Histology and Histol Histopathol (1997) 12: 1179-1188 Histopathology 001: 10.14670/HH-12.1179 From Cell Biology to Tissue Engineering http://www.hh.um.es

Invited Review

Prohormone and proneuropeptide synthesis and secretion

M.J. Perone and M.G. Castro Molecular Medicine Unit, Department of Medicine, University of Manchester, Manchester, UK

Summary. Hormones and in eukaryotic sorting mechanisms which separate proteins destined for cells, are synthesised as large precursor molecules in the the regulated or constitutive secretory pathways from rough endoplasmic reticulum (RER), from where they proteins destined to Iysosomes, mitochondria or plasma are translocated to the Golgi apparatus. The sorting of membrane. How are proteins targeted to specific proteins destined for the regulated secretory pathway organelles or compartments within eukaryotic cells? from those which will be released constitutively takes This can sometimes be explained because particular place in the trans-Golgi network (TGN). In both these amino acid sequences (sorting domains) within proteins pathways, vesicles need to be transported to the plasma can convey the capacity to recognise and interact with a membrane before their contents can be released by specific transporter/carrier protein which can in turn exocytosis. target the protein in question to its cellular destination. Hormones and neuropeptides need to be secreted However, in most cases, these transporter/carrier systems from the cells in which are synthesised to exert their or defined sorting domains have not been identified, biological actions, although they can also play paracrine therefore, other intracellular mechanisms must be and autocrine actions. Prohormones and proneuro­ postulated. must undergo post-translational modifications Neuronal and endocrine tissues are constituted by which occur in determined subcellular compartments highly specialised secretory cells, which synthesise and within eukaryotic cells and are carried out in a strict release hormones and neuropeptides in a regulated succession of intracellular events, which give rise to fashion. Thus, the correct trafficking and sorting of biologically active products. hormones/neuropeptides within the secretory pathway of The biosynthesis of prohormones/proneuropeptides is these cells have profound physiological implications, not mediated by the action of endoproteolytic enzymes and only for single cell physiology, but also for the other post-translational modifying enzymes within the integration of crucial physiological responses within secretory pathway. The major focus of this review will higher organisms. Functions such as reproduction, be the biosynthetic pathway, sorting and intracellular response to stress, food intake, immune response, onset trafficking of prohormone and proneuropeptide of labour are all mediated by these messengers. precursors within the secretory pathway of eukaryotic We will review published evidence which relates to cells. biosynthesis, intracellular sorting and trafficking of prohormones/proneuropeptides within the regulated and Key words: Prohormone, Proneuropeptide, Sorting, constitutive secretory pathways of neuronal and Trafficking, Secretory pathway, Post-translational endocrine cells. We will also discuss evidence from processing work done in our laboratory and work done by other groups which suggests, contrary to what was believed until recently, that biosynthesis, trafficking and sorting Introduction events affecting prohormones/proneuropeptides are not only dependent on their particular amino acid sequences, Eukaryotic cells possess an efficient biochemical but also determined by cell-type specific factors which at machinery for protein synthesis and secretion. present are unknown. Membrane surrounded compartments within these cells, allow a fine regulation of metabolic reactions such as 1. Prohormonelproneuropeptide processing: its role in hormone stability, biological activity and sorting Offprint requests to: Dr. Maria G. Castro, Molecular Medicine Unit, mechanisms Department of Medicine, University of Manchester, Stopford Bldg., Rm 1.302, Manchester M13 9PT, UK It has been previously demonstrated that hormones 1180 Biosynthesis of prohormones

and neuropeptides are synthesised as precursor proteins, Cleavage at single basic amino acid residues may also e.g., prohormones and proneuropeptides. Until recently, occur, e.g., the endoproteolytic cleavage of rat it was believed that higher molecular weight protein by the prohormone convertase I (PC I) to precursors were not biologically active. Work done in yield an 8 leD prodynorphin derived-product and the C­ our laboratory has shown that the full length precursor peptide (Dupuy et aI., 1994). Although, the presence of for corticotrophin-releasing hormone (CRH) is basic amino acids is essential to confer an aqueous biologically active, i.e., it elicits the release of adreno­ environment at the site of cleavage, the secondary corticotrophin (ACTH) from primary cultures of rat structure of the surrounding amino acids also seems to anterior pituitary cells (Morrison et aI., 1995) and it play an important role (Rholam et aI., 1986). To date, induces cell proliferation of AtT20 cells, a corticotrophic several endoproteases involved in the endoproteolytic tumour cell line (Castro et aI., 1995b). Biologically processing of prohormones/proneuropeptides have been active peptides are released from their precursor proteins identified and cloned, and are known as precursor by enzymatic endoproteolytic cleavages. Most pro­ converting enzymes (PCEs) (for review see Smeekens, hormones/proneuropeptides contain within their amino 1993). These belong to the family of subtilisin/Kex2- acid sequence, peptides with different biological serine proteases and exhibit similar amino acid activities. (POMC) is endoproteo­ sequences at different domains such as furinIPACE, PCI, Iytically processed to yield ACTH, B-endorphin and u­ PC2, PACE4, PC4, PC5/PC6 and PC7. However, melanocyte stimulating hormone (Nakanishi et aI., differences have been attributed with respect proprotein 1979). contains multiple copies of the specificity as well as tissue localisation for these same peptide, i.e., six copies of Met- and one enzymes (Seidah et aI., 1994). An aspartyl endo­ copy of Leu-enkephalin (Comb et aI., 1982). The peptidase has been purified from bovine intermediate precursor of FMRF-amide synthesised by the neurons of lobe secretory vesicles, which possesses endoproteolytic the mollusc, Aplysia Cali/arnica contains this activity on POMC in vitro (Loh et aI., 1985); this tetrapeptide repeated 28 times within its amino acid enzyme cleaves mouse and toad POMC in vitro sequence and a single copy of the related peptide FLRF­ generating POMC-derived peptides found in the amide (Taussing and Scheller, 1986). intermediate lobe of the pituitary gland, in vivo (Castro Prohormones and proneuropeptides undergo several et aJ., 1989). modifications before they are released. These If cells need only one PCE or a particular modifications can be co-translational (i.e., removal of the combination of them to cleave a specific prohormonel signal peptide) or post-translational (after they are proneuropeptide still needs to be elucidated; altough the synthesised within the rough endoplasmic reticulum wide distribution of these enzymes in tissues of (RER)). Post-translational modifications are divided into neuronal/endocrine origin suggests that they might be two main categories: 1) proteolytic modifications which able to endoproteolytically process a wide number of involve endoproteases, carboxypeptidases and amino­ propeptides. The cellular machinery for endoproteolytic peptidases, and 2) chemical modifications done by processing and secretion has been conserved during enzymes which covalently modify the lateral chain of evolution, even in cells so evolutionary distant as yeast amino acids within peptides as: glycosylation, and neurons (Bennett and Scheller, 1993). This has been amidation, acetylation, phosphorylation and sulfation. demonstrated by Jung et aI., 1993, who showed that the Many of these enzymes have been biochemically endoproteolytic products of the egg-laying hormone characterised, localised to specific tissues or cellular (ELH) which is a prohormone synthesised within the bag compartments, and in most cases they have been cloned cell neurons of the marine mollusc, Aplysia califarnica, (Seidah et aI., 1990; Smeekens and Steiner 1990; Eipper are similar when this prohormone is expressed in AtT20 et aI., 1992). cells. The expression of heterologous proproteins within Endoproteolytic cleavages are one of the key steps in cells which do not possess a regulated secretory pathway the generation of many hormones/neuropeptides from and therefore do not synthesise the endoproteolytic their protein precursor molecules. After initial cleavage enzymes found in neuroendocrine cells such as PC 1 or of the signal peptide in the lumen of RER (Walter et aI., PC2, does not always yield endoproteolytically 1984) further proteolytic events of most prohormonesl processed products from the precursors, e.g., rat proneuropeptides start within late compartments of the proenkephalin (Lindberg et aI., 1991), mouse prorenin Golgi complex i.e., the trans Golgi network (TGN) (Hosaka et aJ., 1991) and rat procorticotrophin-releasing (Sossin et aI., 1990) and continue in the secretory hormone (proCRH) (Castro et aI., 1995b) in spite of vesicles (Castro et aI., 1989). Neuronal and endocrine these cells expressing furin-like endopeptidases and the cells possess cell-type specific endoproteases which precursors containing furin-like cleavage sites. Furin cleave hormone/ precursor proteins to (also called PACE), PC I and PC2 have been widely generate biologically active peptides. The sites of employed in prohormone/proneuropeptide processing cleavage are paired basic amino acid residues, such as studies (Hosaka et aI., 1991; Zhou et aI., 1993; Paquet et lysine-arginine (KR), RR, RK or KK, although endo­ aI., 1996). Transfection experiments within cells proteolysis at tri- and tetra-basic amino acid residues has expressing the cDNAs encoding for both a prohormone also been reported (for review, see Smeekens 1993). or proneuropeptide and PC 1 or PC2 have shown 1181 Biosynthesis of prohormones

differences in the processing pattern of such precursors. pH and high levels of Ca2+ requirements, both enzymes For example, the expression of exogenous PC2 cDNA have been suggested as putative physiological within AtT20 cells enhances the synthesis of POMC­ carboxypeptidases involved in hormones/neuropeptides derived peptides, such as ACTH 1_13NH2 and CLIP, processing (Song and Fricker, 1995b). which are poorly synthesised in wild type AtT20 cells Aminopeptidases catalyse the hydrolysis of amino expressing low levels of endogenous PC2. Furthermore, acid residues from the H2N-terminus of peptides. It is production POMCl _49, Lyso_ TMSH and B-endorphinl_ well known that aminopeptidases take part in 27 were only observed in Atho cells expressing PC2 degradation of neuropeptides (Turner, 1987). They can (Zhou et aI., 1993). Also, human proenkephalin shows also trim remaining H2N-terminal basic amino acid different endoproteolytic processing when it is co­ residues of peptides generated after endoproteolytic expressed with either PC1, PC2 or furin within GH4C1 cleavage of prohormones. The role of aminopeptidases cells (Breslin et aI., 1993). The corticotrophin releasing in hormone/neuropeptide biosynthesis is crucial, since hormone (CRH) precursor protein is also processed by they modifies the biological activity of neuropeptidesl PC2 within stably transfected CHO cells co-expressing hormones. PC2 and pre-proCRH (Perone et aI., 1996). It has also Several chemical modifications of peptides/hormones been observed that PC2 is capable of endoproteolytically have been described, but only in a few cases the processing proneuropeptide Y within primary cultures of physiological significance of them has been elucidated. rat superior cervical ganglion neurons (Paquet et aI. , Acetylation at the H2N-terminus and its significance 1996). It has been shown that endogenous factors in with respect to biological activity has been studied for a vivo, which may be cell- and species-specific, might number of neuropeptides/hormones. Two pep tides affect the cleavage specificity of these endopeptidases; derived from POMC undergo N-terminal acetylation; these factors can either increase or inhibit processing of i.e., B-endorphin and a-MSH. The presence of the acetyl prohormones or proneuropeptides. It has been group conveys changes in the biological activities of demonstrated that the intact form of the neuroendocrine­ these peptides. Acetylated forms of B-endorphin show a specific polypeptide 7B2 is a potent inhibitor of PC2 but drastic decrease in their binding capacity to the opiod not PC1 (Martens et aI., 1994). On the other hand, receptors f.I and 8, and therefore, they are inactive in their processed 7B2 stimulates POMC cleavage in vitro, central analgesic effects (Akil et aI., 1985). In contrast, probably activating PC2 (Braks and Martens, 1995). acetylated a-MSH increases its stimulating activity on Variations on intracellular factors might modulate melanocytes (Guttman and Boissonnas, 1961) when endoproteolysis, like pH and Ca2+ concentration. The compared to non acetylated a-MSH. Interestingly, H2N­ autocatalytic activation of proPCI and proPC2 requires terminal acetylated forms of POMC peptide products defined pH and Ca2+ concentrations within the TGN such as monoacetylated and di-acetylated MSH and (Shennan et aI., 1995; Guest et aI., 1997). acetyl-B-endorphin are found in the mammalian Deletion of a particular processing site within a intermediate pituitary lobe but not in the anterior prohormone by site direct mutagenesis may affect pituitary (Glembotski, 1982). These findings reflect cleavage at other si tes and also the fate of their tissue-specific differences in the content and probably in prohormone derived-peptides as was reported for ELH the activity of the enzyme/s responsible for the prohormone expressed in AtT20 cells. Deletion of two acetylation of prohormones/proneuropeptides peptide endoproteolytic sites within ELH prohormone causes the products. The acetylation process seems to occur during H2N-terminal products to be sorted to the regulated late stages of the secretory pathway, i.e., within secretory secretory pathway in contrast with the wild type vesicles; this has been described for bovine intermediate prohormone, where the H2N-terminal peptide products pituitary cells (Glembotski 1982). are released via the constitutive route (lung et aI., 1993). It is common to find amidated neuropeptides and This suggests that a strict succession of endoproteolytic hormones at their carboxy-terminal amino acid; often the events are necessary to reach the complete and correct amide group confers full biological activity. Amidation maturation and targeting of prohomones and proneuro­ occurs if glycine is present at the carboxy terminal end peptides. This has also been demonstrated for POMC, of peptides to be amidated (Bradbury et aI., 1982). whereby a concerted action of PCI and PC2 is needed in Generation of amidated peptides needs th~ concerted order to generate the correct peptide products (Zhou et action of two enzymes: the peptidylglycine a­ aI., 1993). hydroxylating monooxygenase (PHM) and the peptidyl­ Carboxypeptidases specifically remove basic amino hydroxyglicine a -amidating lyase (PAL), both generated acids (R or K) from the C-terminus of peptides . after endoproteolytic cleavage from the same precursor Carboxypeptidase E (CPE), also known as CPH and molecule (Perkins et aI., 1990). Amidating activity takes enkephalin convertase is thought to be the unique place principally within secretory granules; although intracellular carboxypeptidase capable of processing recent work has demonstrated that it might also take neuropeptides (Fricker, 1991). Recently, a CPE-like place in the ER (Yun and Eipper, 1995). enzyme called CPD has been purified and characterised Glycosylation is another common chemical modi­ from bovine pituitary (Song and Fricker, 1995a). Based fication found in prohormones/proneuropeptides. The on their localisation within secretory granules, the acidic physiological role of carbohydrate side-chains attached 1182 Biosynthesis of prohormones

to prohormones/proneuropeptides remains uncertain and 1989) have been identified. The physiological role of the existing data, sometimes reflects conflicting results. phosphorylation remains obscure. Moreover, one can not This could be due to the use of high levels of assign any possible organelle sorting role to tunicamycin, the most commonly used inhibitor for phosphorylation due to the ubiquitous intracellular studying protein glycosylation in vitro, which may also distribution of kinase activities, i.e., within Golgi affect protein biosynthesis. There is evidence that complex, ER and secretory granules. It has been shown glycosylation of precursor proteins might affect that the intracellular site where sulfation takes place is endoproteolysis. The endoproteolytic processing of the principally within the Golgi compartment. Carbohydrate H2N-terminal portion of POMC (POMC I_74) provides residues of glycoproteins, proteoglycans and proteins an example for this. In the anterior pituitary, POMC I _74 become sulphated at this stage within the secretory is glycosylated at both Thr45 and Asn65; on the other pathway (Lee and Huttner, 1985). It is also known that hand in the intermediate pituitary, POMC 1-74 is only most sulfated proteins at Tyr residues are secreted which glycosylated at Asn65. It has been shown that POMC 1_ might suggest some function of this chemical 74 is not endoproteolytically processed in the anterior modification in sorting events (Tooze and Huttner, pituitary; whilst in the intermediate pituitary, POMCI_74 1990). is cleaved to generate 'YTMSH and POMC I_48, the latter without any attached glycosylated chain at Thr45 (Birch 2. Hormone/neuropeptide secretion: regulated and et aI., 1991). These findings suggest that the constitutive secretory pathways carbohydrate-chains cause a steric hindrance for the endoproteolytic activity responsible for the cleavage of Sorting and secretion of hormones and neuropeptides POMC I_74 to yield POMC I_48 and 'Y3-MSH. In vitro is a carefully controlled mechanism within endocrine/ experiments using purified bovine l>OMC I_77 and neuronal cells, since hormones and neuropeptides need prohormone endoproteases support this hypothesis to be exported from the cells in which they are (Birch et aI., 1991). A similar inhibitory role was synthesised in order to reach their target tissues and exert attributed to glycosylation in the processing of prorenin; their biological effects. it was found that N-linked glycosylation may reduce Two main routes have been proposed for protein proteolysis of mouse prorenin in transfected AtT20 cells secretion from endocrine and neuronal cells, i.e., (Ladenheim et aI., 1991). An alternative approach was constitutive and regulated secretion (Gumbiner and used to assess the influence of glycosylation on Kelly, 1982). One of the main differences between these endoproteolysis of POMC, by replacing Thr45 by Ala45 two pathways is that proteins which exit the cell using using site directed mutagenesis (Noel et aI., 1991). This the constitutive route are secreted inmediately after they experiments showed that glycosylation does not playa have been synthesised and exocytosis takes place at a critical role in POMCI _74 cleavage. However, the basic constant rate without any external stimulus; although, amino acid residues are important in determining the constitutive secretion is also regulated at multiple extent of endoproteolysis. Additional structural features intracellular levels (reviewed by Mostov, 1995). may also play an important role in POMC cleavages. Constitutive secretion may be blocked by inhibiting The high degree of conservation in the glycosylation protein synthesis (Schmidt and Moore, 1994). On the pattern of POMC amongst several species (Uhler and other hand, within the regulated secretory pathway, Herbert, 1983), suggests that the oligosaccharide secreted proteins can be stored at high concentration for moieties could play essential intracellular functions such long periods of time within secretory vesicles, before as: allow proper folding of proproteins and/or to provide they are released. In cells which possess a regulated adequate "sorting signals" for targeting to appropriate secretory pathway, the exocytosis step, even in the intracellular compartments. In that respect, some abscence of protein synthesis, is triggered by an external nonglycosylated proteins seem to aggregate irreversibly stimulus, which can in turn change cytoplasmic second in association with BiP and are retained in the ER messenger levels. Immunohistochemistry at the light (Hurtley et aI., 1989). On the other hand, glycosylated­ microscopy level, makes it possible to distinguish if a proteins could have crucial extracellular roles by protein is located within the regulated or constitutive modifying the interaction with their or altering secretory pathway, due to its prescence within secretory the half life of the glycosylated peptide. Further research vesicles which are visualized as a fine immunoreactive in eukaryotic cell lines which are deficient in the punctuate pattern (Fig. 1). In contrast, cells which do not enzymes responsible for oligosaccharide assembly have a regulated secretory pathway, do not show this or trimming will provide new insights into the punctate pattern, instead they show a reticular physiological role of glycosylation in endoproteolytic distribution of the immunoreactivity, which is typical of processing, sorting and biological activity of pro­ the localisation of the prohormone within the RER (cells hormones/proneuropeptides. with constitutive secretory pathway: fibroblasts, Fig. 2) Prohormones/proneuropeptides undergo further (Castro et aI., 1995b). At the electron microscopic level modifications like phosphorylation and sulfation. the secretory granules appear with an electron-dense Phosphorylated peptides derived from POMC (Eipper core which might correlate with the high concentration and Mains, 1982) and proenkephalin (Watkinson et al., of the secretory product within these vesicles (Orci et aI., 1183 Biosynthesis of prohormones

1987). between the cytosol and the protein translocation Nascent secretory proteins, lysosomal proteins and apparatus in the ER membrane (Bacher et aI. , 1996). constitutive membrane proteins share the first steps in Signal sequences of prohormones and proneuropeptides their vectorial transport across the intracellular are usually not more than 30 amino acids in length with membranous compartments within eukaryotic cells. strong hydrophobic characteristcs. The signal sequence They must first be targeted to the RER and then, is cleaved off by a resident endopeptidase as the protein translocated towards the lumen of the RER by means of is being synthesised. Although, most of the secretory complex interactions amongst the ribosome-signal proteins known so far possess signal peptides, the fact sequence, the signal recognition particle (SRP) and SRP that some proteins lacking a conventional signal receptor. Translocation usually takes place cotrans­ sequence such us IL-I a, IL-l /3, bFGF and blood lationally. The targeting of the nascent protein to the ER coagulation factor XIIIa are released extracellulary, is regulated by three GTPases, which work as adapters

Fig. 2. Immunocytochemical staining of CHO-K1 cells expressing rat pre-procorticotrophin releasing hormone (CRH) . Panel A, shows immunoreactive (IR)-CRH within the nuclei and the cytoplasm of stably Fig. 1. Immunofluorescence detection of immunoreactive (IR)-ACTH transfected CHO-K1 cells expressing rat pre-proCRH. Panel B, shows within the neuroendocrine cell line AtT20. Panel A, shows IR-ACTH the yuxtanuclear staining of the Golgi apparatus using wheat germ within the cytoplasm and processes (arrows) of AtT20 cells. Note the agglutinin. Comparison between IR-CRH shown in panel A and the punctuate distribution of IR-ACTH within these cells. Panels Band C, Golgi staining shown in panel B, accounts for the localisation of IR-CRH show the nuclei stained with DAPI and the phase contrast micrograph within the Golgi apparatus in transfected CHO cells. Panel C. Phase respectively of the cells shown in panel A. Bar: 30 ~m. contrast micrograph of the cells shown in panel A. Bar 30 ~m . 1184 Biosynthesis of prohormones

suggests that an alternative pathway for protein secretion characterised sortases were chymotrypsinogen A or B might exist (Rubartelli et ai., 1990). It has been (Gorr et aI., 1992). Recently, it has been shown that described that these proteins can exert extracellular sorting of proopiomelanocortin (POMC) to the regulated functions. The possibility that they could be released by secretory pathway occurs by binding to a sorting cell lysis must be excluded, since for their biological receptor, which has been identified as a membrane­ activity they require post-translational modifications associated carboxypeptidase E (also known as only performed by living cells. Cleves et al. (1996) have carboxypeptidase H) (Cool et aI. , 1997). These authors shown an alternative pathway for protein secretion in have also demonstrated that this receptor specifically yeast. This novel secretory mechanjsm is independent of binds proteins which are secreted via the regulated a signal peptide and does not involve the yeast multidrug secretory pathway. resistance-like transporter Ste 6p. The isolation of the The term protein "maturation" within secretory genes involved in this alternative secretory mechanism, granules is employed to described a complex process may provide the tools for the identification of their whereby the clathrin-coat of the secretory granules is mammalian counterparts. lost. Maturation implies, moreover, progressive The trans-most cisternae of the Golgi complex, a acidification, prohormone post-translational modifi­ clathrin-coated compartment, is the site where sorting cations, changes in calcium concentration, correct between secretory and constitutive proteins takes place protein folding and aggregation within the vectorial (Orci et aI., 1987; Tooze and Huttner, 1990). The route from RER towards the secretory granules. It has presence of distinct secretory pathways within the same been demonstrated that protein aggregation might be a cell requires a sorting mechanism to transport secretory mechanism capable of sortjng proteins to the regulated proteins to the correct pathway from the ER to Golgi pathway from resident and constitutively secreted apparatus and finally to secretory granules destined for proteins (Tooze et aI., 1989b); moreover, quality control regulated secretion. Neuronal and endocrine cells, as mechanisms seem to recognise and retain in the early highly specialized secretory cells, which must separate compartments of the secretory pathway, misfolded and hormones and neuropeptides from other proteins such as partially folded proteins in order to prevent further membrane proteins or lysosomal hydrolases. To date, the transport (Hammond and Helenius, 1994). mechanism implicated in sorting of proteins destined to Endoproteolytic processing of prohormones might the regulated secretory pathway is poorly understood. It affect sorting and intracellular fate of their cleavage­ has been proposed that sorting domains of most derived peptides as was demonstrated in the case of ELH regulated secretory proteins reside in their H2N-terminal prohormone in the bag cell neurons of Aplysia. The region, immediately after the signal sequence amjno- and the carboxy-terminal derived peptides from responsible for ER translocation. Comparison of amino ELH prohormone were targeted to and packaged into acid sequences within this region from different different dense core secretory vesicle within the secretory proteins does not show a consensus sequence; regulated secretory pathway (Sossin et aI., 1990). This however, their hydrophobic characteristics and results in different subsets of secretory vesicles secondary structures appear to be the key elements for containing different peptide products derived from the sorting, and these are absent in constitutively secreted same precursor which exert different biological proteins (GOIT and Darling, 1995). Consistent with this activities. hypothesis, it has been reported that the domain Prolactin and growth hormone are both packaged into responsible for the targeting of POMC to the regulated different secretory vesicles within somatomammotrophic secretory pathway resides within its H2N-terminal cells (Fumagalli and Zanini, 1985). Different sorting region which contains an amphipatic loop of 13 amino domains within both hormones might be recognised by acids. Moreover, this conformational signal is dependent the protein sorting machinery of eukaryotic cells, on the integrity of the disulfide bridge between amino whereby these peptides are packaged within different acids Cys8/Cys20 (Cool et aI., 1995). Within pro­ vesicle populations. The corticotrophic neuroendocrine , the information for targeting to the cell line AtT20, is also able to differentially release regulated secretory pathway in transfected mammalian endogenous ACTH and heterologous immunoreactive­ endocrine cell lines is located at the amino-terminal CRH molecules after an extracellular stimulus (Perone region (Sevarino et aI., 1989). Sorting domains within and Castro, unpublished results). This finding suggests precursor molecules could also be recognised by a that ACTH and IR-CRH molecules are packaged into membrane-anchored receptor/carrier at the trans-most different sets of secretory vesicles within AtT20 cells. cisternae of the Golgi complex and this binding could Also, different intracellular mechanisms involving mediate sorting to the regulated secretory pathway. In second messengers could mediate the release of different that respect, a group of 25 kD proteins called «sortases» populations of vesicles containing secretory peptides. that bind selectively prolactin, insulin and human growth The Golgi apparatus in neuroendocrine cells and hormone but did not bind proteins which do not enter the neurons possesses a polarised juxtanuclear localisation regulated secretory pathway, were thought to be the (Lowenstein et aI., 1994). Therefore, it is posible to sorting receptor (Chung et aI. , 1989). Work done by GOIT assume that the direction of hormone secretion is and co-workers has demonstrated that the previously governed by the Golgi apparatus in these cells. Secretory 1185 Biosynthesis of prohormones

vesicles containing hormones might travel towards the the cellular processess awaiting to be released upon cell surface associated and perhaps, guided by stimulation (Fig. I) (Castro et al., 1992; Castro and microtubules and or microfilaments. This assumption is Morrison, 1997). sustained by experiments employing drugs which disrupt Bottger and Spruce (1995) working with another the microtubule-microfilament network of cells in neuropeptide precursor, i.e., proenkephalin have culture. To date, the role of cytoskeleton in the secretion demonstrated nuclear localisation of this precursor in of prohormones/proneuropeptides is unclear. However, rodent and human embryonic fibroblast cell lines (Swiss our studies in AtT20 cells expressing proCRH suggest 3T3 and MRC-5 cells) and in rodent myoblast cells that neither disruption of microtubules nor micro­ (C2C 12 cells). These authors showed that when filaments affects the constitutive release of CRH 1-4 1' proenkephalin was expressed in COS cells, it was Furthermore, we observed that actin filaments playa role localised exclusively in the cytoplasm, while pro­ in mediating the release of CRH 1-41 trough the regulated enkephalin mutated at the first ATG codon or devoid of secretory pathway in transfected AtT20 cells (Perone its signal peptide, was targeted to the nucleus as well as and Castro, unpublished results). To date, nothing is the cytoplasm (BoUger and Spruce, 1995). Results from known about the numerous associated microtubule­ our laboratory using CHO cells expressing proenke­ microfilament proteins which perhaps are the true phalin (Lindberg et aI., 1991) show only cytoplasmic motors in vesicular trafficking within eukarytic secretory localisation for immunoreactive-enkephalin as assessed cells (i.e., endocrine cells, neurons). using immuofluorescence techniques and confocal microscopy (Castro and Lindberg, unplublished data). 3. Cell-type specific targeting of prohormonesl The mechanism of nuclear translocation of prohormonel proneuropeptides proneuropeptides is at present unclear. Certain proteins need a nuclear localisation signal for nuclear import or From the previous section we can conclude that to be associated with chaperone proteins carrying a targeting and sorting domains sequences lack primary nuclear localisation signal (Silver, 1991). Recently it has amino acid sequence homology. Therefore, other factors been reported a sugar dependent process for glyco­ could determine protein sorting, such as tertiary structure protein nuclear import (Duverger et aI., 1995); this and general physicochemical properties (i.e., hydro­ observation opens an interesting avenue to explore the phobicity and hydrophilicity). role of carbohydrate in the mechanism of nuclear Work done in our laboratory and in others has internalisation of prohormones/proneuropeptides. recently shown that cellular factors must also play an Nuclear translocation of prohormones/proneuropeptides important role in determining the intracellular targeting could be due to transport of soluble cytoplasmic forms and final localisation of a given proprotein. Using pre­ of these molecules which may arise through the usage of proCRH as a model proneuropeptide molecule, we alternative initiation codons for translation. This might demonstrated that the intact precursor is translocated to give rise to prohormones/proneuropeptides with the cytoplasm (i.e., secretory pathway), and also to the truncated or extended signal peptide which prevent their nucleus within stably transfected CHO-K I cells (Castro translocation to the ER (Murray, 1996). and Morrison, 1995; Castro et aI., 1995a; Morrison et aI., The results reviewed above point to the crucial role 1995) (Fig. 2). Nuclear proCRH is in close association played by cellular factors in determining the intracellular with double stranded DNA, suggesting that it might play localisation of prohormones/proneuropeptides. This a role in the regulation of gene expression (Castro et aI., draws the attention for the need of additional factors 1995b). The intact pre-proCRH molecule expressed (molecular chaperones), besides the specific sorting within COS-7 cells, in spite of being correctly targeted domains which reside within the primary amino acid to the secretory pathway was not secreted into the sequences of the prohormones/proneuropeptides extracellular medium. The lack of secretion is due to the formation of large precursor aggregates which did not human' ore-DroCRH!1-196! exit the RER of these cells (Morrison et aI., 1992; Castro and Morrison, 1997). When pre-proCRH was expressed RR RERR RK GK within stably AtT20 cells, which possess a regulated N-proCRH(27-122) II rlr;~~~l1 1111 II II secretory pathway and are capable of differentiating in 1 26 CRH(1-41) m _---=;.;.;;.:.1.:.-"":":"-1...... --41 vitro to achieve a neuronal phenotype (Tooze et aI., SP ~1------p-ro~C~RH~(~27~-~19~6~)------~ 1989a), pre-proCRH was targeted to the regulated secretory pathway within these cells; the release of Fig. 3. Structure of the human pre-proCRH molecule. The various proCRH cleavage products, i.e. proCRH 125-151 and domains of the precursor molecule are noted as: SP, signal peptide CRH l _41 (Fig. 3) was stimulated in response to spaning the first 26 amino acids of the precursor molecule; CRH l _41 , extracellular stimulus indicating targeting to secretory corticotrophin releasing hormone (aa: 1-41); N-proCRH27-122, H2N­ vesicles (Castro et aI., 1991). This was confirmed using terminal procorticotrophin releasing hormone (aa: 27-122); proCRH 125_ 151, procorticotrophin releasing hormone (aa: 125-151). Putative sites of immunofluorescence techniques and antibodies specific endoproteolytic cleavage are denoted by amino acids in the single letter for CRH 1-41' Immunoreactive (lR)-CRH was seen as a code. R: arginine, K: lysine; G: glycine; S:serine; E: glutamic acid. Note bright punctuate pattern which accumulates at the tips of that this figure is not drawn to scale. 1186 Biosynthesis of prohormones

themselves. These molecular chaperones could mediate chaperone 7B2 can enhance in vitro POMC cleavage by hormone folding, intracellular trafficking and sorting of neuro­ convertase PC2. FEBS Lett. 371 , 154-158. peptideihormone precursors. Breslin M.B. , Lindberg I. , Benjannet S., Mathis J.P., Lazure C. and Seidah N.G. (1993). Differential processing of proenkephalin by Future prospects prohormone convertases 1(3) and 2 and furin. J. BioI. Chem. 268, 27084-27093. Although our knowledge on the molecular Castro M.G. and Morrison E. (1995). nuclear translocation of neuro­ mechanisms underlying the intracellular sorting, peptides: possible nuclear roles. J. Endocrinol. 146, 9-13. trafficking and endoproteolytic processing of hormone/ Castro M.G. and Morrison E. (1997). Post-translational processing of neuropeptide precursors has advanced rapidly over the proopiomelanocortin in the pituitary and in the brain. Crit. Rev. last decade, there are still many questions which need to Neurobiol. 11, 35-57. be answered. For example, the molecular mechanisms Castro M.G ., Birch N.P. and Loh Y.P. (1989). Regulated secretion of underlying the sorting of prohormones/proneuropeptides proopiomelanocortin converting enzyme and an amino peptidase b­ to the regulated secretory pathway; the accurate like enzyme from dispersed bovine intermediate lobe pituitary cells. identification of the intracellular compartments where J. Neurochem. 52, 1619-1628. post-translational processing takes place; the intra­ Castro M.G., Brooke J., Bullman A. , Hannah M. , Glynn B.P and Lowry cellular localization and release of different products P .J . (1991). Biosynthesis of corticotrophin-releasing hormone derived from a single prohormone/proneuro-peptide; and (CRH) in mouse corticotrophic tumour cells expressing the human the molecular mechanisms underlying ceilltissue­ proCRH gene: intracellular storage and regulated secretion. J. Mol. specific sorting and processing. We hope that what we Endocrinol. 7, 97-104. learn from these mechanisms will provide further insight Castro M.G. , Lowry P.J. and Lowenstein P.R. (1992). Antibodies to into the molecular events which determine neuropeptide precursors expressed as fusion proteins in E. Coli. communication between cells of the endocrine and Their use to examine neuropeptide biosynthetic pathways, nervous systems. intracellular trafficking and physiological activities. Int. J. Biochem. 24, 847-860. Acknowledgements. This work is supported by the Biotechnology and Castro M.G., Morrison E., Tomasec P. , Linton E.A. and Lowenstein P.R. Biological Sciences Research Council, Chemicals and Pharmaceuticals (1995a). Co-localisation of autoimmune antibodies specific for Directorate through a project grant to MGC. We also acknowledge the double stranded DNA with procorticotrophin-releasing hormone support which our laboratory receives from The Wellcome Trust, The within the nucleus of stably transfected CHO-K1 cells. Cell Tissue Cancer Research Campaign, The Parkinson's Disease Society, The Res. 282, 367-376. Royal SOCiety, REMEDI , The Sir Halley Stewart Trust, Sandoz Castro M.G., Tomasec P., Morrison E., Murray C.A., Hodge P., Blanning Foundation for Gerontological Research and The Faculty of Medicine P. , Linton E., Lowry P.J. and Lowenstein P.R. (1995b). Mitogenic Bequest Fund, University of Manchester. We thank Prof. A. M. effects and nuclear localisation of procorticotrophin-releasing Heagerty, Prof S. Tomilson and Dr. J. Brider for their support and hormone expressed within stably transfected fibroblast cells (CHO­ encouragement. K1). Mol. Cell Endocrinol. 107, 17-27. Chung K.-N. , Walter P., Aponte G.w. and Moore H-P.H. (1989). Molecular sorting in the secretory pathway. 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