Characterization of a novel NCAM ligand with a stimulatory effect on neurite outgrowth identified by...

Characterization of a novel NCAM ligand with astimulatory effect on neurite outgrowth identi®ed byscreening a combinatorial peptide library

Lars C. B. Rùnn,1 Marianne Olsen,1 Vladislav Soroka,1 Sùren éstergaard,2 Steen Dissing,3 Flemming M. Poulsen,4

Arne Holm,2 Vladimir Berezin1 and Elisabeth Bock1

1Protein Laboratory, Institute of Molecular Pathology, Panum Institute 6.2., Blegdamsvej 3, DK-2200, Copenhagen N, Denmark2Chemical Department, Royal Agricultural and Veterinary University, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark3Division of Cell Physiology, Department of Medical Physiology, University of Copenhagen, Panum Institute 12.6.,

Blegdamsvej 3, DK-2200 Copenhagen N, Denmark4Institute of Molecular Biology, Department of Protein Chemistry, éster Farimagsgade 2 A, DK-1353 Copenhagen, Denmark

Keywords: axon, cell adhesion, combinatorial chemistry, intracellular calcium, rat

Abstract

The neural cell adhesion molecule, NCAM, plays a key role in neural development and plasticity mediating cell adhesion andsignal transduction. By screening a combinatorial library of synthetic peptides with NCAM puri®ed from postnatal day 10 rat

brains, we identi®ed a nonapeptide, termed NCAM binding peptide 10 (NBP10) and showed by nuclear magnetic resonance

analysis that it bound the NCAM IgI module of NCAM. NBP10 modulated cell aggregation as well as neurite outgrowth inducedspeci®cally by homophilic NCAM binding. Moreover, both monomeric and multimeric forms of NBP10 stimulated neurite

outgrowth from primary hippocampal neurons. The neurite outgrowth response to NBP10 was inhibited by a number of

compounds previously shown to inhibit neurite outgrowth induced by homophilic NCAM binding, including voltage-dependent

calcium channel antagonists, suggesting that NBP10 induced neurite outgrowth by activating a signal transduction pathwaysimilar to that activated by NCAM itself. Moreover, an inhibitor of intracellular calcium mobilization, TMB-8, prevented NBP10-

induced neurite outgrowth suggesting that NCAM-dependent neurite outgrowth also requires mobilization of calcium from

intracellular calcium stores in addition to calcium in¯ux from extracellular sources. By single-cell calcium imaging we furtherdemonstrated that NBP10 was capable of inducing an increase in intracellular calcium in PC12E2 cells. Thus, the NBP10 peptide

is a new tool for the study of molecular mechanisms underlying NCAM-dependent signal transduction and neurite outgrowth, and

could prove to be a useful modulator of regenerative processes in the peripheral and central nervous system.

Introduction

The neural cell adhesion molecule (NCAM) is believed to play an

important role in the formation and plasticity of neuronal connections

(LuÈthi et al., 1994; Rùnn et al., 1995; Berezin et al., 2001).

NCAM is expressed as three major isoforms in the nervous system

of which two, NCAM-180 (NCAM-A) and NCAM-140 (NCAM-B)

are transmembrane, whereas the third, NCAM-120 (NCAM-C), is

linked to the membrane via a glycosyl phosphatidyl inositol (GPI)

anchor. In addition, soluble forms of NCAM can be generated by

truncation and shedding (Olsen et al., 1993). The NCAM protein is

modi®ed by glycosylation. Notably, in the vertebrate nervous system,

NCAM might be the only carrier (Rougon et al., 1986; Cremer et al.,

1994) of polysialic acid (PSA), long linear homopolymers of a2,8

linked sialic acid residues, the expression of which is highly regulated

during development and in synaptic plasticity in the mature nervous

system (Doyle et al., 1992; Becker et al., 1996; Muller et al., 1996).

NCAM mediates cell±cell adhesion through a homophilic

(NCAM±NCAM) mechanism (Thiery et al., 1977; Moran & Bock,

1988). In addition, NCAM binds heterophilically to other cell surface

receptors and extracellular matrix components, including heparin

sulphate proteoglycans (Cole & Glaser, 1986) and the cell adhesion

molecules L1 and TAG-1/axonin-1 (Horstkorte et al., 1993; Milev

et al., 1996). The extracellular part of NCAM is composed of ®ve

immunoglobulin (Ig) homology modules and two ®bronectin type III

(F3) modules. The modules mediating homophilic NCAM binding

have not been identi®ed unequivocally. Reciprocal interactions,

either between the IgIII modules (Rao et al., 1994) or between all ®ve

Ig modules of two NCAM molecules (Ranheim et al., 1996), have

been suggested to be responsible for homophilic NCAM-binding.

Recently, binding between the recombinant NCAM modules IgI and

IgII has been demonstrated by plasmon surface resonance analysis

(Kiselyov et al., 1997) and structurally characterized by means of

nuclear magnetic resonance (NMR) (Thomsen et al., 1996; Jensen

et al., 1999) and X-ray crystallography (Kasper et al., 2000)

suggesting that homophilic NCAM binding is mediated by a double

reciprocal interaction between the IgI- and IgII modules of two

NCAM molecules.

Homophilic NCAM binding initiates a signalling cascade (Kolkova

et al., 2000) involving activation of a ®broblast growth factor

receptor (FGF-R)-dependent pathway (Doherty & Walsh, 1996) and

Correspondence: Dr L.C.B. Rùnn, NeuroSearch A/S, Pederstrupvej 93,DK-2750 Ballerup, Denmark.E-mail: [email protected]

Received 13 May 2002, revised 19 July 2002, accepted 30 August 2002

doi:10.1046/j.1460-9568.2002.02242.x

European Journal of Neuroscience, Vol. 16, pp. 1720±1730, 2002 ã Federation of European Neuroscience Societies

the Ras MAP kinase pathway (Schmid et al., 1999). NCAM has been

hypothesized to bind the FGF-R by interacting with the so-called

CAM homology domain (CHD), a sequence in the FGF-R with

homology to NCAM and the cell adhesion molecules L1 and N-

cadherin. Moreover, NCAM-140 has been shown to interact with the

nonreceptor tyrosine kinase p59fyn. Upon homophilic NCAM bind-

ing, this complex is believed to recruit the focal adhesion kinase

p125FAK. (Beggs et al., 1994, 1997) and subsequently activate the Ras

mitogen-activated protein (MAP) kinase pathway (Schmid et al.,

1999). The FGF-R and the Ras MAP kinase pathways might be

linked by protein kinase C (Kolkova et al., 2000). NCAM-mediated

induction of neurite outgrowth is also dependent on voltage-

dependent calcium channels (VDCCs). Thus, NCAM-dependent

neurite outgrowth can be inhibited by VDCC antagonists (Doherty

et al., 1991). Furthermore, NCAM antibodies in high concentrations

have been shown by spectro¯uorometry to induce an increase in

intracellular calcium (Schuch et al., 1989). Taken together, these

®ndings suggest that NCAM binding activates VDCCs in the plasma

membrane and that the resulting increase in intracellular calcium is

necessary for NCAM-induced neurite outgrowth.

We here report the identi®cation of a nonapeptide, termed NBP10,

identi®ed by screening a combinatorial library of synthetic peptides

with NCAM puri®ed from postnatal day 10 rat brains. We further

show that NBP10 binds the NCAM IgI module and modulates known

NCAM functions, including cell aggregation, neurite outgrowth and

regulation of intracellular calcium.

Materials and methods

Materials

Fura-2 acetomethyl ester (Fura-2-AM), fura-2 pentapotassium salt

and Ca ethylene glycol bis(a-aminoethylether)-N,N¢-tetraacetic acid

(EGTA)/K2EGTA buffers were obtained from Molecular Probes

(Eugene, OR, USA). The calcium channel antagonists w-conotoxin

MVIIA and nifedipine were obtained from Alomone Laboratories

(Jerusalem, Israel). Rabbit FGF-R antiserum (1 : 1000) raised against

a synthetic peptide corresponding to amino acids 119±144 of the

chicken FGF-R situated close to the CAM homology domain and the

p38 MAP kinase inhibitor SB203580 were from Upstate

Biotechnology (Lake Placid, NY, USA). The MEK inhibitor

PD98059 was from New England Biolabs (Beverly, MA, USA).

The intracellular calcium mobilization inhibitor TMB-8 (8-(N,N-

diethylamino)octyl-3,4,5-trimethoxy-benzoate hydrochloride) and the

Src family tyrosine kinase inhibitor PP1 were obtained from

Calbiochem (La Jolla, CA, USA). TentaGel resin was obtained

from Rapp Polymere (TuÈbingen, Germany). Rink amide linker and 9-

¯uorenylmethoxycarbonyl (Fmoc)-protected amino acids were ob-

tained from Novabiochem (LaÈufel®ngen, Switzerland). Dulbecco's

modi®ed Eagles medium (DMEM), Hank's balanced salt solution,

EDTA and B27 supplement were obtained from Gibco BRL (Paisley,

Scotland, UK). Plasticware for cell culture was obtained from NUNC

A/S (Roskilde, Denmark). All other reagents were obtained from

Sigma (St Louis, MO, USA). NCAM from postnatal day 10 rat brain

was puri®ed as described previously (Rasmussen et al., 1982; Krog

et al., 1992).

Cell culture

Fibroblastoid L929 cells, stably transfected with either NCAM-140 or

empty vector, were grown in DMEM supplemented with 10% foetal

bovine serum (FBS), penicillin (100 U/mL) and streptomycin

(100 mg/mL) in a humidi®ed atmosphere at 37 °C and 5% CO2.

For establishment of monolayers for cocultures, L-cells were

dislodged with trypsin (0.5 mg/mL) and EDTA (0.75 mM), seeded

at a density of approximately 55 000 cells/cm2 in 4- or 8-well LabTek

chamber slides with a growth surface of plastic coated with

®bronectin and grown for 24 h.

Dissociated hippocampal cells prepared from rat embryos on

gestational day 18 were seeded in a microwell plate at 50 000 cells in

15 mL medium per well as described previously (Maar et al., 1997;

Rùnn et al., 1999). All animals were handled in accordance with the

national guidelines for animal welfare. Cells were grown in

Neurobasal medium supplemented with B27, 20 mM HEPES, peni-

cillin (100 U/mL), streptomycin (100 mg/mL) and 0.4% w/v bovine

serum albumin. After 24 h in culture, cell aggregation was quanti®ed

by counting the number of cell aggregates per well as described

previously (Maar et al., 1997). Cell aggregates were de®ned as

clusters of cells estimated to be composed of more than 50 cells. The

number of aggregates was counted in a prede®ned circular area of

each microwell corresponding to approximately 0.25 mm2. For

analysis of neurite outgrowth, 5000 cells/well were seeded in 8-

well LabTek tissue culture chamber slides with a growth surface of

Permanox plastic. After 24 h, images of neurons were captured and

analysed by means of computer-assisted microscopy as described

previously (Rùnn et al., 2000b). Brie¯y, images of the cell cultures

were obtained using systematic sampling, the position of the ®rst

image being chosen randomly. A test grid containing six vertical lines

within an unbiased counting frame was superimposed into images of

the cell cultures. The total length of all neurites was estimated

stereologically by counting intersections between neurites and lines

of the test grid. Intersection points and cells within the measuring

area, as de®ned by the grid, were manually dotted on a computer-

monitor using a computer mouse. The counting item for the cells was

the soma of the cell. The number of intersections per cell was

subsequently calculated as a measure of the total neurite length per

cell. The absolute neurite length may be calculated using the

equation:

L � � � d2� I

where d is the vertical distance between the test lines used, L is the

absolute length of neurites and I is the number of neurite intersec-

tions. Estimates of neurite length obtained using the described

stereological method have been reported to be similar to estimates

obtained using tracing of neurites (Rùnn et al., 2000b). In one series

of experiments (Fig. 3D), neurite length was estimated by manually

tracing the extent of the major neurite of each cell by means of

computer-assisted microscopy.

For cocultures, primary hippocampal neurons were seeded on

monolayers of ®broblasts in Neurobasal medium supplemented with

2% v/v FBS. After 24 h, cultures were ®xed and stained for GAP-43

immunoreactivity for selective visualization of neurons as described

previously (Skladchikova et al., 1999). For cocultures, neurite length

was estimated by manually tracing the extent of the major neurite of

each cell by means of computer-assisted microscopy.

The PC12E2 cell line was a gift from Klaus Seedorf, Hagedorn

Research Institute, Denmark (Wu & Bradshaw, 1995). Cells were

grown in DMEM supplemented with 5% v/v FBS and 10% v/v horse

serum (HS). For calcium imaging, PC12E2 cells were dislodged and

seeded at a density of 5000±30 000 cells/cm2 in 4- or 8-well LabTek

chambered coverslides coated with ®bronectin (10 mg/mL) and

grown for 1±5 days. In some cases, neuronal differentiation was

induced by changing the medium to DMEM supplemented with 1%

Novel NCAM ligand stimulates neurite outgrowth 1721

ã 2002 Federation of European Neuroscience Societies, European Journal of Neuroscience, 16, 1720±1730

v/v FBS, 1% v/v HS and nerve growth factor (50 ng/mL) or FGF-2

(10 ng/mL).

Calcium imaging

Cells were washed in Hank's solution comprising (in mM): KCl, 5.4;

NaCl, 137; NaHCO3, 2 mM; MgSO4, 0.8 mM; Na2HPO4, 0.27;

glucose, 5.6 mM; CaCl2, 1.25; KH2PO4, 0.44; and loaded with Fura-

2 AM (2 mM, Molecular Probes, Eugene, OR, USA) dissolved in

dimethylsulphoxide for 35 min at 20 °C in the dark. Hereafter, cells

were washed four times and placed on the stage of an inverted Axiovert

100 TV microscope (Zeiss, GoÈttingen, Germany) equipped with an oil

immersion UV objective (Zeiss Fluar 40 3, 1.3 numerical aperture).

Imaging was performed using a Sensicam 12-bit cooled CCD camera

(PCO, Keilheim, Germany) and a J & M monochromator (J & M,

Aalen, Germany). The software Imaging Workbench (Axon, Foster

City, CA, USA) was used for data acquisition and analysis. Ratio-

images were obtained after background subtraction from images

collected at wavelengths over 510 nm after excitation at 340 and

380 nm, respectively, at sampling rates between 0.1 and 1 Hz.

Calibration was performed using Ca EGTA/K2 EGTA buffers with

known concentrations of free calcium and Fura-2 pentapotassium salt

(5 mM). The concentration of free calcium was estimated according to

the equation, [Ca2+]i = Kd´(R ± Rmin)/(Rmax ± R)´(F380max/F380min),

where R is the ratio of background subtracted ¯uorescence intensities

obtained at excitation at 340 and 380 nm, respectively, Rmax is the

ratio at saturating calcium, Rmin is the ratio at zero free calcium,

F380min is the intensity at saturating free calcium exciting at 380 nm,

while F380max is the intensity at zero free calcium. Rmax was

determined in situ using Fura-2 AM-loaded cells in the presence of

ionomycin (5 mM) and high extracellular calcium (10 mM). Values

determined were: Rmax 9.0; Rmin 0.68; F380max/F380min 7.0. The Kd

used was 236 nM (Groden et al., 1991). Peptides to be tested were

applied directly to the cell culture chambers in a volume corresponding

to half of the volume present in the chamber prior to application to

ensure an even distribution.

Synthesis and screening of peptide libraries

Synthesis of a resin-bound, one-bead one-peptide library was

performed as described previously (Furka et al., 1991; Lam et al.,

1991; Rùnn et al., 1999). Peptides were synthesized on TentaGel resin

with the Rink amide linker using Fmoc-protected amino acids. In each

synthesis step the resin was divided into 19 portions, one for each of

the protein L-amino acids except cysteine (éstergaard et al., 1995). All

peptides had an N-terminal alanine followed by eight random amino

acids. Screenings were carried out by incubating 2 mL resin,

equivalent to approximately 106 beads, with puri®ed biotinylated

NCAM in Tris/HCl buffer (Tris/HCl 0.025 M, pH 7.2, 0.25 M NaCl,

0.1% w/v) Tween 20 containing 0.1% (w/v) gelatin. After visualiz-

ation by a streptavidin-based staining reaction (éstergaard et al.,

1995), stained beads were isolated for microsequencing. Peptides

selected for further analysis were synthesized as monomers,

dendrimers consisting of four peptide monomers coupled to a lysine

backbone, or as BSA-coupled multimeric peptides in which each BSA

molecule carried approximately 20 peptide monomers. BSA-coupled

peptides were synthesized using succinimidyl 3-(2-pyridyldithio)-

propionate coupling. The peptides were at least 95% pure as estimated

by HPLC. All concentrations of multimeric peptides were calculated

according to the amount of peptide monomers they comprised.

NMR spectroscopy

For mapping of the binding of the monomeric NBP10 peptide, spectra

of 15N labelled IgI (0.025 mM) alone or in the presence of the

monomer (NBP10m; 0.25 mM, 0.50 mM, 1.0 mM or 2.0 mM) were

obtained. For mapping of the binding site of the dendrimeric NBP10

peptide, spectra of 15N-labelled IgI (0.025 mM) alone or in the

presence of the dendrimer (NBP10d; 0.20 mM) were obtained.

Samples were prepared in 90%H2O : 10%D2O, 150 mM NaCl,

5 mM sodium phosphate, 0.02% sodium azide, pH 7.34 buffer.1H15N-HSQC NMR spectra were recorded with 12 000 Hz spectral

width, 3792 complex points in t2 and 170 increments in t1 on a Varian

Unity Inova 800 MHz spectrometer at 298 K. The transformation and

analysis of spectra were performed using the MNMR and PRONTO

computer programs, respectively (Kjñr et al., 1994). Putative

interaction sites were identi®ed as chemical shift perturbations

> 0.02 p.p.m. (1H) or 0.1 p.p.m. (15N) upon the addition of

unlabelled peptide to the 15N-labelled IgI module.

Results

Screening of combinatorial libraries of synthetic peptides withNCAM puri®ed from postnatal day 10 rat brain

In order to identify putative ligands of NCAM, combinatorial

libraries of synthetic nonapeptides linked to polystyrene beads were

incubated with biotinylated NCAM puri®ed from postnatal day 10 rat

brain. By means of a streptavidin-based staining reaction, beads

binding NCAM were isolated. The bead-linked peptides were

microsequenced yielding the amino acid sequences shown in

Table 1. No obvious motifs were revealed by comparing the

individual sequences of the isolated, putative NCAM-binding

peptides, possibly re¯ecting a high number of different binding

sites being present in the extracellular part of the NCAM protein.

From the identi®ed sequences, a number of peptides were synthesized

for further analysis, including the nomamer NBP10

(AKKMWKKTW) and the octamer NBP9 (AWKEASWK), both of

which contained lysines ¯anked by tryptophans. As it has been

reported that multimeric forms of peptide ligands identi®ed by means

of phage display peptide libraries have a higher potency for receptor

activation than monomeric forms (Livnah et al., 1996; Cwirla et al.,

1997), the peptides were not only synthesized as monomers but also

as dendrimers composed of four monomers coupled to a backbone

consisting of three lysines and as BSA multimers consisting of

approximately 20 individual monomers coupled to BSA in order to

compare the effects of monomeric and multimeric ligands.

The NBP10 peptide inhibits cell aggregation

An important function of NCAM is to mediate cell adhesion. To

select functional ligands of NCAM from the identi®ed peptide

sequences, we tested the ability of the peptides to inhibit aggregation

of hippocampal cells in primary cultures of hippocampal neurons

grown under conditions permitting cell aggregation (Fig. 1). In this

model system, agents inhibiting the aggregation of cells result in a

higher number of aggregates, with the individual aggregates being

smaller on average. In contrast, agents promoting cell aggregation

result in a lower number of aggregates. Previously, the recombinant

NCAM module Ig1 has been shown to inhibit cell aggregation

potently (Maar et al., 1997; Kiselyov et al., 1997), presumably by

inhibiting NCAM-mediated cell adhesion.

The NBP10 peptide inhibited the formation of aggregates of

primary hippocampal neurons when present either as a monomer

(NBP10m), a dendrimer (NBP10d) or as a BSA-bound multimer

(NBP10BSA). In the absence of peptide, large distinct cell aggregates

were formed (Fig. 1A and F) whereas low concentrations of

monomeric or multimeric NBP10 peptide inhibited aggregation,

1722 L. C. B. Rùnn et al.


leading to the formation of smaller but more numerous aggregates

and indicating an inhibition of cell adhesion (Fig. 1B, C, G and H).

At higher peptide concentrations the morphology of the cultures

changed dramatically leading to the formation of a network of single

cells or very small clusters of cells interconnected by numerous thin

processes (Fig. 1D, E and I). Under these conditions, the number of

aggregates could not be quanti®ed. Thus, the NBP10 monomer

induced the formation of small but distinct aggregates at a

concentration of 60 mM (Fig. 1B and H) whereas cell cultures

grown in the presence of NBP10m at a concentration of 200 mM had a

very different morphology without distinct cell aggregates (Fig. 1D

and I). Similarly, the NBP10 dendrimer induced the formation of

small but distinct aggregates at a concentration of 1 or 2 mM (Fig. 1C

and G) whereas cell cultures grown in the presence of NBP10d at a

concentration of 6 mM had no distinct cell aggregates (Fig. 1E). When

comparing the dose±response relationship of the monomeric, the

FIG. 1. Effect of NBP10 peptides on cell aggregation. (A±E) Micrographs of hippocampal aggregate cultures grown for 24 h in the absence (A) or presence of60 mM NBP10m (B), 1 mM NBP10d (C), 200 mM NBP10m (D) or 6 mM NBP10d (E). Scale bar, 100 mm. (F±I) Micrographs at high magni®cation of culturesgrown in the absence (F) or presence of 2 mM NBP10d (G), 60 mM NBP10m (H) or 200 mM NBP10m (I). Scale bar, 25 mm. (J) Number of aggregates in theabsence or presence of NBP9BSA or NBP10BSA (22 mM). (K) Number of aggregates in the presence of NBP10 as monomer (diamonds), dendrimer (squares)or BSA-bound 20-mer (circles) in the indicated concentrations. Data points are means 6 SEM of 3±7 independent experiments.



dendrimeric and the BSA-bound forms of NBP10, the peptide

dendrimer was most potent with comparable effects at a concentra-

tion approximately 50-times lower than that used with the peptide

monomer (Fig. 1K). This indicated that the binding of multiple

NCAM molecules by a single multimeric peptide ligand could

potentiate the effect, although the presence of multiple binding sites

in the peptide ligand appeared not to be an absolute requirement for

the disaggregating effect as the peptide monomer was also able to

completely prevent the formation of aggregates.

The inhibitory effect of NBP10 on hippocampal cell aggregation

by both monomeric and multimeric peptide forms indicated that the

NBP10 peptide interferes with cell adhesion, a well-known NCAM

function.

NBP10 modulates neurite outgrowth induced by homophilicNCAM binding

We next studied neurite outgrowth induced by homophilic NCAM

binding in cocultures of NCAM-140-transfected ®broblasts and

primary hippocampal neurons. Under these conditions neurite

outgrowth is stimulated speci®cally (by approximately 75%) by

homophilic NCAM binding when neurons are grown on monolayers

of ®broblasts expressing NCAM as compared with cultures in which

neurons are grown on monolayers of ®broblasts without NCAM

expression, in accordance with previous reports (Williams et al.,

1994a; Rùnn et al., 2000a). Furthermore we observed that the

stimulation of neurite outgrowth induced by homophilic NCAM

binding was inhibited in a dose-dependent manner by the BSA-

coupled NBP10 peptide (Fig. 2A) at concentrations similar to those

found to inhibit cell±cell aggregation. In contrast, neither NBP9BSA

nor BSA alone had any effect on NCAM-induced neurite outgrowth

(Fig. 2B). This indicates that NBP10BSA interfered speci®cally with

homophilic NCAM binding, thereby preventing NCAM-induced

neurite outgrowth without interfering with basal neurite outgrowth.

When neurons were maintained on ®broblasts without NCAM

expression, a minor stimulation of neurite outgrowth (~25%) by

NBP10BSA was observed (Fig. 2). Under these conditions, neurite

outgrowth depends on recognition events other than homophilic

NCAM interactions including integrin binding and, possibly,

heterophilic NCAM interactions. Hence, when NCAM on the neurons

is not engaged in homophilic binding, the NBP10 peptide may bind to

NCAM and thereby stimulate outgrowth of neurites, although not as

ef®ciently as the NCAM molecule itself when presented by a

®broblast monolayer.

Stimulation of neurite outgrowth in dissociated cultures ofprimary hippocampal neurons by NBP10

We next examined the effect of NBP10 in dissociated cultures of

primary hippocampal neurons grown on a plastic substratum. Under

these conditions, the dendrimeric NBP10 peptide (NBP10d) had a

strong stimulatory effect on neurite outgrowth at a concentration of

1 mM (Fig. 3A and B). The monomeric NBP10 peptide also

stimulated neurite outgrowth, although with much lower potency

than NBP10d (Fig. 3C). However the maximal effect of NBP10d and

NBP10m was at the same level and both exhibited a bell-shaped

dose±response relationship with a maximal effect at concentrations

around 1 mM (NBP10d) and 100 mM (NBP10m), respectively. The

BSA-coupled NBP10 peptide (NBP10BSA) stimulated neurite out-

growth at a concentration of 22 mM whereas NBP9BSA or BSA alone

had no effect (Fig. 3D).

We then tested the effect of single substitutions in the monomeric

NBP10 sequence (Fig. 3E). Alanine substitution of the amino acid

residue W9 resulted in a statistically signi®cant increase in neurite

outgrowth whereas a phenylalanine substitution to the amino acid

residue A1 resulted in a signi®cant inhibition of the effect. Single

alanine substitutions at other positions in the NBP-10 sequence had

no effect. This indicates that the amino acid residues A1 and W9 are

FIG. 2. Effect of NBP10 on neurite outgrowth induced by homophilic NCAM binding. (A) Length of neurites from primary hippocampal neurons grown onmonolayers of NCAM-transfected ®broblasts (diamonds) or monolayers without NCAM expression (circles) in the presence of NBP10BSA at the indicatedconcentrations. Data are normalized to control cultures of neurons grown on ®broblast monolayers without NCAM expression. Each data point represents themean of 2±5 independent experiments. (B) Length of neurites from hippocampal cells grown on monolayers without NCAM expression (LVN, whitecolumns) or monolayers of NCAM-transfected ®broblasts (LBN, black columns) in the presence of NBP10BSA, NBP9BSA or BSA alone (22 mM). Datapoints represent means of 3±5 independent experiments. *P < 0.05 when compared with LVN control ++P < 0.01 when compared with LBN control, Studentst-test.



probably of importance for the observed neuritogenic effect of the

NBP10 peptide.

NBP10 induces NCAM-dependent signal transduction

We next addressed the involvement of presumed NCAM-dependent

signal transduction pathways in neurite outgrowth induced by the

NBP10 peptide. The dose±response relationship observed was bell-

shaped similar to that for the recombinant NCAM Ig2 module, the

putative endogenous ligand of NCAM Ig1, as well as for a recently

reported synthetic peptide ligand of the NCAM IgI module, C3,

which was identi®ed by screening a combinatorial peptide library

with recombinant NCAM Ig1 (Rùnn et al., 1999). In addition a bell-

shaped dose±response relationship has been reported for FGF-2 and

arachidonic acid (AA), which are believed to be an activator and a

downstream component, respectively, of an NCAM-dependent

signalling pathway (Doherty & Walsh, 1996). Thus, it has been

suggested that neurite outgrowth induced by NCAM±NCAM binding

depends on an interaction of NCAM with the FGF-R followed by

activation of a signalling pathway leading to an increase in the

intracellular concentration of calcium (Williams et al., 1994a;

Doherty & Walsh, 1996). In addition, a p59fyn-dependent activation

of the Ras-MAP-kinase pathway has been implicated in NCAM-

induced neurite outgrowth (Schmid et al., 1999; Kolkova et al.,

2000). We therefore investigated, whether NBP10-induced neurite

outgrowth might depend on activation of similar or identical signal

transduction pathways by testing the effect of a number of

compounds previously reported to inhibit NCAM-dependent signal-

ling (Fig. 3F). The neurite outgrowth response of the NBP10 peptide

was inhibited partially by an FGF-R antibody previously shown to

inhibit speci®cally neurite outgrowth induced by homophilic NCAM-

binding (Williams et al., 1994a), indicating that the neuritogenic

effect of NBP10 could be mediated by an NCAM-dependent

activation of an FGF-R-dependent signalling pathway. NBP10-

induced neurite outgrowth also was partially inhibited by PP1, an

inhibitor of p59fyn and other Src family tyrosine kinases, by

PD98059, an inhibitor of MEK, and SB203580, an inhibitor of

p38MAP kinase, indicating an involvement of MAP kinase signalling

pathways previously shown to be necessary for NCAM-dependent

neurite outgrowth (Kolkova et al., 2000). The correlation between the

effect of the employed inhibitors on NBP10-induced neurite

outgrowth and their previously reported inhibitory effects on

NCAM-induced neurite outgrowth and on neurite outgrowth induced

by the C3 peptide and the recombinant NCAM Ig2 module (Rùnn

et al., 1999) indicates that NBP10 induces neurite outgrowth

speci®cally through NCAM binding.

We also tested the effect of antagonists of calcium mobilization

from intra- and extracellular calcium stores and observed a partial

inhibition of NBP10-induced neurite outgrowth by nifedipine, an L-

type VDCC antagonist, w-conotoxin MVIIA, an N-type VDCC

antagonist and a complete inhibition by TMB-8, an inhibitor of

intracellular calcium mobilization. This suggests that a calcium in¯ux

through plasma membrane VDCCs is involved in NBP10-induced

neurite outgrowth in accordance with previous observations for

neurite outgrowth induced by homophilic NCAM binding in

coculture models (Doherty et al., 1991). However, the present

®nding that NBP10-induced neurite outgrowth is inhibited by TMB-8

indicates that mobilization of calcium from intracellular calcium

stores probably also is involved in NCAM-dependent neurite

outgrowth.

NBP10 increases intracellular calcium in PC12E2 cells

Because NBP10-induced neurite outgrowth appeared to depend on

VDCCs and intracellular calcium stores, we tested whether NBP10

was capable of directly in¯uencing intracellular calcium in neuronal

cells. When NBP10d was applied at a concentration of 50 mM to fura-

2-loaded PC12E2 cells, a sustained increase in the ¯uorescence

following excitation at a wavelength of 340 nm (Fig. 4A and B) was

observed with a concomitant decrease in the ¯uorescence recorded

following excitation at 380 nm (Fig. 4C and D) evidencing a

sustained increase in the intracellular concentration of calcium

(Fig. 4E). When lower concentrations of NBP10 were applied to

PC12E2 cells, only a fraction of the cells showed detectable increases

in intracellular calcium (not shown). These observations further

support the hypothesis that signal transduction and the subsequent

neurite outgrowth response induced by NBP10 peptide and NCAM

rely on an increased intracellular calcium concentration.

NBP10 binds the NCAM IgI module

The NBP10 peptide was identi®ed by screening a peptide library with

intact polysialylated NCAM puri®ed from NBP10 rat brain.

Previously, we have identi®ed a synthetic peptide ligand of the

NCAM IgI module, termed C3 (Rùnn et al., 1999), and showed that it

promotes neurite outgrowth similar to NBP10 with a comparable

potency. The C3 peptide was identi®ed by screening a peptide library

with the recombinant NCAM Ig1 module. Interestingly, the

sequences of NBP10 (AKKMWKKTW) and C3 (ASKKPKRNIKA)

have some similarity. We therefore tested the hypothesis that the

NBP10 peptide is also a ligand of the NCAM IgI module, which is

presumed to be involved in homophilic NCAM binding (Kasper et al.,

2000). We previously determined the structure of the IgI module by

NMR analysis so we used this approach to search for sites in the IgI

module protein that were perturbed upon binding of the NBP10

peptide in order to locate the putative binding site of NBP10 in the IgI

module (Fig. 5). When NMR spectra of 15N-labelled IgI were

obtained in the presence of unlabelled NBP10 in either the

monomeric or dendrimeric form, a number of amino acid residues

of the module displayed chemical shift perturbations indicating that

NBP10 does indeed bind the NCAM IgI module. Moreover, the sets

of amino acids displaying chemical shift perturbations upon binding

of the monomeric and the dendrimeric form of NBP10, respectively,

were very similar. Of the residues exhibiting chemical shifts, only the

TABLE 1. Sequences of putative NCAM-binding peptides identi®ed from a

combinatorial peptide library. Binding sequences were identi®ed from a

combinatorial library of nonapeptides incubated with NCAM puri®ed from

postnatal day 10 rat brain

A ± R K K K P P D NBP1A D Y Y W N K N K NBP2A ± K T N K W W K NBP3A ± ± T K A S S K NBP4A ± K F F K I S S NBP5A L ± K Y ± A G G NBP6A P H K K L ± A A NBP7A P K I K Q P K K NBP8A ± W K E A S W K NBP9A K K M W K K T W NBP10A P ± N K A F F ± NBP11A G H N D K I L M NBP12A F V ± Q K ± F V NBP13A L Y W E L ± G D NBP14A ± N M ± K M M Q NBP15A L ± H K Y P ± L NBP16A ± ± K A W W L L NBP17A ± I I A K L L ± NBP18

The `±' denotes amino acids not determined.



FIG. 3. Effect of NBP10 on neurite outgrowth. (A and B) Micrographs of primary hippocampal neurons grown for 24 h in the absence (A) or presence (B) ofNBP10d (1 mM). Scale bar, 20 mm. (C) Length of neurites from primary hippocampal neurons grown in the presence of NBP10m (circles) or NBP10d(diamonds) in the indicated concentration. Data points are means 6 SEM of 3±4 independent experiments. (D) Length of neurites in the presence ofNBP10BSA, NBP9±BSA or BSA alone (20 mM). Data points are means 6 SEM of 3±4 independent experiments. *P < 0.05, paired t-test. (E) Effect onneurite outgrowth of monomeric NBP10 peptide modi®ed by single substitutions with alanine or phenylalanine. *P < 0.05 when compared with the effect ofNBP10, Students t-test. (F) Effect of putative inhibitors of NCAM-dependent signal transduction on neurite outgrowth induced by NBP10d (1 mM). aFGFR,rabbit antiserum (1 : 1000) raised against a synthetic peptide corresponding to amino acids 119±144 of the chicken FGF receptor situated close to theso-called CAM homology domain; Nif, ni®dipine (10 mM); MVIIA, w-conotoxin MVIIA (1 mM); p38inh, SB203580 (1 mM); MEKinh, PD98059 (10 mM).*P < 0.05; **P < 0.01; ***P < 0.001 when compared with effect of NBP10d, paired t-test.



FIG. 4. Effect of NBP10 on intracellular calcium in PC12E2 cells. (A±D) Micrographs illustrating the effect of NBP10d application (50 mM) on theintracellular calcium concentration in Fura-2-AM-loaded PC12E2 cells. Under these conditions, an increase in intracellular calcium will be re¯ected by anincreased ¯uorescence at an excitation wavelength of 340 nm and a concomitant decrease in ¯uorescence at an excitation wavelength of 380 nm.(A) Fluorescence image obtained by excitation at 340 nm before application of NBP10d. Scale bar, 25 mm. (B) Fluorescence image obtained by excitation at340 nm 50 s after application of NBP10d. (C) Fluorescence image obtained by excitation at 380 nm before application of NBP10d. (D) Fluorescence imageobtained by excitation at 380 nm 50 s after application of NBP10d. (E) Time course of changes in the concentration of intracellular calcium in PC12E2 cellsafter application of NBP10d. The intracellular concentration of calcium was calculated from the ratio between ¯uorescence images obtained by excitation at340 nm (A and B) and 380 nm (C and D). Each trace represents one individual cell; representative of six independent experiments.



side chains of Cys22, Ile33 and Ile50 were buried inside the

hydrophobic core of IgI, whereas the rest of the residues had side

chains accessible to binding with NBP10. These ®ndings strongly

suggest that the NBP10 peptide binds the NCAM IgI module and that

the binding site is localized to the patch of amino acids indicated in

Fig. 5.

Discussion

We have identi®ed a peptide ligand, NBP10, capable of modulating

cell adhesion and inducing neurite outgrowth by screening a

combinatorial library of synthetic peptides with NCAM puri®ed

from rat brain. The NBP10 peptide is very potent as a dendrimer with

a maximal neurite stimulatory effect of approximately 300% of the

control level at a concentration of 1 mM (Fig. 1C), whereas the

monomeric form of the peptide was considerably less potent,

suggesting that the simultaneous binding of multiple NCAM

molecules by the multivalent dendrimer could facilitate the effect.

However NCAM clustering appears not to be an absolute requirement

for neurite outgrowth to be induced as the monomeric peptide has a

comparable maximal effect although at a much higher concentration

than the dendrimer. Future studies on the effect of the NBP10 peptide

on neurons without NCAM expression could further elucidate the

mechanism of action

The NBP10 peptide was found to promote neurite outgrowth with a

bell-shaped dose±response curve under conditions nonpermissive of

homophilic NCAM binding, when neurons were grown as dissociated

cells on a plastic substratum or on a monolayer of ®broblasts without

NCAM expression, but to inhibit neurite outgrowth stimulated

speci®cally by homophilic NCAM±NCAM binding. The neurite

outgrowth induced by NCAM±NCAM binding depends on a

subsequent interaction of NCAM with FGF-Rs, followed by

activation of a signalling pathway leading to an increase in the

intracellular concentration of calcium (Williams et al., 1994a;

Doherty & Walsh, 1996). Stimulation of this signalling pathway by

FGF or arachidonic acid has been reported to stimulate neurite

outgrowth with a bell-shaped dose±response relationship, when

dissociated neurons are grown on simple substrata nonpermissive of

homophilic NCAM±NCAM binding (Williams et al., 1994b), but to

inhibit neurite outgrowth induced by homophilic NCAM±NCAM

binding (Williams et al., 1995) in accordance with the ®ndings for

NBP10 in the present study (Figs 2 and 3). Previously, we have

observed that another ligand of the NCAM Ig1 module, the C3

peptide, also promoted neurite outgrowth from neurons grown on

simple substrata nonpermissive of homophilic NCAM±NCAM bind-

ing but inhibited neurite outgrowth stimulated speci®cally by

homophilic NCAM±NCAM binding in cocultures similar to the

NBP10 peptide described here (Rùnn et al., 1999). A possible

explanation could be that NBP10 works as a partial NCAM mimetic

under conditions in which homophilic, physiological NCAM binding

does not occur, resulting in a stimulation of neurite outgrowth. In

contrast, under conditions permissive of homophilic NCAM binding

NBP10 reduces NCAM-induced neurite outgrowth, presumably

because of interference with the homophilic binding of NCAM on

opposing cells and/or to desensitization of an NCAM-dependent

signalling cascade. Interestingly, the maximal stimulatory effect of

the NBP10 peptide on neurite outgrowth was higher than the maximal

effect observed previously for the C3 peptide although their potencies

are comparable (Rùnn et al., 1999).

By single-cell calcium imaging we further observed that NBP10

induces an increase in intracellular calcium in PC12E2 cells. We

recently observed that the C3 peptide also is capable of increasing

intracellular calcium (Rùnn et al., 2002). It has previously been

shown by means of spectro¯uorimetry that polyclonal NCAM

antibodies can increase the average intracellular concentration of

calcium in a population of PC12 cells or primary cerebellar neurons

(Schuch et al., 1989; Frei et al., 1992; von Bohlen und Halbach et al.,

1992) possibly by activating VDCCs resulting in a calcium in¯ux

from the extracellular space. Moreover, NCAM-antibodies have been

shown to decrease intracellular IP3 levels (Schuch et al., 1989). In

addition NCAM-induced neurite outgrowth has been reported to

depend on L- and N-type VDCCs (Doherty et al., 1991). However,

our ®nding that NBP10-induced neurite outgrowth is also inhibited by

TMB-8 suggests that mobilization of calcium from intracellular

stores also contributes to the increase in intracellular calcium induced

by NCAM binding.

A large sustained increase in intracellular calcium was only

observed after application of NBP10d at a concentration of 50 mM,

whereas the neurite response, presumed to be dependent on VDCCs

and intracellular calcium, was readily observed when NBP10d was

present at a concentration of 1 mM. A seemingly similar discrepancy

has been observed using the C3 peptide (Rùnn et al., 2002). This

could be because of the fact that the neurite response develops over a

period of 24 h whereas the increase in intracellular calcium is

FIG. 5. Mapping of the binding site of NBP10d onto the structure ofNCAM IgI module. Residues marked in yellow designate amino acidresidues of the IgI module exhibiting chemical shift changes > 0.1 p.p.m.(D15N or 5 3 D1H) upon binding to NBP10m. Residues marked in orangedesignate changes > 0.2 p.p.m. Residues marked in red designate changes> 0.3 p.p.m.



measured over a few minutes. A small local increase in intracellular

calcium or a calcium ¯ux without a concomitant rise in bulk calcium

concentration could be suf®cient to induce neurite outgrowth while

being undetectable by the applied fura-2 imaging method. In

accordance with this hypothesis, it has recently been reported that

FGF2 and the neural cell adhesion molecule L1, which like NCAM is

believed to promote neurite outgrowth by activating an FGF-R-

dependent signal transduction pathway, both activate VDCCs without

a rise in bulk calcium (Archer et al., 1999). It might therefore be

expected that the calcium response to the NBP10 peptide exhibits a

linear dose±response relationship, the calcium response increasing

with increasing concentrations of NBP10 peptide, whereas the neurite

outgrowth response to NBP10 is bell-shaped as described above.

By means of NMR analysis, both the monomeric and dendrimeric

forms of the NBP10 peptide were shown to be capable of binding the

NCAM IgI module in solution and the binding site of NBP10d and

NBP10m was mapped to a single large patch of the IgI module.

Previously, we have mapped the binding site of another ligand of

NCAM, the C3 peptide, in the NCAM Ig1 module by NMR (Rùnn

et al., 1999). In addition, we have mapped the binding site of the IgII

module, the putative endogenous ligand of NCAM IgII, in the NCAM

Ig1 module by NMR and X-ray crystallography (Jensen et al., 1999;

Kasper et al., 2000). Interestingly, the residues Phe19, Thr63 and

Tyr65, which showed changes in chemical shift upon binding of the

NBP10 peptide, have been shown to be crucial for IgI±IgII module

binding of NCAM (Jensen et al., 1999). This suggests a direct

competition of the NBP10 peptide with the IgII module for the

binding site in IgI and explains the observed disaggregating effects of

the P10 peptide, as an interaction between the IgI and the IgII

modules of NCAM is presumed to be involved in homophilic NCAM

binding. Although the binding sites of the C3 peptide and the IgII

module do not overlap, the binding site of the NBP10 peptide

partially overlapped with the C3 binding site, the two sites sharing the

amino acid residues Ile33 and Trp54. However, NBP10, C3 and the

IgII module all have a similar stimulating effect on neurite outgrowth,

presumably depending on the same signal transduction pathway.

These ®ndings suggest a key role of the IgI module in NCAM-

dependent adhesion and neurite outgrowth. Moreover, the study

suggests that small peptides could serve as functional mimetics of an

intact NCAM molecule by binding to either one of the various

binding sites of the IgI module of NCAM, namely the NBP10 binding

site identi®ed herein and the previously reported binding sites of C3

and IgII, respectively. Thus, the IgI module of NCAM represents an

interesting target for the development of small mimetics of NCAM,

which could be of value in the treatment of various neurodegenerative

conditions.

In conclusion, by screening of a combinatorial library with whole

NCAM we have identi®ed a peptide, NBP10, that induces neurite

outgrowth upon binding to a novel binding site of the NCAM IgI

module through activation of a signalling pathway similar to that

activated by homophilic NCAM binding. The NBP10 peptide could

be an interesting tool to study NCAM-dependent signalling and

neurite outgrowth. Furthermore, NBP10 could have the potential to

in¯uence NCAM functions important for neuronal regeneration and

synaptic plasticity.

Acknowledgements

Supported by The Danish Biotechnology Programme, The PlasmidFoundation, The Novo Nordisk Foundation, The Carlsberg Foundation, TheDanish Growth & Regeneration Programme, Ministry of Industry andResearch and the EU Fifth Framework Programme.

Abbreviations

BSA, bovine serum albumin; CHD, CAM homology domain; FBS, foetalbovine serum; FGF-R, ®broblast growth factor receptor; F3, ®bronectin typeIII module; GPI, glycosyl phosphatidyl inositol; HS, horse serum; Ig,immunoglobulin homology module; NCAM, neural cell adhesion molecule;NBP, NCAM binding peptide; NMR, nuclear magnetic resonance; PSA,polysialic acid; VDCC, voltage-dependent calcium channel.

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