C methylated molecular weight standards; D, cross-linking of [J25I]IGF-I to OB membrane in presence of cold des(1-3)-IGF-i (1/lg/ml); In, cross-linking of [125I]IGF-I to OB membrane in presence of cold insulin (10/Ig/ml); II, cross-linking of [125I]IGF-I to OB membrane in presence of cold IGFII (1/Zg/ml); I, cross-linking of [125I]IGF-I to OB membrane in presence of cold IGF-I (1/Jg/ml); TB (total binding), cross-linking of [125I]IGF-I to OB membrane. Exposure was for 10 days.

I expression [20]. Recently we identified and characterised the IGFBPs produced by OBs in culture and also located sites of expression of their mRNAs [14, 21]. The most prominent IGFBP in the OB, as in the remainder of the CNS, is IGFBP-2 [12, 14, 22-25]. Similarly to IGFBP-1, IGFBP-2 possesses an R G D sequence [2], which is a recognition site for binding of ECM proteins to their cell membrane receptors, the integrins [26]. It has been shown by Jones et al. that IGFBP-1 stimulates cell migration and binds to the a5fll integrin by means of its R G D (Arg-Gly-Asp) sequence [4]. Mutation of the R G D sequence in IGFBP-1 both prevented it from binding to the integrin and blocked stimulation of cell migration [4]. Reeve and coauthors have described binding of IGF-I and -II to cell surface associated IGFBP-2 in small cell lung carcinoma [7]. Very little is known about I G F - I G F B P - E C M interactions in the rat brain. We therefore decided to investigate if any of the soluble IGFBPs that we previously identified in the conditioned medium of cultured OB [14, 21] were also associated with cell membranes or ECM. Membrane binding studies using [125I]IGF-I and unlabelled IGF-I and des(1-3)IGF-I, an analogue with the same binding affinity as IGF-I for receptors, but a markedly decreased affinity for IGFBPs, suggested the presence of a cell surface associated IGFBP. By affinity cross-linking, we identified a 38-kDa [125I]IGF-IGFBP complex. Western ligand blotting of the solubilised OB membrane and immunoprecipitation indicated that the binding protein was IGFBP-2. Experiments utilising in vitro autoradiography to map IGF binding sites in the OB show that the putative cell associated IGFBP-2 is located adjacent to sites of high density of IGF-I receptors (Russo V. C., Bach L. A. and Werther G. A. 1995,

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MW

RS

MF

CF

30 kDa

FIGURE 3. Autoradiograph of WLB of the membrane fraction and cytosolic fractions of newborn rat olfactory bulb. Lanes are as follows: MW, J4C methylated molecular weight standards; RS adult rat serum; MF, OB membrane fraction; CF, OB cytosolic fraction. Exposure was for 7 days.

manuscript in preparation). Thus we speculate that I G F B P - 2 m a y be delivering I G F s to the receptors or, alternatively, limiting access to receptors. The non I G F - I receptor binding sites for I G F - I in the rat OB represent I G F - I binding to the cell m e m b r a n e associated I G F B P - 2 . However, the mechanisms o f interaction with cell surface are u n k n o w n . We are currently investigating if the association o f I G F B P - 2 to the cell surface is via binding to integrin receptors t h r o u g h its R G D sequence, or if it is associated with glycosaminoglycans t h r o u g h the C-terminal heparin binding d o m a i n [27, 28]. We conclude that I G F B P - 2 is cell m e m b r a n e associated in the rat brain OB and is responsible for the n o n - I G F receptor binding sites in the OB, potentially being involved in the m o d u l a t i o n o f I G F - I availability to its receptors.

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