Heterologous expression of peptide hormone precursors in the yeast Saccharomyces cerevisiae. Evidence for a novel prohormone endoprotease with specificity for monobasic amino acids.

The peptide somatostatin (SRIF) exists as two different molecular species. In addition to the most common form, which is a 14-residue peptide, there is also a 14-amino acid amino-terminally extended form of the tetradecapeptide, SRIF-28. Both peptides are synthesized as larger precursors containing paired basic and monobasic amino acids at their processing sites, which, upon cleavage, generate either SRIF-14 or -28, respectively. In mammals a single prepro-SRIF molecule undergoes tissue-specific processing to generate the mature hormone whereas in some species of fish separate genes encode two distinct but homologous precursors prepro-SRIF-I and -II that give rise to SRIF-14 and -28, respectively. To investigate the molecular basis for differential processing of the prohormones we introduce their cDNAs into yeast cells (Saccharomyces cerevisiae). The signal peptides of both precursors were poorly recognized by the yeast endoplasmic reticulum translocation apparatus, consequently only low levels of SRIF peptides were synthesized. To circumvent this problem a chimeric precursor consisting of the alpha-factor signal peptide plus 30 residues of the proregion was fused to pro-SRIF-II. This fusion protein was efficiently transported through the yeast secretory pathway and processed to SRIF-28 exclusively, which is identical to the processing of the native precursor in pancreatic islet D-cells. Most significantly, cleavage of the precursor to SRIF-28 was independent of the Kex 2 endoprotease since processing occurred efficiently in a kex 2 mutant strain. We conclude that in addition to the Kex 2 protease, yeast possess a distinct prohormone converting enzyme with specificity toward monobasic processing sites.     

The role of paired basic amino acids in mediating proteolytic cleavage of prosomatostatin. Analysis using site-directed mutagenesis

Many small peptide hormones are synthesized as larger precursors in which the mature hormone sequence is flanked by pairs of basic amino acids. These precursors often undergo extensive post-translational modifications; a critical step in this process is proteolytic excision of the hormone at the paired basic residues. To determine the role of paired basic amino acids as recognition signals for cleavage by processing enzymes, we investigated the heterologous expression of prosomatostatin (the pro-somatotropin release inhibiting factor (pro-SRIF). Pro-SRIF is one of the simplest peptide hormone precursors, possessing a single copy of the 14-residue SRIF peptide at its carboxyl terminus preceded by the least common pair of basic amino acids, Arg-Lys. Employing site-directed mutagenesis, we altered the paired basic cleavage site to the more common Arg-Arg and Lys-Arg residues. The native and mutated precursors were expressed in rat pituitary GH3 cells and mouse 3T3 cells using a retroviral vector. Alteration of the paired basic residues had no effect on the specificity of proteolytic cleavage as both the native and mutant precursors were processed with 70 to 80% efficiency in GH3 cells. Surprisingly, when the mutant pro-SRIFs were expressed in 3T3 cells, which do not process the native precursor, the Arg-Arg and Lys-Arg precursors were processed with 16 and 20% efficiency, respectively. The role of an acidic compartment in mediating pro-SRIF cleavage was also investigated using low concentrations of the lysosomotrophic drug Chloroquine. Twenty-five microM Chlorquine completely inhibited pro-SRIF cleavage and intracellular storage; the unprocessed precursor was secreted into the medium. We conclude that (i) exposure to an acidic compartment is required for pro-SRIF maturation, and (ii) the conformation of the processing site, rather than the composition of the basic amino acids, defines cleavage specificity by prohormone processing enzymes.     

Evidence of a novel dipeptidyl aminopeptidase in mammalian GH(3) cells: new insights into the processing of peptide hormone precursors

We investigated whether yeast signals could regulate hormone processing in mammalian cells. Chmeric genes coding for the prepro region of yeast alpha-factor and the functional hormone region of anglerfish somatostatin was expressed in rat pituitary GH(3) cells. The nascent prepro-alpha-factor-somatostatin peptides disappeared from cells with a half-life of 30 min, and about 20% of unprocessed precursors remained intracellular after a 2 h chase period. Disappearance of propeptide was insensitive to lysosomotropic agents, but was inhibited at 15 degrees C or 20 degrees C, suggesting that the hybrid propeptides were not degraded in the secretory pathway to the trans Golgi network or in lysosomes. It appeared that while most unprocessed precursors were constitutively secreted into the medium, a small portion were processed at their paired dibasic sites by prohormone-processing enzymes located in trans Golgi network/secretory vesicles, resulting in the production of mature somatostatin peptides. To test this hypothesis, we investigated the processing pattern of two different hybrid precursors: the 52-1 hybrid precursor, which has a Glu-Ala spacer between the prepro region of alpha-factor and somatostatin, and the 58-1 hybrid precursor, which lacks the Glu-Ala spacer. Processing of metabolically labeled hybrid propeptides to smaller somatostatin peptides was assessed by HPLC. When pulse-labeled cells were chased for up to 2 h, 68% of the initially synthesized propeptides were secreted constitutively. About 22% of somatostatin-related products were proteolytically processed to mature somatostatin, of which 38.7% were detected intracellularly after 2 h. From N-terminal peptide sequence determination of somatostatin-related products in GH(3)-52 and GH(3)-58 cells, we found that both hybrid precursors were accurately cleaved at their dibasic amino acid sites. Notably, we also observed that the Glu-Ala spacer sequence was removed from 52-1 hybrid precursors. The latter result strongly suggests that a novel dipeptidyl aminopeptidase activity - a yeast STE13-like enzyme - is present in the post-trans Golgi network compartment of GH(3) cells. The data from these studies indicate that mechanisms which control protein secretion are conserved between yeast and mammalian cells.     

Secretion of somatostatin by Saccharomyces cerevisiae. Correct proteolytic processing of pro-alpha-factor-somatostatin hybrids requires the products of the KEX2 and STE13 genes

Somatostatin is a 14-amino-acid peptide hormone that is proteolytically excised from its precursor, prosomatostatin, by the action of a paired-basic-specific protease. Yeast (Saccharomyces cerevisiae Mat alpha) synthesizes an analogous peptide hormone precursor, pro-alpha-factor, which is proteolytically processed by at least two separate proteases, the products of the KEX2 and STE13 genes, to generate the mature bioactive peptide. Expression in yeast of recombinant DNAs encoding hybrids between the proregion of alpha-factor and somatostatin results in proteolytic processing of the chimeric precursors and secretion of mature somatostatin. To determine if the chimeras were processed by the same enzymes that cleave endogenous pro-alpha-factor, the hybrid DNAs were introduced into kex2 and ste13 mutants, and the secreted proteins were analyzed. Expression of the pro-alpha-factor-somatostatin hybrids in kex2 mutant yeast resulted in secretion of a high molecular weight hyperglycosylated precursor. No mature somatostatin was secreted, and there was no proteolytic cleavage at the Lys-Arg processing site. Similarly, in ste13 yeast, only somatostatin molecules containing the (Glu-Ala)3 spacer peptide at the amino terminus were secreted. Our results demonstrate that in yeast processing mutants, the behavior of the chimeric precursors with respect to proteolytic processing was exactly as that of endogenous pro-alpha-factor. We conclude that the same enzymes that generate mature alpha-factor proteolytically process hybrid precursors. This suggests that structural domains of the proregion rather than the mature peptide are recognized by the processing proteases.     

Secretion of somatostatin by Saccharomyces cerevisiae. Correct processing of an alpha-factor-somatostatin hybrid

Somatostatin is a 14-amino acid peptide hormone that is proteolytically processed from its precursor, prosomatostatin, by a paired-basic-specific protease localized in the Golgi apparatus and secretory vesicles. Yeast (Saccharomyces cerevisiae MAT alpha) synthesize an analogous peptide hormone precursor, pro-alpha-factor, that contains tandem repeats of alpha factor (13 amino acids) flanked by spacers that include paired basic residues. To investigate the role of these two pro regions in mediating intracellular transport and processing, cloned genes specific for preprosomatostatin and prepro-alpha-factor were used to generate recombinants encoding hybrids between the alpha-factor pro region (amino-terminal) and somatostatin (carboxyl-terminal). These recombinants were inserted into yeast expression vectors under control of either the native alpha-factor promoter or the inducible yeast PHO5 (acid phosphatase) promoter. Yeast transformed with these plasmids expressed the hybrid messenger RNAs constitutively (alpha-factor promoter) or when induced in phosphate-deficient medium (PHO5 promoter). Radioimmunoassay of culture media revealed the secretion of up to 200 ng of immunoreactive somatostatin/10(7) cells. Metabolic labeling with [35S]cysteine, followed by immunoprecipitation with anti-somatostatin antibodies revealed two forms of hybrid precursor intracellularly, one of Mr 25,000, containing core carbohydrates, and a second of Mr 11,000, which was unglycosylated. Translation of mRNA extracted from these transformants in the wheat germ cell-free system revealed that the Mr 11,000 form was the primary translation product, whereas the Mr 25,000 species could be generated in vitro by inclusion of mammalian rough microsomes. The secreted immunoreactive material was shown to be authentic somatostatin by high pressure liquid chromatography analysis and protein sequencing. These results demonstrate that the yeast processing enzymes recognize these chimeric precursors, resulting in the secretion of the mature peptide hormone.     

Retrovirus-mediated expression of preprosomatostatin in rat pituitary GH3 cells: targeting of somatostatin to the regulated secretory pathway

Somatostatin (SRIF) is a 14-amino acid peptide hormone that is synthesized as part of a larger precursor, prepro-SRIF, consisting of a signal peptide and a proregion of 80-90 amino acids; mature SRIF is located at the carboxyl-terminus of the precursor. We have used a recombinant retroviral expression vector encoding anglerfish prepor-SRIF-I to infect rat pituitary GH3 cells. The aim of these studies was to investigate the intracellular storage and secretion of the total pool of endogenous GH compared to that of SRIF. Several clonal lines of GH3 cells expressing high or low levels of SRIF were treated with TRH, forskolin, or depolarizing concentrations of potassium, and the levels of intracellular and secreted GH or SRIF were determined using highly sensitive RIAs. Approximately 65% of the total GH was secreted basally, whereas less than 20% of the SRIF-immunoreactive material was basally secreted. Forskolin treatment or potassium depolarization stimulated GH release, but only about 50% above basal levels. In contrast, SRIF secretion was stimulated approximately 5-fold in response to these secretagogues. Based on its lower basal rate of secretion compared to GH and its enhanced release in response to a variety of secretagogues, we conclude that the heterologously expressed SRIF is preferentially targeted to the regulated pathway in GH3 cells.     

Sequential transport of protein between the endoplasmic reticulum and successive Golgi compartments in semi-intact cells.

The vectorial transport of vesicular stomatitis virus (VSV) G protein between the ER and the cis and medial Golgi compartments has been reconstituted using semi-intact (perforated) cells. The transport of VSV-G protein between successive compartments is measured by the sequential processing of the two N-linked oligosaccharide chains present on VSV-G protein to the endoglycosidase (endo) H-resistant structures which have unique electrophoretic mobilities during sodium dodecyl sulfate-gel electrophoresis. The appearance of a form of VSV-G which contains only one endo H-resistant oligosaccharide chain (GH1) is kinetically and biochemically indistinguishable from the appearance of the Man5, endo D-sensitive form (GD), the latter being a processing reaction diagnostic of transport from the ER to the cis Golgi compartment. These results provide evidence that the cis Golgi compartment may contain in addition to alpha-1,2-mannosidase I, both N-acetylglueosamine transferase I and alpha-1,2-mannosidase II. VSV-G protein is subsequently processed to the form which contains two endo H-resistant oligosaccharides (GH2) after a second wave of vesicular transport. Processing of GH1 to GH2 in vitro occurs only after a lag period following the appearance of GH1; processing is sensitive to N-ethylmaleimide, guanosine-5'-O-(3-thiotriphosphate), and a synthetic peptide homologous to the rab1 protein effector domain, and processing is inhibited in the absence of free Ca2+ (in the presence of EGTA), reagents which potently inhibit ER to cis Golgi transport. These results suggest that VSV-G protein proceeds through at least two rounds of vesicular transport from the ER to the medial Golgi compartment for processing to the GH2 form, providing a model system to study the regulation of the vectorial membrane fission and fusion events involved in vesicular trafficking and organelle dynamics in the early stages of the secretory pathway.     

The influence of mammalian and teleost somatostatins on the secretion of growth hormone from goldfish (Carassius auratus L.) pituitary fragments in vitro

The effect of various vertebrate somatostatins (SRIF) on basal growth hormone (GH) secretion from goldfish pituitary fragments was studied using an in vitro perifusion system. SRIF-14 caused a rapid and dose-dependent decrease in the rate of GH release from goldfish pituitary fragments. The half-maximal effective dose (ED50) of SRIF-14 was calculated as 1.3 nM following exposure to two minute pulses of increasing concentrations of SRIF-14, whereas the ED50 of SRIF-14 calculated after continuous exposure to sequentially increasing doses of SRIF-14 was 65 nM. This difference suggests that the pituitary fragments were less responsive to SRIF-14 in the latter experiment, possibly as a result of previous exposure to SRIF-14. SRIF-28 was found to be equipotent with SRIF-14 in decreasing basal GH secretion from the goldfish pituitary. In contrast, catfish SRIF-22, a uniquely teleost SRIF isolated from catfish pancreatic islets, did not alter GH secretion. These results provide further support for the hypothesis that SRIF-14 or a very similar molecule functions as a GH release-inhibiting factor in teleosts, indicating that this action of SRIF-14 has been fully conserved throughout vertebrate evolution.     

Subcellular sites of processing of precursors to neurosecretory peptides in the bag cells of Aplysia: Inferences from the effects of monensin,FCCP, and chloroquine

The effects of the sodium ionophore monensin were examined in the bag cells of Aplysia californica in order to identify the subcellular sites of processing of precursors to their neurosecretory products. Incubation of bag cells in media containing 10 μM monensin led to a marked disruption of the morphology of the Golgi apparatus without affecting that of other organelles. Exposure of bag cells to monensin led to a significant impairment of processing of the largest precursor and of an intermediate protein which gives rise to the immediate precursors to the final secreted products, the egg-laying hormone (ELH) and the acidic peptide (AP). Furthermore, ELH and AP were never produced in the presence of monensin during the time course of these experiments. When axonal transport was allowed to proceed, the contents of bag-cell terminals indicated that the intermediate protein is the first to be packaged in Golgi-derived vesicles, and in monensin-treated cells may be transported without being processed further. In contrast to these results, the protonophore FCCP-impaired precursor and intermediate cleavage equally, indicating that monensin and FCCP have different effects on intracellular transport and precursor processing. These data are interpreted to indicate that the largest ELH-AP precursor is normally processed within the Golgi apparatus, and that the disruption of this organelle induced by monensin produces the impairment seen in its processing. The impairment of cleavage of the intermediate species, and the blockade of production of AP and ELH, are probably the result of monensin-induced impairment of production of proteolytically competent secretory granules by the Golgi apparatus.     

Role of acidic intracellular compartments in the biosynthesis of Dictyostelium lysosomal enzymes. The weak bases ammonium chloride and chloroquine differentially affect proteolytic processing and sorting

Radiolabel pulse-chase and subcellular fractionation procedures were used to analyze the transport, proteolytic processing, and sorting of two lysosomal enzymes in Dictyostelium discoideum cells treated with the weak bases ammonium chloride and chloroquine. Dictyostelium lacks detectable cation-independent mannose-6-phosphate receptors and represents an excellent system to investigate alternative mechanisms for lysosomal enzyme targeting. Exposure of growing cells to ammonium chloride, which increased the pH in intracellular vacuoles from 5.4 to 5.8-6.1, slowed but did not prevent the proteolytic processing and correct localization of pulse-radiolabeled precursors to the lysosomal enzymes alpha-mannosidase and beta-glucosidase. Additionally, ammonium chloride did not affect transport of the enzymes to the Golgi complex, as they acquired resistance to the enzyme endoglycosidase H at the same rate as in control cells. When the pH of lysosomal and endosomal organelles was raised to 6.4 with higher concentrations of ammonium chloride, the percentage of secreted (apparently mis-sorted) precursor polypeptides increased slightly, but proteolytic processing of intermediate forms of lysosomal enzymes to mature forms was greatly reduced. The intermediate and mature forms of alpha-mannosidase and beta-glucosidase did, however, accumulate intracellularly in vesicles similar in density to lysosomes. In contrast, in cells exposed to low concentrations of chloroquine the intravacuolar pH increased only slightly (to 5.7); however, enzymes were inefficiently processed and, instead, rapidly secreted as precursor molecules. Experiments involving the addition of chloroquine at various times during the chase of pulse-radiolabeled cells demonstrated that this weak base acted on a distal Golgi or prelysosomal compartment to prevent the normal sorting of lysosomal enzymes. These results suggest that although acidic endosomal/lysosomal compartments may be important for the complete proteolytic processing of lysosomal enzymes in Dictyostelium, low pH is not essential for the proper targeting of precursor polypeptides. Furthermore, certain amines may induce mis-sorting of these enzymes by pH-independent mechanisms.     

Effects of ammonia on processing and secretion of precursor and mature lysosomal enzyme from macrophages of normal and pale ear mice: evidence for two distinct pathways

Lysosomal enzymes have been shown to be synthesized as microsomal precursors, which are processed to mature enzymes located in lysosomes. We examined the effect of ammonium chloride on the intracellular processing and secretion of two lysosomal enzymes, beta-glucuronidase and beta-galactosidase, in mouse macrophages. This lysosomotropic drug caused extensive secretion of both precursor and mature enzyme forms within a few hours, as documented by pulse radiolabeling and molecular weight analysis. The normal intracellular route for processing and secretion of precursor enzyme was altered in treated cells. A small percentage of each precursor was delivered to the lysosomal organelle slowly. Most precursor forms traversed the Golgi apparatus, underwent further processing of carbohydrate moieties, and were then secreted in a manner similar to secretory proteins. The lag time for secretion of newly synthesized beta-galactosidase precursor was notably longer than that for the beta-glucuronidase precursor. The source of the secreted mature enzyme was the lysosomal organelle. Macrophages from the pale ear mutant were markedly deficient in secretion of mature lysosomal enzyme but secreted precursor forms normally. These results suggest that ammonia-treated macrophages contain two distinct intracellular pathways for secretion of lysosomal enzymes and that a specific block in the release of lysosomal contents occurs in the pale ear mutant.     

Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.     

An in vivo characterization of the cleavage site specificity of the insulin cell prohormone processing enzymes

Most peptide hormones and neurotransmitters are synthesized as larger precursor proteins, which are post-translationally processed to mature bioactive products. An early event in prohormone maturation is endoproteolytic cleavage, occurring usually at pairs of basic amino acids (e.g. Lys-Arg). Since many of the characteristics of a prohormone endoprotease are unknown, distinguishing these enzymes from other cellular proteases in vitro has been difficult. In this report, the substrate specificity of a model prohormone processing system, the insulinoma cell line Rin m5F, was characterized in vivo to establish a set of criteria by which putative proinsulin endoproteases may be assessed. To determine the role of composition of the paired basic amino acid site in directing cleavage, a series of mutant prohormones containing altered cleavage sites was constructed and expressed in Rin m5F cells. Proopiomelanocortin (POMC) was used as a substrate since this prohormone was previously shown to be processed by these cells. To control for positional effects, all four permutations of lysine and arginine (Lys-Arg, Arg-Arg, Arg-Lys, and Lys-Lys) were introduced at both the efficiently processed cleavage site separating the ACTH and beta-lipotropin (beta-LPH) domains of POMC and at the inefficiently processed site in the beta-endorphin sequence near the COOH-terminus of the precursor. His-Arg and Met-Arg sites were also introduced at the ACTH/beta-LPH junction to assess the requirement for paired lysines and arginines. Identification of POMC-derived peptides demonstrated efficient processing of Lys-Arg and inefficient processing of Lys-Lys and Arg-Lys sites at both positions in the prohormone. The Arg-Arg sequence, however, was processed in a position-dependent manner, being efficiently cleaved between ACTH and beta-LPH but only about 50% processed within beta-endorphin. His-Arg was not cleaved in Rin m5F cells, although surprisingly Met-Arg was partially processed. These results indicate a strict preference of the insulinoma prohormone endoprotease(s) for paired basic amino acids ending in arginine, but that processing efficiency of some sequences may be modulated by location within the precursor molecule.     

Pathways involved in targeting and secretion of a lysosomal enzyme in Dictyostelium discoideum

In Dictyostelium discoideum, the lysosomal enzyme alpha-mannosidase is first synthesized as an N-glycosylated precursor of Mr 140,000. After a 20-30-min lag period, up to 30% of the precursor molecules are rapidly secreted, whereas the rest remain cellular and are proteolytically processed (t 1/2 = 8 min) to mature subunits of Mr 58,000 and 60,000. The secreted precursor is modified more extensively than the cellular form, as is revealed by differences in size, charge, and sensitivity to endoglycosidase H. Subcellular fractionation has shown that, following synthesis in the rough endoplasmic reticulum, the precursor is transported to a low density membrane fraction that contains Golgi membranes. Proteolytic processing takes place in these vesicles, since newly cleaved mature enzyme, but no precursor, co-fractionates with lysosomes. Under conditions that disrupt vesicular membranes, the precursor remains associated with the membrane fraction, whereas the newly processed mature enzyme is soluble. Proteolytic cleavage of the precursor thus coincides with the release of the mature enzyme into the lumen of a lysosomal compartment. These findings suggest a possible mechanism for lysosomal targeting that involves the specific association of enzyme precursors with Golgi membranes.     

The effect of somatostatins (SRIF-14, 25 and 28), galanin and anti-SRIF on plasma growth hormone levels in Coho salmon (Oncorhynchus kisutch, Walbaum)

Porcine galanin, somatostatins (SRIF-25 and SRIF-28) and invariant SRIF-14, known to have inhibitory-stimulatory actions on growth hormone (GH) secretion in higher vertebrates, were tested for their ability to affect plasma GH levels in coho salmon. Peptides were administered by intraperitoneal injection of 10 or 100 ng g⁻¹ body weight. All three SRIFs decreased plasma GH concentrations, their activity following the order SRIF-14 > SRIF-28 > SRIF-25. Galanin and an anti-SRIF produced pronounced, although transient increases in plasma GH.     

Periprandial changes in growth hormone release in goldfish: role of somatostatin, ghrelin, and gastrin-releasing peptide

In goldfish, growth hormone (GH) transiently rises 30 min after meals, returning to baseline at 1 h postmeal. Somatostatin (SRIF) is the major inhibitor of GH release. Three cDNAs encoding pre-pro-SRIF (PSS) have been previously cloned from goldfish brain: PSS-I, which encodes SRIF-14; PSS-II, which is potentially processed into gSRIF-28 that has [Glu(1),Tyr(7)(,)Gly(10)]SRIF-14 at the COOH terminus; and PSS-III, which encodes [Pro(2)]SRIF-14 at its COOH terminus. In goldfish, bombesin (BBS), mimicking the endogenous gastrin-releasing peptide (GRP), acutely suppresses food intake and also stimulates GH release. Ghrelin was recently characterized in goldfish as a GH secretagogue and an orexigen. In this paper, we studied the changes in SRIF mRNA levels during feeding and analyzed the influences of BBS and ghrelin peptides on forebrain PSS expression. The results showed a 60% reduction in PSS-II mRNA after meals, but no changes in the expression of PSS-I and PSS-III were found. Intraperitoneal injections of 100 ng/g body wt of BBS increased GH secretion and decreased PSS-I and PSS-II gene expression. Intraperitoneal injection of goldfish ghrelin (100 ng/g body wt) transiently increased the serum GH levels and increased PSS-I, while decreasing PSS-II mRNA levels. Ghrelin (50 ng/g body wt) blocked the effects of BBS (100 ng/g body wt) on PSS-I but not on PSS-II expression. Coadministration of BBS and ghrelin decreased only the PSS-II gene expression. We conclude that the interactions between BBS/GRP and ghrelin can account for the postprandial variations in serum GH levels and the forebrain expression of PSS-II. Furthermore, we demonstrate that intraperitoneal administration of BBS reduces the ghrelin expression levels in the gut. Thus the inhibition of production of ghrelin in the gut may contribute to the satiety effects of BBS/GRP peptides.     

Expression of human pancreatic polypeptide in heterologous cell lines

Pancreatic polypeptide (PP) is initially synthesized as a larger precursor that requires post-translational processing to produce the biologically active hexatriacontapeptide. These steps include tryptic cleavage at paired basic residues, their subsequent removal by a carboxypeptidase B-like enzyme, and formation of a carboxyl-terminal amide moiety via the action of peptidyl-glycyl alpha-amidating monooxygenase. To examine these reactions further, we utilized the pZIPneo(SVX) retroviral vector to express a cDNA clone encoding human PP in several cell lines including a fibroblast line (psi-2), two endocrine cell lines known to produce amidated peptides (AtT-20 and PC12), and two lines that do not ordinarily produce amidated peptides (RIN5-f and GH3). Transfected psi-2 cells produced an unprocessed precursor of PP that appeared to be secreted constitutively with little remaining in intracellular stores. No post-translational processing of the PP precursor was evident in these cells. By contrast, all 4 endocrine-derived cell lines, regardless of the nature of their endogenous products, were capable of expressing fully processed and carboxyl-terminally amidated PP. Moreover, these lines had the ability to store the processed products. Our results support the notion that post-translational processing of peptide hormone precursors requires storage in secretory granules that contain the appropriate processing enzymes. Furthermore, enzymes such as peptidyl-glycyl alpha-amidating monooxygenase that are required for processing peptides may be a common feature of endocrine-derived cells regardless of the requirement for their activity to process endogenous products.