A cDNA encoding a small common precursor for human pancreatic polypeptide and pancreatic icosapeptide.

A cDNA for the hormone, human pancreatic polypeptide (PP), was isolated by oligodeoxynucleotide screening from a cDNA library constructed from normal human pancreatic mRNA. The primary structure of the precursor protein as deduced from the cDNA sequence is 95 amino acids long and is composed of a typical, but rather long signal peptide of 29 residues, followed by the sequence of the 36 amino acid human pancreatic polypeptide, which again is separated from the human pancreatic icosapeptide sequence by a classic cleavage and amidation site, Gly-Lys-Arg. The precursor terminates in a heptapeptide which is cleaved from the icosapeptide at a monobasic processing site. Both the size and the structure of the PP precursor was supported by the results of peptide analysis of biosynthetically labeled pro-PP isolated from canine PP cells in which processing was prevented by the arginine analogue canavanine. It is concluded that the precursor for mammalian PP gives rise to two peptide products, the well preserved, carboxyamidated PP and an icosapeptide which is preserved only in its COOH-terminal end, plus a small highly variable COOH-terminal oligopeptide.     

Novel organization and processing of the guinea pig pancreatic polypeptide precursor

Studies on New World hystricomorph rodents have revealed interesting structural divergences in the peptide hormones of the islets of Langerhans, particularly with respect to insulin and glucagon. Herein we report the isolation and sequencing of a cDNA encoding the precursor of pancreatic polypeptide (PP) from a guinea pig pancreas cDNA library. The 126-residue precursor sequence is predicted to include a 26-residue NH2-terminal signal peptide followed by the 36-amino acid PP hormonal sequence and a large COOH-terminal extension. The sequence identity between guinea pig and human PP is 89% (32/36 residues), and the predicted sequence is in agreement with that reported by Eng et al. (Eng, J., Huang, C.-G., Pan, Y.-C. E., Hulmes, J. D., and Yalow, R. S. (1987) Peptides 8, 165-168). In contrast, the icosapeptide domain in the guinea pig precursor exhibits only 40% (8/20) identity with the corresponding human precursor domain, and the COOH-terminal extension differs greatly in both sequence and size. The guinea pig precursor lacks the monobasic processing site (Pro-Arg) found at the COOH terminus of the icosapeptide domain in human, ovine, canine, and feline proPP. An icosapeptide is thus not likely to be liberated as such from this precursor. Of particular interest in guinea pig proPP is the substitution of serine for arginine at the dibasic amino acid processing site on the COOH-terminal side of the PP domain. Results of radioimmunoassays of gel-filtered protein fractions from a guinea pig pancreas extract indicate that efficient proteolytic cleavage takes place at this Lys-Ser site and that mature guinea pig PP is normally carboxyamidated.     

Effect of amino acid analogs on the processing of the pancreatic polypeptide precursor in primary cell cultures

Amino acid analogs, which can be incorporated into nascent peptide chains were used in cultures of endocrine cells from canine pancreas to study the effect on processing of the metabolically labeled precursor for pancreatic polypeptide. Analogs for basic amino acids, canavanine, and aminoethylcysteine prevented the di-basic processing of the prohormone. The polar leucine analog, beta-hydroxyleucine, only partially perturbed the function and cleavage of the signal peptide but efficiently and unexpectedly blocked the dibasic cleavage of the prohormone. Other nonbasic amino acid analogs, beta-hydroxynorvaline and azetidine-2-carboxylic acid, which only could be incorporated into the prohormone at a distance from the processing site, also prevented dibasic cleavage of the prohormone. Although there are no phenylalanine residues in the prohormone, analogs for this amino acid, fluoro-phenylalanine and particularly phenylserine, could also block the processing of the prohormone at the dibasic site. This effect was prevented by addition of a small quantity of phenylalanine. It is concluded that amino acid analogs can interfere with precursor processing through altering both the primary and the secondary structure of the precursor but also through incorporation into cosynthesized protein(s) which are necessary for the precursor processing.     

Cat pancreatic eicosapeptide and its biosynthetic intermediate. Conservation of a monobasic processing site.

Pancreatic eicosapeptide is synthesized together with the hormone pancreatic polypeptide in a common precursor in the major endocrine cell type of the duodenal pancreas. This processing has been previously demonstrated in man and in the dog. In the present study the cat pancreatic eicosapeptide and a C-terminally extended form of this were isolated and characterized from acid/ethanol extracts of pancreas by gel filtration and reverse-phase h.p.l.c. The sequence homology in the C-terminal part of the eicosapeptides from different species was shown to continue to the other side of the monobasic cleavage site in the extended intermediate form, whereas the end of the extension differed both in chain length and amino acid sequence. It is concluded that the processing site in the intermediate form of the pancreatic eicosapeptide is an example of a proline-directed monobasic cleavage site that has been conserved during evolution.     

Immunocytochemical localisation of the icosapeptide fragment of the PP precursor: a marker for ‘true’ PP cells?

Antisera were raised against the icosapeptide fragment of the pancreatic polypeptide (PP) isolated from the canine pancreas. They were used for the immunocytochemical study of the cellular localisation and distribution of the icosapeptide in the gut and pancreas of various mammals. The results indicate that PP and the icosapeptide coexist in the majority of the PP-immunoreactive cells in the pancreas of cat, dog, pig, monkey and man and in all the PP-immunoreactive cells in the stomach of the cat and dog. The icosapeptide does not seem to occur in cells or nerves containing PP-related peptides, such as peptide YY or neuropeptide Y. PP-immunoreactive cells devoid of the icosapeptide could be demonstrated in the large intestine. These cells are probably distinct from the pancreatic PP cell type, and the PP-immunoreactive material probably represents the homologous peptide YY rather than PP. The present findings support the view that the icosapeptide is part of the PP precursor and hence, only the cells containing immunoreactive icosapeptide in addition to immunoreactive PP are to be considered ‘true’ PP cells. The icosapeptide antisera did not stain PP cells in mouse, rat and guinea-pig, suggesting marked species variation in the amino acid sequence of the icosapeptide portion of the PP precursor.     

Prohormone Thiol Protease and Enkephalin Precursor Processing: Cleavage at Dibasic and Monobasic Sites

Production of active enkephalin peptides requires proteolytic processing of proenkephalin at dibasic Lys-Arg, Arg-Arg, and Lys-Lys sites, as well as cleavage at a monobasic arginine site. A novel “prohormone thiol protease” (PTP) has been demonstrated to be involved in enkephalin precursor processing. To find if PTP is capable of cleaving all the putative cleavage sites needed for proenkephalin processing, its ability to cleave the dibasic and the monobasic sites within the enkephalin-containing peptides, peptide E and BAM-22P (bovine adrenal medulla docosapeptide), was examined in this study. Cleavage products were separated by HPLC and subjected to microsequencing to determine their identity. PTP cleaved BAM-22P at the Lys-Arg site between the two basic residues. The Arg-Arg site of both peptide E and BAM-22P was cleaved at the NH₂-terminal side of the paired basic residues to generate [Met]-enkephalin. Furthermore, the monobasic arginine site was cleaved at its NH₂-terminal side by PTP. These findings, together with previous results showing PTP cleavage at the Lys-Lys site of peptide F, demonstrate that PTP possesses the necessary specificity for all the dibasic and monobasic cleavage sites required for proenkephalin processing. In addition, the unique specificity of PTP for cleavage at the NH₂-terminal side of arginine at dibasic or monobasic sites distinguishes it from many other putative prohormone processing enzymes, providing further evidence that PTP appears to be a novel prohormone processing enzyme.     

Structural characterization of peptides derived from prosomatostatins I and II isolated from the pancreatic islets of two species of teleostean fish: the daddy sculpin and the flounder

The primary structures of three peptides from extracts from the pancreatic islets of the daddy sculpin (Cottus scorpius) and three analogous peptides from the islets of the flounder (Platichthys flesus), two species of teleostean fish, have been determined by automated Edman degradation. The structures of the flounder peptides were confirmed by fast-atom bombardment mass spectrometry. The peptides show strong homology to residues (49-60), (63-96) and (98-125) of the predicted sequence of preprosomatostatin II from the anglerfish (Lophius americanus). The amino acid sequences of the peptides suggest that, in the sculpin, prosomatostatin II is cleaved at a dibasic amino acid residue processing site (corresponding to Lys61-Arg62 in anglerfish preprosomatostatin II). The resulting fragments are further cleaved at monobasic residue processing sites (corresponding to Arg48 and Arg97 in anglerfish preprosomatostatin II). In the flounder the same dibasic residue processing site is utilised but cleavage at different monobasic sites takes place (corresponding to Arg50 and Arg97 in anglerfish preprosomatostatin II). A peptide identical to mammalian somatostatin-14 was also isolated from the islets of both species and is presumed to represent a cleavage product of prosomatostatin I.     

The processing of peptide precursors. 'Proline-directed arginyl cleavage' and other monobasic processing mechanisms

The classical conversion site in precursors of regulatory peptides is a sequence of two basic amino acids. During recent years, however, a group of monobasic cleavage sites has emerged. In certain cell systems it has been shown that the monobasic cleavage mechanism is both a specific mechanism which only attacks a particular basic residue, and a distinct mechanism which can be separated from the dibasic cleaving mechanism within the same cell. The vast majority of monobasic cleavages occur at single arginines although cleavage after a lysine residue has also been demonstrated. There is no 'consensus sequence' of amino acids surrounding the single basic residue which is the apparent signal for proteolytic processing. However, in approximately one third of the cases, a proline residue is found either just before or just after the basic residue. On the basis of this 'proline-directed arginyl cleavage' it is discussed how the conformation of the peptide backbone might be important for this type of cleavage. Finally, it is suggested that tissue-specific expression of different processing enzymes, e.g. dibasic and monobasic specific forms, might explain the tissue-specific processing of precursors like the pro-opiomelanocortin and the CKK and somatostatin precursor.     

Canine pancreatic polypeptide complementary deoxyribonucleic acid sequence: pancreatic polypeptide and insulin messenger ribonucleic acid distribution in the lobes of the pancreas

The structure of the canine prepropancreatic polypeptide (preproPP) cDNA was determined. The nucleotide sequence conservation between human and canine preproPP is very high for the signal peptide (82%) and the region coding for the 36 amino acid pancreatic polypeptide (PP) (92%). The overall sequence homology for the C-terminal portion of proPP containing the icosapeptide and a C-terminal extension peptide is only 63% whereas the 3'-untranslated regions of human and canine PP mRNA share 73% homology after alignment for maximal homology. The only sequence conservation in icosapeptide is the region coding for the last 10 amino acids of the icosapeptide. Comparison of PP immunoactivity and PP mRNA concentrations in extracts of the developmentally distinct uncinate process and splenic lobes of the canine pancreas revealed the same ratio of mRNA concentrations (16 +/- 6.5) and PP peptide concentrations (18 +/- 7.0) in the uncinate process compared to the splenic lobe (n = 6). However, a similar comparison of insulin C-peptide (CP) immunoactivity and insulin mRNA concentration revealed a smaller ratio of CP immunoactivity (0.37 +/- 0.05) than insulin mRNA (0.58 +/- 0.10) between the same lobes (P less than 0.0074, n = 6). This increased steady state CP concentration relative to insulin mRNA in splenic lobe compared to the uncinate process was not observed for PP peptide and mRNA.     

Consensus sequence for processing of peptide precursors at monobasic sites

Many regulatory peptide precursors undergo post-translational processing at mono- and/or dibasic residues. Comparison of amino acids around the monobasic cleavage sites suggests that these cleavages follow certain sequence motifs and can be described as the rules that govern monobasic cleavages: (i) a basic amino acid it present at either 3, 5, or 7 amino acids N-terminal to the cleavage site, (ii) hydrophobic aliphatic amino acids (leucine, isoleucine, valine, or methionine) are never present in the position C-terminal to the monobasic amino acid at the cleavage site, (iii) a cysteine is never present in the vicinity of the cleavage site, and (iv) an aromatic amino acid is never present at the position N-terminal to the monobasic amino acid at the cleavage site. In addition to these rules, the monobasic cleavages follow certain tendencies: (i) the amino acid at the cleavage site tends to be predominantly arginine, (ii) the amino acid at the position C-terminal to the cleavage site tends to be serine, alanine or glycine in more than 60% of the cases, (iii) the amino acid at either 3, 5, or 7 position N-terminal to the cleavage site tends to be arginine, (iv) aromatic amino acids are rare at the position C-terminal to the monobasic amino acid at the cleavage site, and (v) aliphatic amino acids tend to be in the two positions N-terminal to and the two positions C-terminal to the cleavage site, except as noted above. When compared with a large number of sequence containing single basic amino acids, these rules and tendencies are capable of not only correctly predicting the processing sites, but also are capable of excluding most of the single basic sequences that are known to be uncleaved. Many or these rules can also be applied to correctly predict the dibasic and multibasic cleavage sites suggesting that the rules and tendencies could govern endoproteolytic processing at the monobasic, dibasic and multibasic sites.     

Specificity of the dynorphin-processing endoprotease: comparison with prohormone convertases

The cleavage specificity of a monobasic processing dynorphin converting endoprotease is examined with a series of quench fluorescent peptide substrates and compared with the cleavage specificity of prohormone convertases. A dynorphin B-29-derived peptide, Abz-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-Arg-Ser-Glneddnp (where Abz is o-aminobenzoyl and eddnp is ethylenediamine 2,4-dinitrophenyl), that contains both dibasic and monobasic cleavage sites is efficiently cleaved by the dynorphin converting enzyme and not cleaved by two propeptide processing enzymes, furin and prohormone convertase 1. A shorter prorenin-related peptide, Dnp-Arg-Met-Ala-Arg-Leu-Thr-Leu-eddnp, that contains a monobasic cleavage site is cleaved by the dynorphin converting enzyme and prohormone convertase 1 and not by furin. Substitution of the P1' position by Ala moderately affects cleavage by the dynorphin-processing enzyme and prohormone convertase 1. It is interesting that this substitution results in efficient cleavage by furin. The site of cleavage, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry, is N-terminal to the Arg at the P1 position for the dynorphin converting enzyme and C-terminal to the Arg at the P1 position for furin and prohormone convertase 1. Peptides with additional basic residues at the P2 and at P4 positions also serve as substrates for the dynorphin converting enzyme. This enzyme cleaves shorter peptide substrates with significantly lower efficiency as compared with the longer peptide substrates, suggesting that the dynorphin converting enzyme prefers longer peptides that contain monobasic processing sites as substrates. Taken together, these results suggest that the cleavage specificity of the dynorphin converting enzyme is distinct but related to the cleavage specificity of the prohormone convertases and that multiple enzymes could be involved in the processing of peptide hormones and neuropeptides at monobasic and dibasic sites.     

Identification of a calcium-dependent microsomal proteinase responsible for monobasic cleavage of chicken proalbumin

The location and nature of the endoproteolytic activity involved in processing of proproteins has been studied in chicken liver microsomes. A membrane-bound, calcium-dependent proteinase was found to cleave chicken proalbumin with a monobasic cleavage site approx. 10-times faster than human proalbumin, which has a dibasic cleavage site. The mutant (human) proalbumin Christchurch (Arg(-1)----Gln), with a potential monobasic site, was not processed. The enzyme, which had a pH optimum of between 5.0 and 7.0, was not inhibited by serine or aspartyl proteinase inhibitors but was affected by some inhibitors of cysteine proteinases. The convertase was specifically inhibited by the reactive centre variant alpha 1-antitrypsin Pittsburgh, but not by normal alpha 1-antitrypsin.     

Exons of the human pancreatic polypeptide gene define functional domains of the precursor

Pancreatic polypeptide is a 36-amino acid peptide which inhibits pancreatic exocrine function. We have previously determined from the nucleotide sequence of a cDNA that pancreatic polypeptide is derived from a 95-amino acid precursor, prepropancreatic polypeptide. Pulse-chase studies have suggested that the precursor is cleaved to produce three peptides: pancreatic polypeptide, an icosapeptide, and a smaller peptide. In the present study, we have used the cloned cDNA as a hybridization probe to isolate the pancreatic polypeptide gene from a human bacteriophage genomic library. The nucleotide sequence of 2.8 kilobases of DNA representing the entire human pancreatic polypeptide gene was determined. The gene contains four exons and three introns. Exon 1 encodes the 5'-untranslated region of the mRNA, exon 2 encodes the signal sequence and the sequence of pancreatic polypeptide, exon 3 encodes the icosapeptide, and exon 4 encodes a carboxyl-terminal heptapeptide and the 3'-untranslated region of the mRNA. By Southern blot analysis, the gene detected in a pancreatic polypeptide-producing islet cell tumor was indistinguishable from that in normal human leukocytes. The structure of the human pancreatic polypeptide gene is consistent with the hypothesis that prepropancreatic polypeptide generates three distinct peptides, each encoded by a separate exon. Increased expression of pancreatic polypeptide in the islet cell tumor does not appear to be correlated with major alterations in pancreatic polypeptide gene structure.     

Expression and posttranslational processing of preprodynorphin complementary DNA in the mouse anterior pituitary cell line AtT-20

A recombinant plasmid containing the rat prodynorphin cDNA was introduced into the mouse anterior pituitary corticotroph cell line AtT-20. These cells normally express and posttranslationally process proopiomelanocortin, but not prodynorphin. Stable transformants were isolated and analyzed for the expression and processing of prodynorphin. The stably transformed AtT-20 cells that expressed a 1.3-kilobase prodynorphin mRNA also expressed prodynorphin protein and processed it to dynorphin peptides. The peptides included leucine-enkephalin, beta-neoendorphin, dynorphin-A8, and dynorphin-B, as identified by gel filtration and reverse phase HPLC followed by RIA using peptide-specific antisera. These results demonstrate that AtT-20 cells efficiently and accurately process prodynorphin at both dibasic sites and monobasic cleavage sites, indicating that the AtT-20 cells contain enzymes capable of cleaving the precursor not only at dibasic residues but also at monobasic residues. The release of prodynorphin-derived peptides paralleled secretion of endogenous proopiomelanocortin-derived peptides when stimulated by CRF, a natural secretagogue for ACTH.     

Differential processing of hormone precursor. Independent production of somatostatins 14 and 28 in transfected neuroblastoma 2A cells.

Neuro 2A cells infected with a retroviral vector carrying human prosomatostatin cDNA expressed and processed correctly the precursor into somatostatins-14 and -28 [(1989) EMBO J. 8, 2911-2916]. In order to study the mechanisms by which the active hormone sequences arise, site directed mutagenesis was performed on either the dibasic (ArgLys) or monobasic (Arg) cleavage sites involved in the production of somatostatins-14 and -28, respectively. Radioimmunochemical analysis of the somatostatin-related products indicated that replacement of either Arg-2-Lys-1 by Asn-2-Asn-1 or of Arg-15 by Asn-15 resulted in the exclusive production of either somatostatin-28 or -14, respectively. Moreover only prosomatostatin[1-76] was detected and no somatostatin-28[1-12] could be measured in cell extracts. Selective suppression of either somatostatin-14 or somatostatin-28 release by mutation did not affect the level of production of the other hormone but resulted in a correlative increase of unprocessed prosomatostatin. It is concluded that in this cell type (i) somatostatin-14 is exclusively generated by dibasic cleavage at the Arg-2-Lys-1 site of the intact precursor with concomitant production of prosomatostatin[1-76], and (ii) no direct interactions between the monobasic and dibasic processing domains occur.     

Consensus sequence for precursor processing at mono-arginyl sites. Evidence for the involvement of a Kex2-like endoprotease in precursor cleavages at both dibasic and mono-arginyl sites.

Many peptide hormones and neuropeptides are produced from larger, inactive precursors through endoproteolysis at sites usually marked by paired basic residues (primarily Lys-Arg and Arg-Arg), or occasionally by a monobasic residue (primarily Arg). Based upon data concerning processing of prorenin and its mutants around the native Lys-Arg cleavage site expressed in mouse pituitary AtT-20 cells, we present the following sequence rules that govern mono-arginyl cleavages: (a) a basic residue at the fourth (position -4) or the sixth (position -6) residue upstream of the cleavage site is required, (b) at position -4, Arg is more favorable than Lys, and (c) at position 1, a hydrophobic aliphatic residue is not suitable. These rules are compatible with those proposed by comparison of precursor sequences around mono-arginyl cleavage sites. We also provide evidence that precursor cleavages at mono-arginyl and dibasic sites can be catalyzed by the same Kex2-like processing endoprotease, PC1/PC3.     

Proteolysis of ProPTHrP(1-141) by "prohormone thiol protease" at multibasic residues generates PTHrP-related peptides: implications for PTHrP peptide production in lung cancer cells

The parathyroid hormone-related protein (PTHrP) precursor requires proteolytic processing to generate PTHrP-related peptide products that possess regulatory functions in the control of PTH-like (parathyroid-like) actions and cell growth, calcium transport, and osteoclast activity. Biologically active peptide domains within the PTHrP precursor are typically flanked at their NH2- and COOH-termini by basic residue cleavage sites consisting of multibasic, dibasic, and monobasic residues. These basic residues are predicted to serve as proteolytic cleavage sites for converting the PTHrP precursor into active peptide products. The coexpression of the prohormone processing enzyme PTP ("prohormone thiol protease") in PTHrP-containing lung cancer cells, and the lack of PTP in cell lines that contain little PTHrP, implicate PTP as a candidate processing enzyme for proPTHrP. Therefore, in this study, PTP cleavage of recombinant proPTHrP(1-141) precursor was evaluated by MALDI mass spectrometry to identify peptide products and cleavage sites. PTP cleaved the PTHrP precursor at the predicted basic residue cleavage sites to generate biologically active PTHrP-related peptides that correspond to the NH2-terminal domain (residues 1-37) that possesses PTH-like and growth regulatory activities, the mid-region domain (residues 38-93) that regulates calcium transport, and the COOH-terminal domain (residues 102-141) that modulates osteoclast activity. Lack of cleavage at other types of amino acids demonstrated the specificity of PTP processing at basic residue cleavage sites. Overall, these results demonstrate the ability of PTP to cleave the PTHrP precursor at multibasic, dibasic, and monobasic residue cleavage sites to generate active PTHrP-related peptides. The presence of PTP immunoreactivity in PTHrP-containing lung cancer cells suggests PTP as a candidate processing enzyme for the PTHrP precursor.     

FMRF-amide immunoreactivity in the mammalian gastroenteropancreatic neuroendocrine system

The presence of FMRF-amide, a cardioactive tetrapeptide, was studied by immunocytochemistry in human and rat gastric antrum and pancreas, and in the ovine, bovine, canine and rabbit pancreas. In human and rat gastric antrum, numerous cells contained FMRF-amide immunoreactive material. By staining of serial sections and by double staining, colocalization of immunoreactivity for gastrin and FMRF-amide was observed in part of the gastrin cells. In the pancreas of these and the other species, immunoreactivity for FMRF-amide was located both in acinar and islet endocrine cells. Colocalization of FMRF-amide and pancreatic polypeptide was found in a proportion of pancreatic polypeptide cells in the pancreas. FMRF-amide immunoreactivity never colocalized with the other neurohormonal peptides which occur in the gastric antrum and the pancreas. Our observations show that neuroendocrine cells occur in the gastric antrum and pancreas which are exclusively immunoreactive or gastrin and for pancreatic polypeptide respectively. In addition cells occur which show immunoreactivity for FMRF-amide as well as for gastrin in the gastric antrum and with antiserum to FMRF-amide as well as for pancreatic polypeptide in the pancreas. It is concluded that FMRF-amide antibodies probably recognize a substance in G and PP cells which is not identical but may be structurally related to gastrin and pancreatic polypeptide.     

FMRF-amide immunoreactivity in the mammalian gastroenteropancreatic neuroendocrine system

Summary The presence of FMRF-amide, a cardioactiv tetrapeptide, was studied by immunocytochemistry in human and rat gastric antrum and pancreas, and in the ovine, bovine, canine and rabbit pancreas. In human and rat gastric antrum, numerous cells contained FMRF-amide immunoreactive material. By staining of serial sections and by double staining, colocalization of immunoreactivity for gastrin and FMRF-amide was observed in part of the gastrin cells. In the pancreas of these and the other species, immunoreactivity for FMRF-amide was located both in acinar and islet endocrine cells. Colocalization of FMRF-amide and pancreatic polypeptide was found in a proportion of pancreatic polypeptide cells in the pancreas. FMRF-amide immunoreactivity never colocalized with the other neurohormonal peptides which occur in the gastric antrum and the pancreas.Our observations show that neuroendocrine cells occur in the gastric antrum and pancreas which are exclusively immunoreactive or gastrin and for pancreatic polypeptide respectively. In addition cells occur which show immunoreactivity for FMRF-amide as well as for gastrin in the gastric antrum and with antiserum to FMRF-amide as well as for pancreatic polypeptide in the pancreas. It is concluded that FMRF-amide antibodies probably recognize a substance in G and PP cells which is not identical but may be structurally related to gastrin and pancreatic polypeptide.In honour of Prof. P. van Duijn     

Identification,localization and receptor characterization of novel mammalian substance P-like peptides

Hemokinin-1 (HK-1) is a novel substance P (SP)-like peptide that is encoded by the preprotachykinin C (PPT-C) gene recently identified in mouse B cells and shown to be a potentially important regulator of B cell development (Nat. Immunol. 1 (2000) 392). We have now isolated and characterized the human and rat orthologs of PPT-C and examined activities of human and mouse HK-1 on the three tachykinin receptors, neurokinin-1-3 (NK1-3). The rat PPT-C polypeptide is highly homologous to mouse PPT-C and contains the same processing sites to generate predicted HK-1. The human PPT-C polypeptide is also homologous to mouse PPT-C, however, it contains two potential monobasic cleavage sites rather than a single dibasic cleavage site at the amino-terminal end of the predicted HK-1 peptide. Thus, human PPT-C has the potential to generate full length predicted HK-1 as well as a truncated version (HK-1(4-11)). Polymerase chain reaction analysis revealed that both human and mouse PPT-C were expressed in a variety of tissues with strong signals detected in the skin of both species and in the mouse brain. Binding and functional analysis indicated that human and mouse HK-1 peptides were nearly identical to SP in their overall activity profile on the three NK receptors with the most potent affinity for the NK1 receptor. The results indicate that PPT-C encodes another high affinity ligand of the NK1 receptor which may play an important role in mediating some of the physiological roles previously assigned to the NK1 receptor.