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: ln.dicou@utmb.edu (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, E. Dicou Non membrane-anchored precursors 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. 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REVIEW ARTICLE Biologically active, non membrane-anchored precursors – an overview Eleni Dicou Institut de Pharmacologie. (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