Table Of ContentCurrent Topics in
Microbiology
165 and Immunology
Editors
R. W. Compans, Birmingham/Alabama· M. Cooper,
Birmingham/Alabama . H. Koprowski, Philadelphia
I. McConnell, Edinburgh' F. Melchers, Basel
V. Nussenzweig, New York . M. Oldstone,
La Jolla/California . S. Olsnes, Oslo . M. Potter,
Bethesda/Maryland . H. Saedler, Cologne . P. K. Vogt,
Los Angeles· H. Wagner, Munich· I. Wilson,
La Jolla/California
Neuronal Growth
Factors
Edited by M. Bothwell
With 14 Figures
Springer-Verlag
Berlin Heidelberg New York London
Paris Tokyo Hong Kong Barcelona
Professor MARK BOTHWELL, MD
Department of Physiology and Biophysics
University of Washington
Seattle, WA 98195
USA
ISBN-13:978-3-642-75749-5 e-ISBN-13: 978-3-642-75747-1
001: 10.1007/978-3-642-75747-1
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Softcover reprint of the hardcover lst edition 1991
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Preface
A host of environmental factors regulate the embryonic
development of neurons. In adults, the survival of neurons, the
regeneration of damaged axons, and plastic changes in axonal
arborization are also controlled by a complex array of
environmental cues. An important category of regulatory
influences involves target-derived hormone-like peptides act
ing on neuronal cell surface receptors. Nerve growth factor
(NGF) was the first such factor to be characterized (LEVI
MONTALCINI and HAMBURGER 1953) and has served as the model
against which all similar factors are compared. A number of
factors with properties similar to NGF have been described
with activities toward differing populations of neurons. Many of
these factors have been characterized only poorly at the
biochemical level. However, several factors have been charac
terized to the extent that molecular clones are available and
complete amino acid sequences are known. These include: three
structurally related factors, NGF itself (SCOTT et al. 1983),
brain-derived neurotrophic factor (BDNF) (LEIBROCK et al.
1989), and neurotrophin-3 (NT-3) (MAISONPIERRE et al. 1990;
HOHN et al. 1990); ciliary neurotrophic factor (CNTF) (LIN et al.
1989) and a second set of structural homologs; acidic and basic
fibroblast growth factors (aFGF, bFGF) (ABRAHAM et al. 1986;
JAYE et al. 1986). Investigators have cloned the receptors for
NGF (JOHNSON et al. 1986; RADEKE et al. 1987) and FGF (LEE
et al. 1989).
As the NGF and FGF systems have been the most
extensively characterized, the biology of these two peptide
factors, or, more precisely, families of peptide factors, will be
described in detail in this volume. It is our expectation that the
information obtained from the NGF and FGF systems will be
invaluable for understanding the functional properties of the
numerous similarfactors which remain to be characterized. The
NGF and FGF systems also serve to illustrate an important
point: NGF was originally discovered as a neurotrophic factor.
However, as will be discussed in the following pages, it is
VI Preface
becoming clear that NGF and the NGF receptor are involved in
regulation of a variety of non-neural cell types. Conversely,
while aFGF and bFGF were originally discovered as mitogens
for non-neural cell types, it is now clear that they have potent
neurotrophic activity. Epidermal growth factor is another
mitogenic growth factor which has been reported to have
neurotrophic activity (MORRISON et al. 1988). It is likely that
many of the growing list of mitogenic growth factors will have
specific regulatory functions for specific populations of post
mitotic neurons.
Mark Bothwell
References
Abraham JA, Whang JL, Tumolo A, Mergia A, Friedman J, Gospodarowicz D,
Fiddes JC (1986) Human basic fibroblast growth factor: nucleotide sequence
and genomic organization. EMBO J 5: 2523-2528
Hohn A, Leibrock J, Bailey K, Barde V-A (1990) Identification and characteriz
ation of a novel member of the nerve growth factor/brain-derived neuro
trophic factor family. Nature 344: 339-341
Jaye M, Howk R, Burgess W, Ricca G, Chiu I, Ravera M, O'Brien S, Modi W,
Maciag T, Drohan WN (1986) Human endothelial cell growth factor:
cloning, nucleotide sequence and chromosome localization. Science
233: 541-545
Johnson D, Lanahan A, Buck CR, Sehgal A, Morgan C, Mercer E, Bothwell M,
Chao M (1986) Expression and structure of the human NGF receptor. Cell
47:545-554
Lee PL, Johnson DE, Cousens LS, Fried VA, Williams LT (1989) Purification
and complementary DNA cloning of a receptor for basic fibroblast growth
factor. Science 245: 57-60
Leibrock J, Lottspeich F, Hohn A, Hofer M, Hengerer B, Masiakowski P,
Thoenen H, Barde V-A (1989) Molecular cloning and expression of brain
derived neurotrophic factor. Nature 341: 149-154
Levi-Monta1cini R, Hamburger V (1953) A diffusable agent of mouse sarcoma
producing hyperplasia of sympathetic ganglia and hypemeurotization of
viscera in the chick embryo. J Exp Zoo1123: 233-278
Lin L-F, Mismer D, Lile JD, Armes LG, Butler ET, Vannice JL, Collins F (1989)
Purification, cloning and expression of ciliary neurotrophic factor (CNTF).
Science 246: 1023-1025
Maisonpierre PC, Belluscio L, Squinto S, Ip NY, Furth ME, Lindsay RM,
Yancopoulos GD (1990) Neurotrophin-3: a neurotrophic factor related to
NGF and BDNF. Science 247: 1446-1451
Morrison RS, Keating RF, Moskal JR (1988) Basic fibroblast growth factor and
epidermal growth factor exert differential trophic effects on CNS neurons. J
Neurosci Res 21: 71-79
Radeke MJ, Misko TP, Hsu C, Herzenberg LA, Shooter EM (1987) Gene
transfer and molecular cloning of the rat nerve growth factor receptor.
Nature 325: 593-597
Scott J, Selby M, U rdea M, Quiroga M, Bell GI, Rutter W (1983) Isolation and
nucleotide sequence of a cDNA of mouse nerve growth factor. Nature
302:538-540
Table of Contents
M. FAHNESTOCK: Structure and Biosynthesis
of Nerve Growth Factor. . . . . . . . . . . . . 1
A. H. Ross, U. R. REDDY, G. SOBUE, H. HOTTA, and
U. R. REDDY: Biochemical Characterization of the
Nerve Growth Factor Receptor in Neural-Related
Tumors. . . . . . . . . . . . . . . . . . . . . . . . . . 27
M. V. CHAo: The Membrane Receptor
for Nerve Growth Factor. . . . . . . . . . . . . . . . . 39
M. BOTHWELL: Tissue Localization of Nerve Growth
Factor and Nerve Growth Factor Receptors. . . . 55
F. H. GAGE, M. H. TUSZYNSKI, K. S. CHEN,
A. M. FAGAN, and G. A. HIGGINS: Nerve Growth
Factor Function in the Central Nervous System. . 71
J. A. WAGNER: The Fibroblast Growth Factors:
An Emerging Family of Neural Growth Factors. . . 95
S. HALEGOUA, R. C. ARMSTRONG, and N. E. KREMER:
Dissecting the Mode of Action of a Neuronal
Growth Factor . . . . . . . . . . . . . . . . . . . . . . 119
Subject Index . . . . . . . . . . . . . . . . . . . . . . . 171
List of Contributors
(You will find the addresses at the beginning of the respective
contribution)
ARMSTRONG A. C. . 119 HIGGINS G. A. . 71
BOTHWELL M .. .. 55 HOTTA H .. . . 27
CHAoM. v. 39 KREMER N. E. 119
CHEN K. S .. 71 REDDY U. R. . 27
FAHNESTOCK M .. .. 1 Ross A. H. 27
FAGAN A. M .. 71 TUSZYNSKI M. H. 71
GAGEF.H. .. 71 SOBUE G .. 27
HALEGOUA S .. 119 WAGNER J. A. 95
Structure and Biosynthesis of Nerve Growth Factor
M. FAHNESTOCK
1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 Structure of NGF ....................................... . 2
2.1 Murine NGF .......................................... . 2
2.1.1 The PS ubunit ......................................... . 2
2.1.2 The y Subunit. ......................................... . 3
2.1.3 The IX Subunit ......................................... . 4
2.2 High Molecular Weight NGF ................................ . 4
3 Sources of NGF ........................................ . 5
3.1 Mastomys NGF ............... : ........................ . 6
3.2 Snake Venom NGF ...................................... . 7
3.3 Human NGF .......................................... . 8
3.4 Other Sources of NGF .................................... . 9
3.5 Comparison of High Molecular Weight NGFs ...................... . 9
4 NGF Biosynthesis ....................................... . 10
4.1 Transcriptional Regulation .................................. . 10
4.2 Transcription of p-NGF ................................... . 11
4.3 Translation of p-NGF . .................................... . 12
4.4 Precursor Processing ..................................... . 12
4.5 The Role of the y-Subunit .................................. . 13
4.6 Biosynthesis of the y-Subunit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
4.7 Biosynthesis of the IX-Subunit ................................. . 14
4.8 The Role of Other Kallikreins in NGF Processing .................... . 14
4.9 Biosynthesis of NGF from M astomys natalensis. . ................... . 15
5 Proteolysis of Mature p-NGF ................................ . 17
5.1 Processing at the N-Terminal ................................ . 17
5.2 Processing at the C-Terminal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
5.3 NGF in Blood and Saliva .................................. . 18
5.4 Regulation of NGF Activity in the 7S Complex ..................... . 19
6 Future Prospects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
6.1 The High Molecular Weight Complex ........................... . 19
6.2 Processing of p-NGF ..................................... . 19
7 Summary ............................................ . 20
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
1 Introduction
The first description of nerve growth factor (NGF) by LEVI-MONTALCINI and
HAMBURGER (1953) was of a factor from mouse sarcoma cells (S-180 and S-37)
that elicited extensive growth of chick embryo neurons. Shortly thereafter, a
Molecular Biology Department SRI International, 333 Ravenswood Avenue, Menlo Park,
CA 94025, USA
Current Topics in Microbiology and Immunology, Vol. 165
© Springer· Verlag Berlin' Heidelberg 1991
2 M. Fahnestock
serendipitous use of snake venom in the purification of this factor led COHEN to
the discovery that NGF was present in moccasin snake venom as well (COHEN
and LEVI-MoNTALCINI 1956). The finding that the mouse submaxillary gland-a
homologue of snake venom glands-stores even larger quantities of NGF than
does the snake venom gland (COHEN 1960) led to the extensive analyses of mouse
salivary NGF that continue today.
The ability to elicit nerve fiber outgrowth from cultured chick embryo sensory
or sympathetic ganglia is still the most reliable bioassay for NGF activity.
Despite a large number of tissues shown to exhibit this activity, there are few
sources of mammalian NGF sufficiently rich for purification and molecular
experimentation. The submaxillary gland of the male mouse has been the
traditional source; the ability to purify significant amounts of this protein led to
the development of antibodies for immunoassay (SUDA et al. 1978; KORSCHING
and THOENEN 1983; LARKFORS and EBENDAL 1987) and to the cloning of the gene
for murine NGF (SCOTT et al. 1983; ULLRICH et al. 1983). However, the limited
quantities of NGF present in other species and tissues as well as the poor
immunological cross-reactivity among NGFs from different species have ham
pered experimentation. In recent years, comparative study of NGF has
intensified due to the highly conserved nucleic acid sequences of NGF among
species. This review will focus on the comparative aspects of the structure,
biosynthesis, and processing of NGF from a number of sources.
2 Structure of N G F
2.1 Murine NGF
The structure, synthesis, and biological activity of NGF have been extensively
studied in the mouse, but little is known about its processing or regulation. High
molecular weight NGF isolated from mouse submaxillary glands has a
sedimentation coefficient of 7S, from which a molecular weight of 140000 was
calculated (V ARON et al. 1968). The complex consists of three types of subunits
(SERVER and SHOOTER 1977): j3, r, and Ct..
P
2.1.1 The Subunit
The most extensively studied subunit, j3-NGF, is responsible for the nerve
growth-promoting activity. cDNA clones for the j3 subunit have been obtained by
two research groups (ULLRICH et al. 1983; SCOTT et al. 1983), and the genomic
sequence including promoter also has been determined (SELBY et al 1987b;
ZHENG and HEINRICH 1988). j3-NGF is present in the 7S complex as a
noncovalently linked dimer of molecular weight 26000; strong denaturing agents
separate j3-NGF into two identical chains 118 amino acids long (VARON and
SHOOTER 1970). Both monomers and dimers are biologically active (FRAZIER
Structure and Biosynthesis of Nerve Growth Factor 3
et al. 1973a; STACH and SHOOTER 1974). Each chain contains three intrachain
disulfide bonds, and reduction of these bonds causes complete loss of biological
activity (GREENE and SHOOTER 1980; THOENEN and BARDE 1980). Although
tyrosine residues are not required for activity, tryptophan residues are important
for biological activity, since oxidation of Trp-21 causes loss ofNGF activity and
receptor binding (FRAZIER et al. 1973b; COHEN et al. 1980). A potential glycosyl
ation site exists in the mature NGF protein at Asn-45. Although the molecule was
thought not to be glycosylated (ANGELETTI et al. 1973), a glycosylated form exists
in the mouse submaxillary gland in small amounts (MURPHY et al. 1989). The
biological activity of the glycosylated form does not differ from the nonglycosy
lated form. The isoelectric point of the fJ subunit is 9.4, making fJ-NGF the most
basic of the three subunits.
r
2.1.2 The Subunit
The two y subunits of the 7S NGF complex are responsible for its proteolytic
activity (GREENE et al. 1969). Both the amino acid and nucleotide sequences of the
y subunit have been determined, and they demonstrate that it belongs to the
kallikrein family of trypsin-like serine proteases (THOMAS et al. 1981a; ULLRICH
et al. 1984; EVANS and RICHARDS 1985). Like other kallikreins, the y subunit has a
molecular weight of 26000 and is glycosylated.
The y subunit displays a high degree of electrophoretic heterogeneity; up to
six different forms have been identified (STACH et al. 1976; THOMAS et al. 1981b).
The different forms of the y subunit contain either two or three chains connected
by disulfide bridges. The two and three chain forms arise by endoproteolytic
cleavage of a single chain protein at two sites. C-terminal basic residues located at
these internal cleavage sites are removed from some molecules to create further
heterogeneity. This heterogeneity is not due to the variability of the tissue source,
however, since each submaxillary gland contains all six forms (SMITH et al. 1968).
The esterase activities of the different forms of the y subunit are all equivalent
(SERVER and SHOOTER 1977). Although no biological significance has been
demonstrated for the existence of six different forms, a 7S complex formed with y3
(a three chain form) is the most stable (THOMAS et al. 1981b; NICHOLS and
SHOOTER 1983). Since cleavage of they subunit into its most modified form (y3)
occurs during the secretion of 7S NGF into saliva (BURTON et al. 1978), this
processing may increase the stability of the complex in saliva.
The y subunit has been implicated in the processing of the fJ-NGF precursor
(see Sect. 3). The C-terminal amino acid sequence of the fJ-NGF precursor is Arg
Arg-Gly. The y subunit cleaves the bond between the two Arg residues, liberating
a dipeptide, and remains bound to the C-terminal Arg residue of fJ-NGF (BERGER
and SHOOTER 1977; BOTHWELL and SHOOTER 1978). Removing the C-terminal Arg
with carboxypeptidase B prevents the y subunit from binding to fJ-NGF and thus
prevents formation ofthe 7S complex (MOORE et al. 1974). It has been postulated,
therefore, that the y subunit binds to the C-terminal of fJ-NGF at its active
site.
