Báo cáo khoa học: Biologically active, non membrane-anchored precursors – an overview ppt

REVIEW ARTICLE
Biologically active, non membrane-anchored precursors –
an overview
Eleni Dicou
Institut de Pharmacologie Mole
´
culaire et Cellulaire du CNRS, UMR6097, Valbonne, France
Introduction
Precursor proteins mature through proteolytic cleavage
within the cell. In most cases, these precursors are bio-
logically inert and their existence is limited to the cyto-
plasmic compartments where processing of secretory
proteins takes place.
There are two sorting mechanisms in precursor ⁄ pro-
hormone secretion. The first is the constitutive path-
way, in which newly synthesized proteins continuously
pass through the trans-Golgi network and are trans-
ported in vesicles to the plasma membrane for immedi-
ate release. The second is the regulated secretory
pathway, in which dense-core secretory granules that
contain a condensed cargo of pro-hormones depend
on an extracellular stimulus for the release of the
stored contents in a controlled manner. This pathway
is operative in neuroendocrine cells and neurons.
Growth factors that derive from membrane-
anchored precursors constitute an important exception
to this general model. The membrane-anchored growth
factor precursors are biologically active and, once they
reach the cell surface, they can contact and activate
cognate receptors on adjacent cells. Thus, cleavage of
their extracellular domain into soluble forms consti-

tutes a process of conversion of one active form into
Keywords
bioactive precursors; chromogranins;
precerebellin; proapoA-I; proCHR;
proenkephalin; progastrin; proGRP;
proneurotrophins; PTH-P
Correspondence
E. Dicou, Department of Biochemistry and
Molecular Biology, University of Texas
Medical Branch, Galveston, TX 77555-1072,
USA
Fax: +1 409 772 8028
Tel: +1 409 772 3686
E-mail:
(Received 27 November 2007, revised 15
February 2008, accepted 28 February 2008)
doi:10.1111/j.1742-4658.2008.06366.x
Peptides function as chemical signals between cells of multicellular organ-
isms via specific receptors on target cells. Many hormones, neuromodula-
tors and growth factors are peptides. Peptide hormones and other
biologically active peptides are synthesized as higher molecular weight pre-
cursor proteins (pro-hormones), which must undergo post-translational
modification to yield the bioactive peptide(s). In many instances, more than
one biologically active peptide is generated from one and the same precur-
sor. In most cases, these precursors are biologically inert and their existence
is confined to the membrane-enclosed subcellular compartments where pro-
cessing of the pro-hormones takes place. A class of growth factors that
derive from membrane-anchored precursors which themselves are biologi-
cally active constitute an exception to this model. The list of the mem-
brane-anchored biologically active precursors has been the subject of

specialized reviews. The present review focuses on precursors other than
membrane-anchored precursors, which were found to be biologically active
and which often display different biological activities, and may mediate
their effects via receptors independent from those of their generated pep-
tides.
Abbreviations
ABCA1, ATP-binding cassette A1; ACTH, adrenocorticotrophic hormone; apo, apolipoprotein; BNDF, brain-derived neurotrophic factor; CCK
2
-
R, cholecystokinin 2 receptor; Cg, chromogranin; CRH, corticotrophin-releasing hormone; GRP, gastrin-releasing peptide; HDL, high-density
lipoprotein; IL, interleukin; LCAT, lecithin:cholesterol acetyltransferase; LPS, lipopolysaccharide; MNC, mononuclear cell; NGF, nerve growth
factor; Penk, proenkephalin A; PPR, PTH ⁄ PTHrP receptor; PTH, parathyroid hormone; PTHrP, parathyroid hormone-related protein; SCLC,
small cell lung carcinoma; TNF, tumor necrosis factor.
1960 FEBS Journal 275 (2008) 1960–1975 ª 2008 The Author Journal compilation ª 2008 FEBS
another rather than a process of pro-hormone activa-
tion. The list of known membrane-anchored growth
factor precursors includes more than 10 members that
belong to the epidermal growth factor gene super-
family, precursors for tumor necrosis factor (TNF)-a,
colony-stimulating factor-1 and the c-kit receptor
ligand [1,2].
The present article provides an overview of the non
membrane-anchored, biologically active precursors,
which may have biological functions and act via recep-
tors that are distinct from those of their cleaved pep-
tides. These include the precursor of cerebellin, the
family of chromogranins ⁄ secretogranins, proapolipo-
protein (apo)A-I, procorticotrophin-releasing hormone,
progastrin, progastrin-releasing peptide, parathyroid
hormone (PTH)-related protein, proenkephalin and the

proneurotrophins (Fig. 1). The present list includes
only well-documented cases of biologically active
precursors.
Precerebellin
Precerebellin, Cbln1, is the prototype for a family of
four brain-specific proteins (Cbln1–Cbln4) that was
initially identified for harboring a naturally occurring
16-amino acid peptide, cerebellin [3]. The peptide cere-
bellin is abundant in Purkinje cells of the cerebellum
and cartwheel neurons in the dorsal cochlear nucleus
Fig. 1. Preprohormone amino acid sequences deduced from cDNAs. h, human; m, mouse.
E. Dicou Non membrane-anchored precursors
FEBS Journal 275 (2008) 1960–1975 ª 2008 The Author Journal compilation ª 2008 FEBS 1961
[4]. During rat development, precerebellin mRNA lev-
els mirror the levels of the cerebellin peptide. Its levels
increase in parallel with synapse formation during the
immediate postpartum period and decrease with subse-
quent synapse loss during remodelling. In murine
mutants such as staggerer and weaver that have per-
turbed Purkinje cell synaptogenesis, cerebellin levels
are diminished.
However, it has become increasingly apparent that
Cbln1 is not only a precursor, but also a signalling
molecule that is secreted from cerebellar granule cells,
which form synapses with Purkinje cells [3,5]. Electro-
physiological and anatomical analyses of mutant mice
lacking the cbln1 gene have indicated that Cbln1 is
essential for synaptic integrity and plasticity in the cer-
ebellum and, in particular, in the matching and main-
tenance of pre- and postsynaptic structures and the

induction of long-term depression [5]. Consequently,
cbln1-null mice display severe motor discoordination
and ataxic gait. Interestingly, these abnormalities are
shared by mutant mice lacking the d
2
glutamate recep-
tor and it has been proposed that GluRd2 and Cbln1
may engage in a common signalling pathway crucial
for synapse integrity and plasticity.
The cerebellin peptide is flanked by Val–Arg and
Glu–Pro residues. Therefore, cerebellin is not liber-
ated from precerebellin by the classical dibasic amino
acid proteolytic-cleavage mechanism usually seen in
neuropeptide precursors. The cerebellin peptide and
an N-terminal truncated version, des-Ser
1
-cerebellin,
are present in the cerebella from diverse vertebrate
species, suggesting that cerebellin is not a random
by-product of proteolysis. Although abundant in the
cerebellum, cerebellin was also detected in the hypo-
thalamus, in ventromedial hypothalamic nuclei [6],
where it was implicated as a possible target of the
orphan nuclear receptor steroidogenic factor-1 and,
thus, may play a role in the development and ⁄ or
migration of ventromedial hypothalamic neurons.
Cerebellin was also shown to stimulate norepineph-
rine release and enhance adrenocortical steroid secre-
tion of the adrenal gland [7]. It is found enriched in
synaptosomes and is released in a calcium-dependent

manner after depolarization, suggesting that it may
act as a neurotransmitter [8].
Although cerebellin has features of a neuropeptide,
the precursor Cbln1 belongs to the C1q ⁄ TNF super-
family of secreted proteins, which suggests that it is
the biologically active molecule and that the proteo-
lytic events generating cerebellin serve another func-
tion. Although precerebellin has no collagen motif, the
C-terminal two-thirds of the protein shows significant
similarity (52%) to the globular (noncollagen-like)
region of the B chain of human complement compo-
nent C1q (gC1q). The gC1q signature domain, also
found in many noncomplement proteins, has a com-
pact jelly-role b-sandwitch fold similar to that of the
multifunctional TNF ligand family [9]. The members
of the ‘C1q ⁄ TNF’ superfamily are involved in pro-
cesses as diverse as host defense, inflammation, apop-
tosis, autoimmunity, cell differentiation, organogenesis,
hibernation and insulin-resistant obesity.
Because most of the C1q signature domain proteins
exist as an assembly of trimeric complexes, the exis-
tence of a precerebellin family (Cbln1–Cbln4) was
identified [10–13], suggesting that precerebellins are
secreted proteins that function as heteromeric com-
plexes. Cbln1 was recently shown to form a trimer via
its C-terminal C1q domain and a hexamer consisting
of two trimers connected via N-terminal disulfide
bonds [14]. Interestingly, cleavage at the N-terminus or
C-terminus of the cerebellin peptide influences the state
of assembly of Cbln1 complexes [14]. Each member

has a C-terminal C1q domain and an overall amino
acid sequence similarity with each other (60–80%) and
they can form homomeric and heteromeric complexes
in mammalian cells in vitro [15]. However, although
the different Cbln subtypes are often coexpressed in
certain brain regions, they have distinct patterns
of spatial and temporal expression in the adult and
developing brain, indicating distinct roles for each
member [13].
It is not yet known whether the cerebellin peptide or
the precerebellins interact with specific receptors. It is
conceivable that the precerebellin complexes interact
with a membrane receptor and activate an intracellular
signal transduction cascade in a manner analogous to
TNF-a.
Chromogranins/secretogranins
The granin family comprises another example of pre-
cursors that have biological activities distinct from
their cleaved peptides. The three classic granins are
chromogranin (Cg)A, CgB and secretogranin II, in
addition to four other less well known members, secre-
togranins III–VI [16,17]. The members of the granin
family are uniquely acidic proteins ubiquitous in secre-
tory cells of the nervous, endocrine and immune sys-
tems. They are proposed to play roles, first, in the
formation and condensation of secretory granules by
virtue of the ability of the granins to aggregate in the
low pH, high calcium environment of the trans-Golgi
network and, second, as a result of post-translational
proteolytic processing, as pro-hormones that generate

bioactive peptides.
Non membrane-anchored precursors E. Dicou
1962 FEBS Journal 275 (2008) 1960–1975 ª 2008 The Author Journal compilation ª 2008 FEBS
CgA has been proposed to act as an ‘on ⁄ off’ switch
in the biogenesis of dense-core granules by a mecha-
nism involving upregulation of protease nexin-1, a ser-
ine protease inhibitor [18]. Downregulation of CgA
using antisense RNAs in the PC-12 rat pheochromo-
cytoma cells leads to profound loss of dense-core
secretory granules and impaired secretion of pro-opio-
melanocortin. Although transfection of CgA in a CgA
deficient PC-12 clone rescued the regulated secretory
phenotype, CgB expression was found to be important
in the induction of secretory granule formation in non-
endocrine CgB-transfected 3T3 and COS-7 cells [19].
Granins, besides their function in the biogenesis of
granule formation, also function as helper proteins in
the sorting of peptide precursors [20] or as inhibitors
of precursor processing [21]. CgB, but not CgA, was
shown to have a nuclear localization in addition to its
localization in the cytoplasm and was implicated in a
transcription control role. In gene array assays, CgB
induced or suppressed transcription of many genes,
including those of transcription factors [22].
Assays for granins, especially CgA, are of great clin-
ical use because circulating granins have served as
diagnostic markers for a variety of neuroendocrine
tumors [16] and in chronic heart failure [23]. More
recently, two surprising functions were attributed to
CgA: the regulation of catecholamine-containing

dense-core chromaffin granule formation and the con-
trol of blood pressure in CgA knockout mice where
transgenic expression of the human CgA restored
blood pressure [24].
The presence of numerous paired basic amino acids
in granins suggests that they also give rise to peptides
as a result of post-translational proteolytic processing.
Indeed, a variety of peptides derived from CgA, CgB
and other granin members have been identified and
shown to have autocrine, paracrine and endocrine
activities [25]. Among them, vasostatins I and II
derived from CgA inhibit vasoconstriction, PTH secre-
tion, myocardial inotropy, vascular leakage and micro-
bial growth [17]; chromacin and catestatin, two other
fragments generated from CgA, as well as chrombacin
and secretolytin derived from CgB, also exert bacterio-
lytic and antifungal effects. Pancreastatin from CgA
inhibits insulin release from pancreatic-islet beta cells
and modulates insulin responses in adipocytes and
hepatocytes whereas parastatin, containing the catesta-
tin region of CgA, also inhibits PTH secretion. Other
granin-derived peptides are secretoneurin cleaved from
secretogranin II, which stimulates dopamine release
from nigrostriatal neurons, and 7B2 from secretogra-
nin V, which activates pro-hormone convertase PC2
[16,17].
Two interesting examples of precursor ⁄ cleaved pep-
tide opposing actions implicate vasostatin I and catest-
atin. CgA has anti-adhesive effects on fibroblasts and
smooth muscle cells in vitro but its fragments (e.g.

after cleavage by plasmin) exert pro-adhesive effects
[26,27]. In hypertension, CgA is overexpressed whereas
catestatin, a catecholamine release-inhibitory fragment,
is diminished via blocking of the nicotinic cholinergic
receptor [24]. Intraperitoneal injection of catestatin in
CgA
)
⁄
)
mice resulted in the substantial reduction of
their elevated blood pressure, analogous to the hista-
mine-related hypotensive effect of intravenous injection
of catestatin in rats [28]. However, to date, the recep-
tors and⁄ or the mechanisms of action of CgA and its
derived peptides remain elusive.
ApoA-I
ApoA-I, the major protein of serum high-density lipo-
protein (HDL), is a key element of the reverse choles-
terol transport pathway, a process that removes
cholesterol from extrahepatic tissues, including the ves-
sel wall, thus protecting against the development of
atherosclerosis [29]. In this pathway, apoA-I defines
the particle structure and stability of the HDL, pro-
motes cholesterol efflux and activates lecithin:choles-
terol acetyltransferase (LCAT). It is synthesized mainly
in hepatic and intestinal cells as a 267 amino acid pre-
proprotein [30]. The 18 amino acid leader sequence is
cleaved during transit through the Golgi and a 249
amino acid proprotein is released into the plasma
where the six amino acid propeptide (RHFWQQ) is

proteolytically cleaved extracellularly to yield mature
apoA-I. The pro-segment of apoA-I is unusual in that
it terminates with a Gln-Gln dipeptide rather than a
pair of basic amino acids. Therefore, proapoA-I is
itself the secretory form and proteolytic processing of
proapoA-I to apoA-I occurs extracellularly.
ProapoA-I is biologically active and, in several
in vitro studies, was shown to be functionally and
structurally indistinguishable from mature apoA-I
purified from plasma. ProapoA-I secreted in a baculo-
virus–insect cell system was found to bind lipid, and
thus meet the essential criterion for its classification as
an apolipoprotein, and to stimulate LCAT activity as
effectively as purified plasma apoA-I [31]. However,
using recombinant proapoA-I expressed in Escherichia
coli, the ability of proapoA-I to bind to and reorganize
phospholipid as compared to native apoA-I and the
ability of the proform of apoA-I to form reconstituted
HDL particles, as well as its capacity for LCAT acti-
vation, were found to be very similar to the mature
recombinant or native apoA-I forms [32]. Although
E. Dicou Non membrane-anchored precursors
FEBS Journal 275 (2008) 1960–1975 ª 2008 The Author Journal compilation ª 2008 FEBS 1963
most of newly secreted apoA-I and 3% of the plasma
apoA-I is proapoA-I, the biological function of pro-
apoA-I is not yet clear. When the synthesis and secre-
tion of pro- and mature forms of apoA-I from a
baculovirus–insect cell expression system were
compared in parallel experiments, the amount of the
pro-form of apoA-I synthesized and secreted was

several-fold higher than that of the mature form of
apoA-I. Furthermore, their ability to bind to plasma
HDL subfractions differed. Twice as much proapoA-I
was found to be associated with preb
1
-HDL and
preb
2
-HDL subfractions compared to the mature form
but proapoA-I was found to be decreased in a
1
-HDL
and a
2
-HDL. It is apparent, therefore, that the pro-
peptide is important for the effective synthesis and
secretion of apoA-I and that its deletion stimulates
conversion of preb-HDL to a-HDL [33].
A familial HDL deficiency, which is associated with
an increased risk of coronary heart disease, has been
characterized by reduced levels of apoA-I that were
not caused by reduced apoA-I production. The hyp-
ercatabolism of the mature form, but not the pro-
form, was responsible for the HDL deficiency [34].
This comprises evidence to suggest that the pro- and
mature forms can be distinguished during HDL metab-
olism in vivo. ProapoA-I has also been linked to Tang-
ier disease, a disease with abnormally low levels of
apoA-I and HDL. In Tangier disease, proapoA-I is
present in approximately equivalent concentrations

compared to mature apoA-I and this is not due to a
deficiency of the converting enzyme activity [35]. It is
thought that the differences in the levels of proapoA-I
versus apoA-I are a consequence of the rapid rate of
catabolism of apoA-I in Tangier disease due to its lack
of lipidation [36].
Other potential roles for the propeptide were pro-
posed following the observation that deleting the pro-
peptide from preproapoA-I altered the efficiency of
in vitro cotranslational translocation ⁄ processing, thus
suggesting that the propeptide plays a role in the
optimal folding of the precursor protein; it ‘helps’ the
nascent preprotein to assume an optimized conforma-
tion so that it may efficiently enter the secretory
apparatus [37]. The propeptide also appears to play a
role in intracellular transport and to facilitate trans-
port of apoA-I out of the endoplasmic reticulum
[38].
The proapoA-I cleavage appears to be an intermedi-
ate step in the formation of biologically active preb
1
-
HDL. Recently, the apoA-I proprotein convertase was
identified as the bone morphogenetic protein-1 and
shown to stimulate the conversion of newly secreted
proapoA-I to its phospholipid-binding form [39].
The mechanism of the formation of functional HDL
from secreted lipid-free apoA-I has implicated the
ATP-binding cassette A1 (ABCA1) transmembrane
lipid transporter, which is responsible for the transfer

of phospholipid from cell membranes to circulating
HDL [40,41]. Notably, in Tangier disease, ABCA1
activity is congenitally deficient. The absence of func-
tional ABCA1 in Tangier disease, or its significant
reduction in familial HDL deficiency patients, results
in the failure of newly synthesized apoA-I to acquire
lipid, leading to rapid catabolism of lipid-poor nascent
HDL particles [42].
The scavenger receptor type B class I was identified
as a high affinity HDL receptor that recognizes apoA-I.
Other receptors have also been postulated to be apoA-I
or HDL receptors, although the physiological relevance
of these findings remains to be established [30].
Procorticotrophin-releasing hormone
Corticotrophin-releasing hormone (CRH) is one of the
main actors in the stress response in invertebrates and
vertebrates [43]. Studies mainly performed in mam-
mals have demonstrated that CRH mediates the
release of adrenocorticotrophic hormone (ACTH)
from the pituitary, and this in turn leads to the release
of glucocorticoids from the adrenal gland. CRH is a
41 amino acid peptide, produced as the C-terminal
portion of a 196 amino acid CRH precursor
(proCRH). After removal of the signal peptide and
C-terminal amidation, this precursor, proCRH(27–
194), has a molecular mass of approximately 19 kDa.
ProCRH contains two potential cleavage sites,
CS1(124–125) and CS2(151–152). Cleavage at CS2
would give rise to proCRH(27–151) and mature CRH
whereas cleavage at CS1 would result in two other

peptides: an N-terminal fragment proCRH(27–124)
and the 8 kDa proCRH(125–151). ProCRH is
expressed mainly in the hypothalamus and placenta.
In the human normal term placenta, most of the
CRH exists as unprocessed proCRH and pro-
CRH(125–194) with very little in the form of CRH,
except in pre-eclampsia, a disorder characterized by
high blood pressure. In the maternal plasma, CRH is
the only one of the proCRH fragments to be main-
tained in significant amounts in the maternal circula-
tion [44].
ProCRH itself was shown to exert important biolog-
ical effects. Stably transfected CHO-K1 fibroblast cells
expressing rat preproCRH synthesize and release the
intact precursor, whereas no endoproteolytic products
derived from proCRH were detectable in the extracel-
lular medium. ProCRH has a nuclear localization in
Non membrane-anchored precursors E. Dicou
1964 FEBS Journal 275 (2008) 1960–1975 ª 2008 The Author Journal compilation ª 2008 FEBS
these transfected cells and appears to be in close asso-
ciation with DNA ⁄ chromatin [45]. ProCRH stimulated
the proliferation and DNA synthesis rate of the trans-
fected CHO-K1 cells compared to wild-type CHO-K1
cells. Furthermore, treatment of mouse corticotrophic
tumor cells (AtT20 ⁄ D16-16) with conditioned medium
from transfected CHO-K1 cells expressing proCRH
stimulated both DNA synthesis and cell proliferation,
providing evidence of a mitogenic role for proCRH on
a corticotrophic cell population [45]. ProCRH was also
effective in inducing ACTH release from primary cul-

tures of rat anterior pituitary cells, therefore acting as
an ACTH secretagogue in vivo [46].
ProCRH was also shown to be biologically active
within the immune system where it exerts an immuno-
modulatory action. ProCRH, as well as CRH, has
been detected in human lymphocytes [47]. ProCRH
exerted an inhibitory effect on basal and lipopolysac-
charide (LPS)-stimulated release of interleukin (IL)-6
by human peripheral blood mononuclear cells (MNCs)
[48]. The dose of proCRH (nm range) effective for
inhibiting the release of IL-6 from MNC was the same
as that stimulating ACTH release from primary cul-
tures of rat anterior pituitary cells [46]. This dose of
proCRH is also consistent with the dose of CRH nor-
mally used to stimulate ACTH release from cortico-
trophic cells, which further indicates a physiological
role for the intact precursor.
It is interesting to note the opposing effects of
proCRH and CRH on IL-6 release from MNCs.
ProCRH has an inhibitory effect whereas CRH stimu-
lates basal IL-6 release from MNCs [49]. By contrast,
both have a stimulatory action, inducing ACTH
release from primary cultures of pituitary cells [46],
which suggests a dissociation between immunoregula-
tory and endocrine activities. It has been suggested
that cellular components of the immune system may
be able to distinguish between closely related or trun-
cated peptides, whereas the classic neuroendocrine tar-
get cells might not [50].
A proCRH gene displaying a high degree of homol-

ogy with other proCRH genes known in vertebrates
has been isolated from the catfish Ameiurus nebulosus
[51]. Interestingly, only one protein with a molecular
mass of 18 kDa, which is comparable to that of the
putative catfish proCRH peptide, was detected in all
tissues examined. These results suggest that, in A. neb-
ulosus, the proCRH does not require further process-
ing to be active and provide further evidence that
proCRH can exert itself important biological effects.
Upon the stress response, besides activation of the
hypothalamic-pituitary-adrenal axis, the immune sys-
tem is also suggested to be actively involved. A rapid
increase in proCRH levels was found in the central
nervous system of the catfish A. nebulosus after 15 min
of treatment with LPS [51]. LPS is an immunologic
challenger and could be considered as a stressor. In
this case, the increased proCRH could be a conse-
quence of a response to LPS in which both immune
and neuroendocrine systems are required for restoring
body homeostasis [51]. It is noteworthy that a close
phylogenetic relationship and a high degree of conser-
vation of proCRH and the CRH fragment is observed
from invertebrates to vertebrates [52].
Progastrin
The hormone gastrin, first identified as a stimulant of
gastric acid secretion [53], exists in two forms (17 and
34 amino acids, respectively), which share a common
C-terminal sequence ending in an amidated phenylala-
nine residue. Both forms derive from a larger precur-
sor molecule, the 101 amino acid preprogastrin, which

is rapidly converted to progastrin by cleavage of an
NH
2
-terminal signal peptide between residues 21 and
22. Amidated gastrin is believed to be the main biolog-
ically active form, but recent studies have raised the
possibility that non-amidated precursor forms of gas-
trin, such as glycine extended gastrin (G-Gly) and
progastrin, may also have growth factor properties
[54].
Progastrin itself appears to act as a growth factor
for normal colon, as transgenic mice expressing pro-
gastrin in the liver have increased circulating concen-
trations of progastrin and a hyperplastic colonic
mucosa [55]. Human colon cancers and colon cancer
cell lines have been shown to express progastrin [56],
and a possible autocrine growth factor role has been
suggested, as in the case for gastrins [57]. In addition,
progastrin may act as a co-carcinogen in the develop-
ment of colorectal carcinoma because, following treat-
ment with azoxymethane, increased numbers of
aberrant crypt foci and tumors were observed in the
colonic mucosa of transgenic mice overexpressing
progastrin compared to wild-type mice [58].
Recombinant human progastrin(1–80) stimulated
proliferation and migration of the mouse gastric cell
line IMGE-5 [59]. Progastrin(1–80) was also shown to
exert direct antiapoptotic effects on intestinal epithelial
cells and upregulated cytochrome c oxidase [60].
Under physiological conditions, only processed

forms are present as the major circulating forms of
gastrins in humans and rodents. The full length pro-
gastrin is generally not detected in the circulation. In
patients with colorectal cancers and hypergastrinemia,
elevated levels of circulating progastrin were measured,
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FEBS Journal 275 (2008) 1960–1975 ª 2008 The Author Journal compilation ª 2008 FEBS 1965
and it has been suggested that they may play a role in
colon carcinogenesis [56]. Progastrin and G-Gly repre-
sent 90–100% of the gastrin peptides produced by
colon tumor and are found in 80–90% of colorectal
polyps in humans.
Elevated levels of progastrin in the circulation of
transgenic mice overexpressing progastrin in the intes-
tinal mucosal cells resulted in significant alterations in
the emotional behaviour of these mice. There was a
significant increase in the aggression, locomotor activ-
ity and anxiety-like behavior of the transgenic mice
compared to wild-type mice [61].
Amidated gastrins exert their effect through activa-
tion of their cognate receptors, cholecystokinin 2
receptors (CCK
2
-R). Low-affinity gastrin-binding sites
(K
d
= 1.0 lm) termed CCKC-R bind progastrin and
gastrins [62]. More recently, high affinity binding sites
were identified that were distinct from CCK
2

-R and
CCKC-R [63,64]. The observations that recombinant
progastrin did not bind to the CCK
2
-R and that
antagonists to this receptor did not reverse the prolif-
erative effects of progastrin suggested that progastrin
stimulated proliferation independently of the CCK
2
-R,
probably via receptors specific to progastrin. Biologi-
cally active recombinant human progastrin was found
to contain a tightly bound calcium ion and constitutes,
with the exception of proinsulin, comprising a first
example of selective, high affinity binding of metal ions
to a pro-hormone [63]. More recently, annexin II was
identified as a high affinity progastrin binding protein
[65]. A possible role of annexin II in mediating the
growth factor effects of progastrin was determined by
downregulating the expression of annexin II using an
antisense strategy.
In response to progastrin, there is activation of Src
(which is an oncogene linked to colon cancer), the
phosphatidyl inositol 3¢-kinase ⁄ Akt pathway (which is
involved in the regulation of proliferation and sur-
vival), Janus-activated kinase 2, signal transducer and
activator of transcription 3 (which is recognized as an
oncogene implicated in many cancers) and extracellu-
lar-signal regulated kinases [66,67]. Progastrin, there-
fore, is another example of a pro-hormone that is itself

biologically active and mediates effects via receptors
independent from those of its cleaved peptides.
Progastrin-releasing peptide
Gastrin-releasing peptide (GRP) is a 27 amino acid
peptide with an amidated C-terminus and is a member
of the bombesin family of neuropeptides. Bombesin
was originally isolated from the skin of the frog,
whereas GRP is the homologous peptide in mammals.
It was initially characterized for its potent stimulation
of gastrin release [68]. The widespread distribution of
GRP, with significant amounts present in the central
nervous system and throughout the gastrointestinal
tract, suggests that it has more general actions. It is
now known to perform many other functions, includ-
ing stimulation of the secretion of a variety of gastro-
intestinal hormones and pancreatic enzymes, as well as
the control of intestinal transit, smooth muscle con-
tractility, metabolism and behaviour; it is also known
to regulate the immune system and to modulate
smooth muscle contractility [69,70].
In particular, GRP has been recognized as the pro-
totypical autocrine growth factor, based on the detec-
tion of GRP and its cognate receptor in small cell
lung carcinoma (SCLC) and on the anti-proliferative
effect of GRP antibodies [71]. GRP is also a potent
mitogen for several other types of carcinomas, such
as colorectal, pancreas, prostate and breast tumors
[72]. GRP(1–27) is subsequently cleaved and amidated
to form GRP(18–27).
The precursor of GRP, proGRP, is a 125 amino

acid protein and was shown to be biologically active
[73]. It was found to stimulate proliferation of the
colon cancer cell line DLD-1 as efficiently as GRP(18–
27.) It also activates mitogen-activated protein kinase
phosphorylation in these cells, as does GRP(18–27).
This stimulation was reversed by the addition of an
agonist of the GRP receptor, GRP-R, in the case of
GRP, but not of proGRP. Interestingly, proGRP dif-
fered from GRP in that it failed to stimulate inositol
production whereas GRP significantly stimulated inosi-
tol production and this effect was reversed by the addi-
tion of the GRP-R antagonist. GRP mediates its
effects via two receptors: the GRP-R and the BRS-3
receptors. The proGRP appears to act through an
independent receptor because, in binding experiments,
proGRP did not compete with labelled bombesin for
binding to GRP-R, nor did it compete with labeled
BRS-3 agonist for binding to BRS-3. A GRP-R antag-
onist blocked the effect of GRP, but not proGRP, on
mitogen-activated protein kinase stimulation. ProGRP
was found to be present in several endometrial, pros-
tate and colon cancer cell lines and in resected colorec-
tal tumors [73].
GRP was expected to serve as a useful tumor mar-
ker for SCLC patients; however, the instability of
GRP in blood made its measurement difficult in clini-
cal situations. ProGRP (31–98), a region common to
three isoforms of human proGRP, is stable in blood
and can be conveniently measured by ELISA. Neuron-
specific enolase and carcinoembryonic antigen were

also reported to be useful markers for patients with
Non membrane-anchored precursors E. Dicou
1966 FEBS Journal 275 (2008) 1960–1975 ª 2008 The Author Journal compilation ª 2008 FEBS
SCLC. However, proGRP was found to be superior in
terms of sensitivity [74]. Assays for circulating proGRP
have also been used more recently as a tumor marker
for prostate and medullary thyroid cancer [75,76]. The
possibility remains for using antibodies or antagonists
to proGRP in the treatment of colorectal and other
cancers that express proGRP. Thus, proGRP is
another example of a pro-hormone giving rise to bio-
active peptides with independent receptors and differ-
ent bioactivities.
PTH-related protein
Parathyroid hormone-related protein (PTHrP) has
been identified as an oncoprotein that is involved in
the pathogenesis of the paraneoplastic syndrome of
humoral hypercalcimia of malignancy. It is structurally
related to PTH, the major regulator of calcium homeo-
stasis. Unlike PTH, PTHrP does not circulate in
appreciable amounts in normal subjects but is pro-
duced by most cells and tissues in the body. The peri-
natal lethality of PTHrP knockout mice emphasizes
the importance of this peptide system in normal life.
Although PTHrP was discovered as a hypercalcemic
factor, one of its primary roles might be to regulate
differentiation, proliferation and death [77,78]. The
dominant role of PTHrP as a developmental factor
has been well established in bone, skin and mammary
gland. Such a role also appears to be relevant in most

other organs, including the cardiovascular system and
the kidney [79].
Following translation, PTHrP enters the secretory
pathway and, in cell types that possess the regulated
secretory pathway, such as pancreatic islet cells and
atrial cardiocytes, it is packaged into secretory gran-
ules and is subject to regulated secretion. In tissues
that lack the regulated secretory pathway, such as
squamous carcinoma cells and fibroblasts, it is secreted
constitutively. This duality of secretory mechanisms
indicates that PTHrP is unusual with respect to other
precursors in that it is both a neuroendocrine peptide
and a growth factor or cytokine. During its transit
through the secretory pathway, the precursor is endo-
proteolytically processed at basic residues to yield a
family of mature secretory forms of the peptide [80].
PTHrP(1–36), displays smooth muscle relaxant proper-
ties and growth factor effects similar to PTHrP;
PTHrP(38–94 ⁄ 95⁄ 101) regulates calcium transport;
PTHrP(107–139), known as osteostatin, modulates
osteoclast activity; and PTHrP(141–173) stimulates the
growth of bone cells and collagen synthesis. Interest-
ingly, the generated peptides may also have opposing
effects among themselves. For example, PTHrP(1–36)
stimulates bone resorption whereas PTHrP(107–139)
inhibits bone resorption.
The best-studied biological effects of PTHrP are
mediated through the binding of its NH
2
terminus to a

G-protein-coupled receptor, PTH ⁄ PTHrP (PPR) that it
shares with PTH [81]. PPR signals through both the
adenyl cyclase and phospholipase C second messenger
pathways. Pharmacological evidence supports the exis-
tence of specific receptors for mid-region and carboxy-
terminal PTHrP peptides; however, further research is
required for their identification [81].
Recent studies have demonstrated that some of the
biological actions of PTHrP are cell surface receptor
independent and mediated through ‘intracrine’ mecha-
nisms [77,82]. The site between residues 87–107 of the
PTHrP constitutes a nuclear ⁄ nucleolar targeting
sequence and is implicated in the role of PTHrP in cell
cycle progression and apoptosis. Such an intracrine
mechanism has also been reported to increase cell pro-
liferation. This aspect raises new concepts in cellular
protein trafficking. However, the molecular mecha-
nisms and the molecular targets of nuclear PTHrP
remain unknown. The PTHrP nuclear import appears
to be mediated by the transport receptor importin b
[83]. PPR has been detected in the nucleus in various
cells and, hence, an active PTHrP ⁄ PPR system may be
functional at the nuclear compartment.
Thus, in a single cell type, PTHrP may inhibit or
stimulate proliferation or apoptosis, depending on
whether it acts through the auto ⁄ paracrine pathway or
through the intracrine pathway [77,78,82].
Another role suggested for PTHrP might be related
to its nuclear localization. PTHrP binds mRNA and
this binding competes with a peptide corresponding to

the nuclear ⁄ nucleolar targeting sequence, implying that
PTHrP may act as a nuclear export factor for mRNA
[84]. In a recent study, the role of PTHrP as an angio-
genesis inhibitor on hair growth was proposed [85].
Proenkephalin A
Proenkephalin A (Penk) is one of the three opioid pre-
cursor molecules (pro-opiomelanocortin, prodynor-
phin, proenkephalin) which, upon complete processing
by cleavage at sites of dibasic residues, yield four cop-
ies of the pentapeptide [Met]enkephalin, and one copy
each of the pentapeptide [Leu]enkephalin, the hepta-
peptide [Met]enkepalin–Arg
6
–Phe
7
and the octapeptide
[Met]enkephalin–Arg
6
–Gly
7
–Leu
8
. Enkephalins are
naturally occurring peptides exhibiting opiate-like
activity. Enkephalins and opioid receptors have been
identified in the brain, spinal cord, sympathetic ganglia
and adrenal medulla, as well as in sympathetic and
E. Dicou Non membrane-anchored precursors
FEBS Journal 275 (2008) 1960–1975 ª 2008 The Author Journal compilation ª 2008 FEBS 1967
parasympathetic neurons to the heart, spleen, vas def-

erens, stomach, intestine, lung, pancreas and liver [86].
Extended enkephalin-containing peptides that are bio-
logically active have been detected, as derived from
incomplete processing. However, the biological signifi-
cance of Penk remained elusive for some time due to a
lack of appropriate antibodies because antibodies to
the small enkephalin peptides exhibited minimal or no
cross-reactivity with the full-length precursor. The sub-
sequent generation of monoclonal antibodies to human
Penk-b-galactosidase fusion protein synthesized in
E. coli facilitated the detection of the precursor [87].
The discrepancy between significant levels of Penk
mRNA but negligible amounts of mature enkephalin
peptides in bovine cerebellum [88] was confirmed using
monoclonal antibodies to the enkephalin precursor by
the immunofluorescent detection of Penk in subpopu-
lations of rat cerebellar neurons and in the absence of
mature enkephalin peptides [89]. Penk was found to be
present at significant levels in astroglia cells [89,90] and
lymphocytes [91], and was released into the medium by
cultured astrocytes [92]. These observations suggest a
biological role for Penk itself.
A possible involvement of Penk in decision-making
events in growth control was demonstrated by its
nuclear localization in fibroblast and myoblast cells
[93]. In cells that are in transition to growth arrest,
nuclear Penk responded promptly to mitogen with-
drawal and cell–cell contact by unmasking transiently
antigenic domains, which indicated the acknowledg-
ment of growth arrest and differentiation signals by

nuclear Penk.
Opioids are known to affect survival and prolifera-
tion and their growth-promoting effects were found to
be mediated through Akt and Erk signalling cascades
[94]. In addition, morphine has been shown to have
antitumor activity in vivo, mediated in part through
phosphorylation and activation of p53 [95]. More
recently, Penk was implicated in apoptosis regulation.
It was shown to physically associate with two tran-
scription factors: p53, known for its pro-apoptotic
function and its role as a tumor suppressor, and the
RelA(p65) subunit of nuclear factor-kappa B, follow-
ing UV-C irradiation and assisting in apoptosis
through transcriptional repression of p-53 and nuclear
factor-kappa B gene targets [96]. In addition, Penk
associates with high affinity to the transcriptional co-
repressor histone de-acetylase, which suggests that it
may be a component of a transcriptional repression
complex that contributes to a pro-apoptotic outcome.
Penk, as well as the other opioid peptide precursors,
was shown to display sequence similarity with several
eukaryotic transcription factors [97].
A consensus regulated secretory pathway sorting sig-
nal has been identified in Penk, which is similar to the
sorting signal motif identified in pro-opiomelanocortin
and proinsulin. The mechanism involves the binding of
the two acidic residues in the RSP sorting signal motif
to the two basic residues of the sorting receptor car-
boxypeptidase E to effect sorting at the trans-Golgi
network [98]. Enkephalins interact with the d-opioid

peptide receptors, although whether Penk interacts
with the same receptors remains open to future investi-
gation. The availability of recombinant Penk should
facilitate the search for other biological activities of
Penk.
Proneurotrophins
The neurotrophins [nerve growth factor (NGF), brain-
derived neurotrophic factor (BDNF), NT-3, NT-4] are
members of a family of homologous proteins that play
a critical role in the development, maintenance and
regeneration of the nervous system. These factors exist
in solution as noncovalently linked homodimers. The
biological effects of the neurotrophins are mediated by
the Trk family of tyrosine kinase receptors (TrkA,
TrkB, TrkC), and the low affinity receptor p75
NTR
,
which is a member of the TNF receptor superfamily
[99–101]. Unlike the nonselective p75
NTR
receptor,
which has a similar affinity for all neurotrophins, each
Trk receptor selectively binds a different neurotrophin.
Neurtotrophins are initially synthesized as precur-
sors that are subsequently proteolytically processed to
release mature neurotrophin. An NGF precursor form
of 31 kDa was initially detected in the rat thyroid
[102], and NGF precursors of 31 kDa and 24 kDa
were observed in the rat hippocampus [103]. Following
the initial observation that proNGF was the predomi-

nant form in the rat thyroid with respect to NGF
[102], it has been well documented that proNGF forms
predominate in both central and peripheral tissues
whereas the mature NGF peptide is rare [104]. Several
studies have suggested that the prodomain facilitated
protein folding and promoted correct processing of
biologically active NGF [105,106].
However, subsequently, proNGF and proBDNF
were found to be secreted into conditioned media
when they were expressed in heterologous cells
[107–109], suggesting that they may act as ligands dis-
tinct from the mature peptides. Purified recombinant
proNGF was shown to bind the p75
NTR
with higher
affinity than NGF and to induce apoptosis [109].
Later, it was found that proNGF binds simultaneously
to p75
NTR
and sortilin, a member of the Vps10p fam-
ily of receptors, in a ternary complex. Thus, sortilin
Non membrane-anchored precursors E. Dicou
1968 FEBS Journal 275 (2008) 1960–1975 ª 2008 The Author Journal compilation ª 2008 FEBS
acts as a cell-surface coreceptor with p75 to mediate
proNGF induced cell death [110]. ProBDNF also
induced neuronal apoptosis by binding to the
p75
NTR
⁄ sortilin complex, and proBDNF is secreted by
cultured neurons [111]. Production of proNGF in vivo

by basal forebrain astrocytes was demonstrated after
kainic-acid induced seizures, indicating local produc-
tion of proneurotrophins under pathological conditions
[112]. Upregulation of proNGF and p75
NTR
after
spinal cord injury was shown to induce p75-mediated
death of oligodendrocytes, and proNGF present in the
injured spinal cord lysates induced apoptosis in culture
[113]. Furthermore, using an axotomy model for the
induction of death of rat corticospinal neurons in vivo,
proNGF was shown to be secreted in the cerebrospinal
fluid of the lesioned animals and was capable of trig-
gering apoptosis in culture [114]. Consequently, a plau-
sible role of proneurotrophins is to eliminate damaged
cells that express p75
NTR
.
Thus, it is widely agreed that the Trk receptors pro-
mote cell survival and enhance synaptic transmission
upon binding of the mature neurotrophins; by con-
trast, the proneurotrophins preferentially bind to the
p75
NTR
⁄ sortilin complex to induce apoptosis. This
dual system of ligand ⁄ receptor assures neuronal fate.
The duality of function of proneurotrophin ⁄ neurotro-
phin in the context of cell survival and death also
extends to the expression of plasticity in the brain.
NGF and especially BDNF play important roles in

long-term potentiation via the Trk receptors. In a
recent study, proBDNF was shown to enhance hippo-
campal long-term depression, whereas BDNF facili-
tates long-term potentiation [115].
Evidence that the pro-region may be important for
intracellular processing and secretion was provided in
a recent study of a single nucleotide polymorphism,
which converts a valine to methionine at codon 66
in the 5¢ pro-region of the human BDNF [116]. This
substitution affected intracellular trafficking and
activity-dependent secretion of BDNF, leading to
impairment in hippocampal function. Sortilin was
shown to interact specifically with BDNF in a region
encompassing the methionine substitution and to
control BDNF sorting to the regulated secretory
pathway [117]. Interestingly, in another study, a sort-
ing motif within the mature BDNF was found to
interact with the sorting receptor carboxypeptidase E
and the substitution of two acidic residues with ala-
nine resulted in attenuation of the regulated secretion
of BDNF [118]. Thus, elements present both in the
pro-region and the mature protein appear to control
the sorting of the BDNF to the regulated secretory
pathway.
From the evidence provided above, it is clear that
the precursors (proneurotrophins) and their generated
peptides (neurotrophins) have a differential ability to
bind to selective receptors and mediate distinctive bio-
logical actions.
Paradoxically, up to now, the processing of the

proNGF and proNT-3 has been limited to the study
of the liberation of the NGF and NT-3 peptides. How-
ever, the NGF precursor sequence contains four sites
of dibasic amino acids and can yield two additional
peptides of 29 amino acids (LIP1) and 38 amino acids
(LIP2), whereas a 37 amino acid peptide can also be
liberated from proNT-3 (elenin). ProBDNF cannot
generate any other peptide except the BDNF.
Chemically synthesized peptides that reproduce their
sequences were shown to be biologically active. They
significantly inhibited the mitogenic activity of estro-
gen, insulin-like growth factor and endothelial growth
factor in MCF-7 breast cancer cells [119,120]. LIP1
and LIP2 induced F-actin rearrangement and TrkA
phosphorylation in PC-12 cells [121], which suggests
that they mediate their action via the TrkA receptor,
and enhanced cholinergic enzyme activities (choline
acetyltransferase and acetylcholinesterase) in vivo in
the cortex, septum and hippocampus of the neonatal
hypothyroid rat [122]. LIP1 and LIP2 bind and induce
Akt phosphorylation in N11 microglial cells [119].
LIP1 binds to sortilin with an approximately six-fold
lower affinity than neurotensin, a ligand of sortilin,
and thus may antagonize proNGF in certain cell con-
ditions [119].
LIP1, LIP2, and elenin were neuroprotective against
N-methyl-d-aspartate cytotoxicity in cultures of corti-
cal neurons, and LIP1 and LIP2 also protected against
ibotenate induced lesions in vivo [119]. Furthermore,
high levels of LIP1 and LIP2 were detected in the sera

and synovial fluid of rheumatoid arthritis patients, sug-
gesting that they are circulating peptides with a cyto-
kine-like role [123]. Thus, these peptides will further
extend the list of the known members of the neurotro-
phin family and again modify the known neurotrophin
family landscape.
Conclusions
The present review has assembled information on ten
biologically active precursors that are not membrane-
anchored precursors. All of the cited cases have been
well-documented, and isolated reports of biologically
active precursors for certain neuropeptides or hor-
mones have not been included in this list. Nonethe-
less, this review does not claim to be an exhaustive
list on the subject. In general, from the above cited
E. Dicou Non membrane-anchored precursors
FEBS Journal 275 (2008) 1960–1975 ª 2008 The Author Journal compilation ª 2008 FEBS 1969
examples, it seems that, if a pro-hormone is found in
a secreted form, this is a good indication that it may
be biologically active. Furthermore, the availability
of a recombinant pro-hormone is a prerequisite that
facilitates research on its biological activities and
binding properties.
All of the above mentioned biologically active pre-
cursors are sorted via the regulated secretory pathway,
except for proapoA-I, which is sorted constitutively,
and PTHrP and proNGF, which use a dual secretory
mechanism (i.e. regulated ⁄ constitutive).
For some of the precursors, such as CgA, Penk and
BDNF, a sorting mechanism implicating carboxypepti-

dase E has been proposed. It remains to be seen
whether the other pro-hormones listed also have CPE
motifs or whether they share other common sorting
signal motifs.
Proteolytic processing of the above precursors to
generate cleaved peptides occurs intracellularly with
the exception of proapoA-I, which occurs extracellu-
larly. All the precursors act via auto ⁄ paracrine mecha-
nisms with the exception of PTHrP, which also acts
through an intracrine pathway.
Some of these precursors, such as progastrin, pro-
GRP and PTHrP, have growth factor properties
whereas others, such as like proNGF, proBDNF and
Penk, are pro-apoptotic.
A nuclear localization was observed for certain pre-
cursors, such as the CgB, proCRH, PTHrP and Penk,
which may suggest an involvement in decision-making
events in growth control or transcriptional control.
Some of these biologically active precursors are
linked to diseases and used as diagnostic markers (e.g.
proapoA-I is associated with Tangier’s disease, CgA is a
diagnostic marker of a variety of neuroendocrine mark-
ers and chronic heart failure, progastrin is found to be
elevated in colorectal cancers and hypergastrinemia,
and proGRP is a useful marker for SCLC patients and
for prostate and medullary thyroid cancer).
All of the above precursors have distinct biological
roles compared to those of their cleaved peptides but,
in some cases, their roles oppose those of their cleaved
products (e.g. CgA action opposes that of vasostatin I

and catestatin; proCRH has opposing effects to CRH
in immunoregulatory activities; proNGF and proB-
DNF exert pro-apoptotic actions that oppose the sur-
vival effects of the mature neurotrophins).
Receptors for some of them, such as precerebellin,
chromogranins and proapoA-I, remain unknown or
undefined, whereas progastrin and proGRP appear to
use receptors independent from the gastrins and GRP.
PTHrP shares the same receptor as PTH and the
PTH-like peptide. Penk may share the same receptors
as the enkephalins, although this is not clear. ProNGF
and proBDNF mediate their effects by the
p75
NTR
⁄ sortilin receptors, whereas the mature neuro-
trophins bind to the Trk receptors.
Finally, for all these precursors, proteolysis occurs
at sites with dibasic residues to liberate smaller pep-
tides, with two exceptions: the cerebellin peptide,
which is flanked by Val–Arg and Glu–Pro residues,
and the six amino acid prosegment of the proapoA-I,
which terminates with a Gln–Gln dipeptide.
It is anticipated that future research will continue to
enlarge the current list of biologically active precur-
sors.
Acknowledgements
The author thanks G. S. Baldwin, M. G. Castro,
K. B. Helle, A. Shulkes and D. Sviridov for their com-
ments. E. Dicou is charge
´

e de recherche in the CNRS
(Centre National de la Recherche Scientifique).
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