Characterization of the cDNA encoding the prohormone convertase PC2 and localization of the mRNA in the brain of the frog Rana ridibunda

Molecular Brain Research 63 Ž1998. 1–13

Research report

Characterization of the cDNA encoding the prohormone convertase PC2 and localization of the mRNA in the brain of the frog Rana ridibunda Didier Vieau 1, Franc¸oise Gangnon 2 , Sylvie Jegou, Jean-Michel Danger, Hubert Vaudry ´

)

European Institute for Peptide Research (IFRMP no. 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U413, UA CNRS, UniÕersity of Rouen, 76821 Mont-Saint-Aignan, France Accepted 25 August 1998

Abstract A number of precursors for neuropeptides have recently been cloned in amphibians, but little is known concerning the endoproteases responsible for the processing of these precursors. Here we report on the molecular cloning of the cDNA encoding the proprotein convertase PC2 and the distribution of the corresponding mRNA in the European green frog Rana ridibunda. The full cDNA structure Ž2125 bp. was obtained from the analysis of the PCR products combined with the sequence from a clone isolated from a frog pituitary cDNA library. The deduced amino acid sequence revealed that frog PC2 comprises 636 amino acid residues including a 22-residue signal peptide. RT-PCR analysis showed that PC2 is expressed not only in the brain and pituitary but also in various peripheral organs including the pancreas, stomach, intestine, liver, kidney and testis. In situ hybridization histochemistry revealed that, in the central nervous system, PC2 mRNA is widely distributed, the highest concentrations being found in the pallium, the anterior preoptic area, the hypothalamus and the medial amygdala. High levels of PC2 mRNA were also detected in the intermediate lobe of the pituitary. The overall distribution of PC2 mRNA in the frog brain is consistent with its involvement in the processing of a number of neuropeptide and hormone precursors. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Proprotein convertase; Proteolytic processing; In situ hybridization; Neuropeptides; Frog brain

1. Introduction Neuropeptides and peptide hormones are synthesized as inactive precursors which are subsequently converted to their mature forms by selective endoproteolytic cleavage, generally at pairs of basic amino acid residues. This activation process is catalyzed by a family of serine proteases called proprotein convertases ŽPCs. related to the bacterial subtilisins and the Kex2 protease of the yeast Saccharomyces cereÕisiae w22x. During the last decade, seven members of the PC family have been cloned in mammals. On the basis of their tissue distribution and intracellular localization, PCs can be Abbreviations: PC, proprotein convertase; CNS, central nervous system; PCR, polymerase chain reaction; POMC, proopiomelanocortin; CLIP, corticotropin-like intermediate lobe peptide; a-MSH, a-melanocytestimulating hormone; bp, base pair; aa, amino acid ) Corresponding author. Fax: q 33-235-14-6946; E-mail: [email protected] 1 This author has equally contributed to this work. 2 This author has equally contributed to this work.

classified into four subfamilies: Ž1. furin and the recently discovered PC7 Žalso called SPC7, C8 and LPC. exhibit an ubiquitous tissue distribution and process precursors which are targeted to the cell surface via the constitutive secretory pathway w7,31,43,50x; Ž2. PACE4 and PC5 Žalso called PC6., which are expressed in both endocrine and nonendocrine cells, process precursors in both the constitutive and the regulated secretory pathways w17,23,29,35x; Ž3. PC1 Žalso known as PC3. and PC2, which are exclusively found in endocrine, neuroendocrine and neural cells, are mainly involved in the cleavage of neuropeptide precursors w46,47,51,52x; and Ž4. PC4 which is almost exlusively synthesized in testicular germ cells and ovaries w36,48x. We have recently undertaken a program of identification of neuropeptides in the European green frog Rana ridibunda, a species which has been widely used as a model for the investigation of the biochemical mechanisms of neuroendocrine communication w1,27,56x. This program involves the purification and amino acid sequence determination of novel neuropeptides w13,58,59x and the molecular

0169-328Xr98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 2 3 5 - 6

2

D. Vieau et al.r Molecular Brain Research 63 (1998) 1–13

cloning of their precursors w2,57x. However, the proprotein convertases PC1 and PC2, which are likely responsible for the processing of these neuropeptide precursors, have not yet been characterized in frog. We describe herein the molecular cloning and nucleotide sequence analysis of the cDNA encoding frog PC2. The distribution of PC2 mRNA in various tissues has been determined by RT-PCR and the localization of PC2 gene expression in the brain and pituitary has been investigated by in situ hybridization.

2. Materials and methods

labeled fluorescent primers. Automated DNA sequence determination was performed using a Li-cor DNA sequencer model 4000 L. Screening of the library allowed characterization of the fPC2 sequence from nucleotide Žnt. 381 down to the polyŽA. tail. A homologous 23-mer antisense primer, 5X-GCTTCAGCAACATTCAAATCAAG-3X was used along with a SP6 primer Žpresent in the cDNA library vector. in a PCR reaction in order to amplify the 5X region of fPC2 from the library. The PCR conditions were as above except that the annealing temperature was 558C. A 506-bp fragment was subcloned into the pGEM-T vector and three clones were completely sequenced. 2.3. ReÕerse transcriptase-polymerase chain reaction

2.1. Animals Adult male frogs Ž R. ridibunda. weighing 30–40 g were obtained from a commercial source ŽCouetard, ´ Saint-Hilaire de Riez, France.. The animals were housed in glass tanks supplied with circulating water and maintained at constant temperature Ž8 " 0.58C. under a 12-h lightr12-h dark cycle Žlights from 0600 to 1800 h., for at least one week before use. Animal manipulations were performed according to the recommendations of the French Ethical Committee and under the supervision of authorized investigators. 2.2. Cloning of frog PC2 Purified DNA from a frog pituitary cDNA library ŽCDM7ramp, kindly provided by Dr. Y. Anouar. was amplified by polymerase chain reaction ŽPCR. in a Robocycler Gradient 40 ŽStratagene, La Jolla, CA, USA.. After 3 min of denaturation at 948C, 30 cycles of amplification were performed under low stringency conditions: denaturation for 1 min at 948C, annealing for 1.5 min at 408C, and extension for 1 min at 728C, using degenerate oligonucleotides ŽGenosys, Cambridge, UK. selected within the highly conserved catalytic region surrounding the serine active site of PCs. The sequences of the sense and antisense primers were: 5X-ATCTACAGŽCrT.GCŽArC.AGŽArC. TGGGGCCC-3X and 5X-CAGŽArG.TGŽCrT.TGCATGTC ŽTrC.CŽTrG.CCAGGT-3X , respectively. The amplified PCR products were electrophoresed on a 1.5% agarose gel and 450-bp fragments Žthe expected size based on the known mammalian PC2 cDNA sequences., subcloned directly into the pGEM-T vector ŽPromega, Madison, WI, USA. and sequenced. One of the subcloned 450-bp fragments corresponding to the DNA sequence encoding frog PC2 ŽfPC2. was labeled by random-primed incorporation of w32 PxdCTP and used to screen 10 5 recombinants of the frog cDNA library under high stringency hybridization. Twenty three positive clones were obtained, purified by a second screening and sequenced using T7 and SP6 5X-end

Total RNA from various frog tissues Žwhole brain, pituitary, spinal cord, liver, kidney, pancreas, stomach, testis and intestine. was isolated by the acid guanidium thiocyanate–phenol–chloroform method w11x. Equivalent amounts of total RNA Ž5 mg. were reverse transcribed with Superscript II RT ŽLife Technologies, Paisley, UK.. PCR amplification was performed Ždenaturation, 1 min at 948C; annealing, 1 min at 508C; and extension, 1.5 min at 728C. for 30 cycles using the sense primer 5X-GTGGATGGGCCCAGAGAACTG-3X Žnt 876–896. and the antisense primer 5X-CCGCAGATGTTCCTGAGTGAC-3X Žnt 1179– 1199.. The amplified PCR product Ž324 bp. was resolved by 1.5% agarose gel electrophoresis. 2.4. In situ hybridization Sense and antisense riboprobes were prepared by in vitro transcription of the 450-bp fPC2 fragment subcloned into pGEM-T, in the presence of w35 SxUTP ŽAmersham, Les Ulis, France. and T7 or T3 RNA polymerases, respectively, by using a riboprobe kit ŽPromega.. Frogs were anaesthetized with 1% triaminobenzoic acid ethyl ester ŽMS 222, Sigma, St. Louis, MO, USA. and perfused transcardially with 4% paraformaldehyde. The brains and pituitaries were postfixed for 3 h at 48C in the same solution, transferred into 0.1 M phosphate buffer containing 15% saccharose for 12 h, and frozen in isopentane at y308C. Coronal sections Ž12-mm thick. were cut in a cryomicrotome Ž2800 Frigocut, Leica, Nussloch, Germany. and collected on 0.5% gelatinr0.05% chrome alumr0.01% polylysine-coated slices. Sections were incubated in 0.1 M triethanolamine ŽpH 8.0. for 5 min, rinsed in 2 = standard saline citrate ŽSSC; 0.15 M NaClr0.015 M sodium citrate, pH 7.0. and covered with prehybridization buffer Ž50% formamider0.6 M NaClr0.01 M Tris–HClr0.02% Ficollr0.02% polyvinylpyrrolidoner0.02% BSAr0.001 M EDTAr550 mgrml yeast tRNA.. Hybridization was performed overnight at 608C in the same buffer Žexcept for salmon sperm whose concentration was 60 mgrml. supplemented with 0.01 mM dithiothreitol, 10% dextran sulfate and 10 7 cpmrml heat-denatured antisense fPC2 RNA

D. Vieau et al.r Molecular Brain Research 63 (1998) 1–13 Fig. 1. Nucleotide and deduced amino acid sequence of the cDNA encoding frog PC2. The active site Asp ŽB., His ŽB., and Ser ŽB., the important Asp ŽI. residue, the three potential N-glycosylation sites Ž)., and the potential Tyr sulphation site Ž`. are indicated. The polyadenylation signal is underlined. The arrow indicates the presumptive site of signal peptide cleavage. The potential zymogen activation sites are boxed. The sequence of frog PC2 has been submitted to EMBL under the accession number AF090986. 3

4 D. Vieau et al.r Molecular Brain Research 63 (1998) 1–13

Fig. 2. Comparison of the amino acid sequences of PC2 in various species. Black boxes indicate residues conserved in all species while those conserved in frog and several other species are shaded.

D. Vieau et al.r Molecular Brain Research 63 (1998) 1–13

probe, as previously described w2x. Briefly, tissue slices were washed in 2 = SSC at 608C and treated with Rnase A Ž50 mgrml. for 60 min at 378C. Five final high-stringency washes were performed in 0.01 = SSC containing 14 mM 2-mercaptoethanol and 0.05% sodium pyrophosphate. The slices were dehydrated in ethanol and exposed onto Hyperfilm-BETAmax ŽAmersham. for 2 weeks. Control sections were hybridized with 35 S-labeled sense fPC2 probe. Tissue slices were dipped into a Kodak NTB2 liquid emulsion at 408C, exposed for 20 days and developed. In order to identify anatomical structures, brain and pituitary sections were stained with hematoxylin and eosin. Nomenclature of frog brain structures was based on the atlas of Northcutt w37,39x as previously described w10x.

3. Results 3.1. cDNA cloning and sequence of frog PC2 PCR amplification of cDNA prepared from a pituitary cDNA library with degenerate primers resulted in a 450-bp band, consistent with the expected length of the PC2 cDNA fragment. High stringency screening of 10 5 clones of the frog pituitary cDNA library with the 450-bp fPC2 probe led to the isolation of 23 positive clones with inserts ranging from 900 to 1800 bp. In order to characterize the 5X-region of fPC2 cDNA, PCR amplification was performed using an internal fPC2 antisense primer and a Sp6 oligonucleotide as an anchored primer. The 2125 nucleotide sequence depicted in Fig. 1 comprises an open reading frame of 1908 bp encoding a protein of 636 amino acids including a 22-residue signal peptide. Cleavage of this signal peptide would thus generate a 614-residue pro-enzyme with a calculated molecular weight of 68,166 Da and an isoelectric point of 5.64. Comparison of the nucleotide sequence and the deduced amino acid sequence with those of known convertases, confirmed that this cloned cDNA actually corresponds to fPC2. The protein contains the Asp165, His 206 , and Ser 382 residues found in the catalytic triad of serine proteinases of the subtilisin family. In addition, fPC2 as all PC2 identi-

5

fied so far, possesses an Asp 308 instead of the usual Asn308 found in other convertases. Frog PC2 also possesses three potential N-linked glycosylation sites ŽAsn373 –Cys–Thr, Asn512 –Ser–Thr and Asn522 –Met–Thr. and one potential sulphation site ŽTyr 170 . which is conserved in all PC2 structures w4x. fPC2 lacks the canonical integrin binding site ŽArg–Gly–Asp. present in all mammalian convertases with the exception of PC7 w7,31,50x. 3.2. Comparison of the nucleotide and peptide sequences of PC2 in Õarious species The amino acid sequence of fPC2 was compared to those of the PC2 identified in five other species by using a multiple alignment analysis software. The overall sequence identity of fPC2 to its Lymnaea stagnalis w53x, Aplysia californica w41x, Xenopus laeÕis w6x, mouse w46x, and human w51x counterparts were 61%, 60%, 90%, 86% and 87% at the protein level and 58%, 59%, 78%, 72% and 72% at the nucleotide level. The catalytic domain Žresidues 108–410. was the most preserved region while the Nterminal prosegment was far less conserved ŽFig. 2.. 3.3. Identification of PC2 mRNA in Õarious tissues The presence of PC2 mRNA in the brain and peripheral organs was investigated by RT-PCR amplification. PC2 mRNA was readily detected in all tissues examined, particularly in the brain and pituitary but also in the pancreas, stomach, intestine, liver, kidney and testis ŽFig. 3.. 3.4. Distribution of PC2 mRNA in the frog brain and pituitary In situ hybridization histochemistry revealed that PC2 mRNA was widely distributed in the frog brain. In the telencephalon, a strong hybridization signal was observed in all the subdivisions of the pallium. Ventrally, the ventral and dorsal nuclei of the striatum contained a high concentration of PC2 mRNA. A moderate signal was observed in the nucleus accumbens and in the lateral and

Fig. 3. RT-PCR analysis of PC2 mRNA expression in frog tissues using oligonucleotides within the catalytic domain.

6

D. Vieau et al.r Molecular Brain Research 63 (1998) 1–13

Fig. 4. Distribution of PC2 mRNA by in situ hybridization in the frog brain and pituitary. Frontal brain sections were hybridized with antisense ŽA–F. or sense ŽG. frog PC2 RNA probes and exposed onto X-ray films for 2 weeks. The anatomical structures are presented on the left hemisections. The rostro-caudal levels of the sections are indicated on the sagittal section. Abbreviations are indicated in Table 1.

D. Vieau et al.r Molecular Brain Research 63 (1998) 1–13

7

Fig. 5. Cytological localization of PC2 mRNA on emulsion-coated tissue slices. Hybridization was carried out with the antisense ŽA, C, D, E. or sense ŽB. fPC2 mRNA probe. A, B, pallium; C, anterior preoptic area; D, ventral hypothalamic nucleus; E, pars intermedia ŽP I. and pars distalis ŽP Dis. of the pituitary. Arrows indicate strongly hybridizing cells and arrowheads point to unlabeled cells. Scale bar s 10 mm.

8

D. Vieau et al.r Molecular Brain Research 63 (1998) 1–13

medial septum ŽFig. 4A.. The medial amygdala was intensely labeled ŽFig. 4B.. In the diencephalon, a high concentration of PC2 mRNA was found in the anterior preoptic area ŽFig. 4B.. Caudally, autoradiographic labeling was visualized in most diencephalic nuclei including the suprachiasmatic nucleus, the magnocellular preoptic nucleus and the anterior thalamic nucleus ŽFig. 4C.. No hybridization signal could be detected in the corpus geneticulatum thalamicum and in the ventral thalamic nucleus ŽFig. 4C.. The hypothalamic regions bordering the infundibular recess contained a dense accumulation of PC2 mRNA. In particular, intense labeling was observed in the nucleus of the periventricular organ and the ventral hypothalamic nucleus ŽFig. 4D.. The posterior tuberculum also showed a strong hybridization signal ŽFig. 4D.. In the mesencephalon, all layers of the optic tectum exhibited a positive signal, the superficial layer being however less intensely labeled than the internal layers ŽFig. 4E.. PC2 mRNA was found in both the anterodorsal and anteroventral tegmental nuclei. A weak hybridization signal was also detected in the torus semicircularis. In the metencephalon, a high concentration of PC2 mRNA was detected in the Purkinje and granule cell layers of the cerebellum. No hybridization signal was seen in the molecular cell layer of the cerebellum. In the rhombencephalon, PC2 mRNA was visualized in the nuclei of the abducent, facial, stato-acusticus and glossopharyngeal nerves, as well as in the reticularis inferior nucleus and the fasciculus solitarius. No hybridization signal was detected in the raphe nucleus ŽFig. 4F.. In the pituitary, a high density of PC2 mRNA was found in the pars intermedia while the pars nervosa and the pars distalis were virtually devoid of hybridization signal ŽFig. 4E.. Control sections treated with the sense fPC2 probe were totally devoid of labeling ŽFig. 4G.. Emulsion-coated slices in the brain regions which exhibited intense labeling, such as the pallium ŽFig. 5A., the anterior preoptic area ŽFig. 5C. and the ventral hypothalamic nucleus ŽFig. 5D., revealed that several cells strongly expressed the PC2 gene while others were virtually devoid of PC2 mRNA. Within the pituitary, intense cytoautoradiographic labeling was visualized in all melanotrope cells of the pars intermedia ŽFig. 5E.. In contrast, no detectable fPC2 mRNA was found in the anterior lobe ŽFig. 5E. and in the pars nervosa Žnot shown..

4. Discussion Although several neuropeptides have been recently identified in the brain of the frog R. ridibunda w8,9,13,18,28,58,59x, the proprotein convertases which are responsible for the post-translational processing of the

precursors of these neuropeptides have not yet been characterized. The present report describes the molecular cloning of the cDNA and the distribution of the mRNA encoding frog PC2, one of the convertases that play a crucial role in the endoproteolytic cleavage of neuropeptide precursors. 4.1. Primary structure of frog PC2 Previous studies have shown that, in mammals, PC2 is actively expressed in the pars intermedia of the pituitary where it is involved, together with PC1, in the processing of POMC and the biosynthesis of a-MSH. We have thus used a frog pituitary cDNA library to generate by PCR a specific probe which has been subsequently used to isolate cDNA clones. The fPC2 cDNA consists of a single open reading frame of 1908 bp that is predicted to encode a 636-amino acid protein with a putative 22-residue signal peptide, a prosegment, a subtilisin-like catalytic domain, a P-domain and a C-terminal region. Alignment of the peptide sequence of fPC2 with those of PC2 previously identified in other species revealed that the structure of the protein has been highly conserved during evolution, in particular within the catalytic segment Žresidues 108–410.. It has been shown that PCs are first synthesized as inactive precursor enzymes Žzymogens. which usually undergo autocatalytic excision of their N-terminal prosegment via cleavage at a specific Lys–Arg sequence. The observation that fPC2 possesses two clusters of basic residues at positions 76–79 and 104–107 suggests that fPC2, as all mammalian PCs, is first synthesized as a zymogen with an 85-residue prosegment which would be cleaved at the Arg-Lys-Lys-Arg-107 y sequence to release the active enzyme. As shown in Fig. 1, the mature enzyme would therefore contain 529 aa, three potential Asn-linked glycosylation sites Žresidues 373, 512 and 522. and one potential Tyr sulphation site Žresidue 170. which is conserved in all PC2 structures w4x. Similar to Lymnaea w53x and Aplysia PC2 w41x, but different from Xenopus PC2 w6x, we noticed the absence of the characteristic canonical integrin binding Arg–Gly–Asp ŽRGD. sequence found in all mammalian convertases, with however the exception of PC7 w7,31,50x. The physiological significance of this structure is still unclear although it has been recently shown that RGD mutations prevent PC1 from entering the secretory granules and result in the mis-sorting of PC1 mutants towards the constitutive secretory pathway w30x. The equivalent sequence in fPC2 is Arg–Gly–Asn Žresidues 516–518.. The carboxy terminal domain of fPC2, which contains a potential amphipathic a-helical segment, is remarkably well conserved among species, suggesting that this region is functionally important. In carboxypeptidase E, this amphipathic a-helix has been shown to play an important role in membrane association and sorting of the enzyme w33x. Consistent with this observation, it has been recently shown

D. Vieau et al.r Molecular Brain Research 63 (1998) 1–13

9

Table 1 PC2 mRNA and cell bodies expressing various neuropeptides in the brain of the frog R. ridibunda Structure

PC2 mRNA

Neuropeptides

Telencephalon LP Lateral pallium DP Dorsal pallium MP Medial pallium LS Lateral septum DS Dorsal striatum MS Medial septum VS Ventral striatum NDB Nucleus of the diagonal band of Broca NA Nucleus accumbens MA Medial amygdala LA Lateral amygdala Ea Anterior entopeduncular nucleus Ep Posterior entopeduncular nucleus BN Bed nucleus of the pallial commissure

qqq qqq qqq q qq q qq q q qqq qq q q qq

Enk w32x, NPY w15x, SRIF w25,57x ANF w38x, Enk w32x, NPY w15x, SRIF w25,57x ANF w38x, Enk w32x, NPY w15x, SRIF w25,57x Neuro B w42x, Tach w54x Enk w32x, SRIF w25,57x, Tach w54x ANF w38x, Enk w32x, mGnRH w12x, Neuro B w42x, SRIF w25,57x, Tach w54x Enk w32x, Neuro B w42x, SRIF w25,57x, Tach w54x Neuro B w42x Gal w26x, PACAP w61x, SRIF w25,57x Enk w32x, Gal w26x, Neuro B w42x, NPY w15x, PACAP w61x, SRIF w25,57x, Tach w54x a MSH w5x, PACAP w61x, SRIF w25,57x

Diencephalon Poa Anterior preoptic area

qqq

Mg OV SC DH VH

Magnocellular preoptic nucleus Organum vasculosum Suprachiasmatic nucleus Dorsal hypothalamic nucleus Ventral hypothalamic nucleus

qqq qq qqq qq qqq

ANF w38x, AVT w24x, CGRP w34x, CRF w40,55x, Enk w32x, Gal w26x, mGnRH w12x, a MSH w5x, MT w19,24x, NPY w15x, PACAP w61x, SRIF w25,57x, Tach w54x, TRH w24x Gal w26x, SRIF w25,57x

LH TP NPv E TE Hc Hd Hv BM B NB Vld Vlv VM A Vs CP La Lpd

Lateral hypothalamic nucleus Posterior tuberculum Nucleus of the periventricular organ Epiphysis Thalamic eminence Habenular commissure Dorsal habenular nucleus Ventral habenular nucleus Bed nucleus of the stria medullaris Neuropil of Bellonci Nucleus of Bellonci Ventrolateral thalamic nucleus, dorsal part Ventrolateral thalamic nucleus, ventral part Ventromedial thalamic nucleus Anterior thalamic nucleus Superficial ventral thalamic nucleus Corpus geniculatum thalamicum Lateral thalamic nucleus, anterior division Lateral thalamic nucleus, posterodorsal division Lateral thalamic nucleus, posterodorsal division Central thalamic nucleus Posterior thalamic nucleus Subcommissural organ Uncinate neuropil Optic chiasma Optic nerve Median eminence

0 qqq qqq 0 0 0 qqq qqq 0 q q q q q q 0 0 qqq qqq

Lpv CNT P CO U OC ON ME

Mesencephalon OT Optic tectum 6 Tectal lamina six NPC Nucleus of the posterior commissure NLM Nucleus lentiformis mesencephali NMLF Nucleus of the medial longitudinal fasciculus

NPY w15x, SRIF w25,57x PACAP w61x

CGRP w34x, Gal w26x, PACAP w61x, SRIF w25,57x, TRH w24x Neuro B w42x ANF w38x, DSIP w60x, Enk w32x, Gal w26x, a MSH w5x, Neuro B w42x, NPY w15x, PACAP w61x, SRIF w25,57x, Tach w54x AVT w24x, SRIF w25,57x, Neuro B w42x Gal w26x, Neuro B w42x, PACAP w61x Enk w32x, Gal w26x, a MSH w5x, Neuro B w42x, NPY w15x, PACAP w61x, SRIF w25,57x SRIF w25,57x Neuro B w42x, Tach w54x Neuro B w42x, Tach w54x Neuro B w42x, Tach w54x

ANF w38x ANF w38x, SRIF w25,57x Neuro B w42x, PACAP w61x, SRIF w25,57x PACAP w61x Neuro B w42x Enk w32x

0 qq qq q 0 0 0 qq

NPY w15x, SRIF w25,57x ANF w38x, Neuro B w42x, NPY w15x, PACAP w61x, SRIF w25,57x

qqq qqq 0 0 qq

Enk w32x, Gal w26x, Neuro B w42x, NPY w15x NPY w15x, SRIF w25,57x

Gal w26x, a MSH w5x

PACAP w61x, SRIF w25,57x

D. Vieau et al.r Molecular Brain Research 63 (1998) 1–13

10 Table 1 Žcontinued. R. ridibunda Structure

PC2 mRNA

Mesencephalon BON Basic optic nucleus NPM Nucleus profundus mesencephali PtG Pretectal gray PtrG Pretoral gray III Oculumotor and trochlear nuclei TS Torus semicircularis AD Anterodorsal tegmental nucleus AV Anteroventral tegmental nucleus PD Posterodorsal tegmental nucleus PV Posteroventral tegmental nucleus RIS Nucleus reticularis isthmi NIP Nucleus interpeduncularis NI Nucleus isthmi Cer Nucleus cerebelli

0 0 qq q q q q qq qq qq q qq qqq qq

Metencephalon MC Molecular cell layer of the cerebellum PC Purkinje cell layer of the cerebellum GC Granule cell layer of the cerebellum CAL Auricular lobe of the cerebellum

0 qq qqq qq

Rhombencephalon Pch Choroid plexus SL Sulcus limitans Gc Griseum central rhombencephali Ra Nucleus raphes Ri Nucleus reticularis inferior Rm Nucleus reticularis medius FS Fasciculus solitarius V Nucleus of the trigeminal nerve VI Nucleus of the abducent nerve VII Nucleus of the facial nerve VIII d Dorsal nucleus of the stato-acusticus nerve VIII v Ventral nucleus of the stato-acusticus nerve

0 0 qqq 0 0 0 q q q q q q

Hypophysis PN PI Pdis

0 qqqq 0

Pars nervosa Pars intermedia Pars distalis

Neuropeptides

SRIF w25,57x SRIF w25,57x UII w10x Enk w32x, Gal w26x, PACAP w61x, SRIF w25,57x Neuro B w42x, NPY w15x, SRIF w25,57x ANF w38x, Neuro B w42x, NPY w15x, PACAP w61x, SRIF w25,57x Neuro B w42x Neuro B w42x ANF w38x, CRF w40,55x, Enk w32x, NPY w15x, SRIF w25,57x NPY w15x

DSIP w60x

Neuro B w42x, NPY w15x, SRIF w25,57x Neuro B w42x

Gal w26x UII w10x UII w10x

a MSH w5x DSIP w60x

q, low density; qq, moderate density; qqq, high density; qqqq, very high density; 0, no PC2 mRNA hybridization signal. Abbreviations used: ANF, atrial natriuretic factor; AVT, arginine vasotocin; CGRP, calcitonin gene-related peptide; CRF, corticotropin-releasing factor; DSIP, delta sleep-inducing peptide; Enk, enkephalin; Gal, galanin; mGnRH, mammalian gonadotropin-releasing hormone; MT, mesotocine; Neuro B, neuromedin B; NPY, neuropeptide Y; PACAP, pituitary adenylate cyclase-activating polypeptide; SRIF, somatostatin; Tach, tachykinin; TRH, thyrotropin-releasing hormone; UII, urotensin II.

that sorting of PC2 into the regulated secretory pathway depends on its carboxyl terminal region w14x. 4.2. Distribution of PC2 mRNA RT-PCR analysis showed that the PC2 gene is expressed in all tissues examined including brain, pituitary, pancreas, stomach, liver, kidney, intestine and testis. These results are in agreement with those reported in mammals using Northern blot analysis w49x. In situ hybridization histochemistry revealed the widespread distribution of PC2 mRNA in the frog brain and pituitary. The highest densi-

ties of PC2 mRNA were observed in the lateral thalamic nucleus, and in various hypothalamic nuclei including the magnocellular preoptic nucleus, the suprachiasmatic nucleus, the ventral hypothalamic nucleus and the nucleus of the periventricular organ. High concentrations of PC2 mRNA were also found in the pallium, the amygdala, the striatum, the optic tectum and the tegmental area. In the pituitary, the PC2 gene was strongly expressed in the pars intermedia of the pituitary while the neural and the distal lobes exhibited no significant mRNA expression. Comparison between the location of PC2 hybridization signal and the distribution of immunoreactive peptides in the brain of R. ridibunda Žor the closely related species R.

D. Vieau et al.r Molecular Brain Research 63 (1998) 1–13

esculenta. revealed that most areas which actively express PC2 are also enriched with cell bodies containing biologically active neuropeptides ŽTable 1.. In particular, the preoptic nucleus, the suprachiasmatic nucleus, the ventral hypothalamic nucleus and the nucleus of the periventricular organ which contain a number of neuronal populations expressing various neuropeptide precursors also exhibited a high concentration of PC2 mRNA. This observation is consistent with the notion that PC2 plays a major role in the processing of neuropeptides. However, a few regions expressing high levels of PC2 mRNA, such as the lateral thalamic nucleus and the granule cell layer of the cerebellum, do not contain any known neuropeptides. The expression of PC2 in these brain regions suggests that the convertase may be involved in the processing of precursors for novel neuropeptides that have not yet been identified. Reciprocally, a few brain regions which are enriched with peptidergic neurons appeared to be virtually devoid of PC2 mRNA. For instance, the lateral hypothalamic nucleus, which contains somatostatinergic neurons w25,57x, did not exhibit any PC2 mRNA. This observation suggests that, in these neuronal populations, neuropeptide precursors are processed by PC1 or other PCs. Consistent with this hypothesis, knock-out experiments have recently shown that mice lacking the PC2 gene, although having reduced growth rate and fertility, do not show major physiological and behavioural abnormalities w21x. These observations suggest the existence of redundancy Žandror plasticity. in the convertase action on neuropeptide precursors. The distribution of PC2 mRNA in the frog brain is remarkably similar to that previously reported in mammals w45x. In both rat and frog, the highest density of PC2 is found in regions known to be enriched with neuropeptides such as the arcuate nucleus and the magnocellular neurons of the supraoptic nucleus. However, some discrete differences were observed, notably in the raphe nucleus which does not express the PC2 gene in frog but contains a moderate density of PC2 mRNA in rat. In the amphibian, as in the mammmalian brain, PC2 transcripts were found in neurons to the exclusion of other types of cells such as the ependymal cells bordering the ventricles or the choroid plexus. Consistent with this observation, it has been reported that, in glial cells which do not possess dense core secretory vesicles, neuropeptide precursors are likely processed by furin andror other convertases such as PACE4 w20x. In the pituitary, the expression pattern of PC2 was very similar in frog to that previously reported in rat w16,45x. High levels of PC2 mRNA were observed in the pars intermedia which strongly suggests that, in the amphibian as in mammalian melanotrope cells w3x, PC2 is responsible for the production of a-MSH. No PC2 mRNA was detected in the pars distalis, indicating that the enzyme is not involved in the processing of precursors for adenohypophysial hormones. Similarly, PC2 is not expressed in the

11

pars nervosa in which pituicytes, the predominant cell type, exhibit structural and functional similarities with astroglial cells w44x. In conclusion, the characterization of the frog PC2 cDNA has revealed that this enzyme has been remarkably well conserved during evolution. The pattern of distribution of PC2 mRNA in the brain and in the intermediate lobe of the pituitary is consistent with the view that PC2 plays an important role in the post-translational processing of polypeptide precursors.

Acknowledgements This work was supported by grants from INSERM ŽU413. and the Conseil Regional de Haute-Normandie. We ´ gratefully acknowledge P. Bizet for excellent technical assistance. We thank Drs Y. Anouar for providing the frog pituitary cDNA library and I. Boutelet for stimulating comments on the manuscript.

References w1x A.C. Andersen, M.C. Tonon, G. Pelletier, J.M. Conlon, A. Fasolo, H. Vaudry, Neuropeptides in the amphibian brain, Int. Rev. Cytol. 138 Ž1992. 89–208 and 315–326. w2x Y. Anouar, S. Jegou, D. Alexandre, I. Lihrmann, J.M. Conlon, H. ´ Vaudry, Molecular cloning of frog secretogranin II reveals the occurrence of several highly conserved potential regulatory peptides, FEBS Lett. 394 Ž1996. 295–299. w3x S. Benjannet, N. Rondeau, R. Day, M. Chretien, N.G. Seidah, PC1 ´ and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues, Proc. Natl. Acad. Sci USA 88 Ž1991. 3564–3568. w4x S. Benjannet, N. Rondeau, L. Paquet, A. Boudreault, C. Lazure, M. Chretien, N.G. Seidah, Comparative biosynthesis, covalent post´ translational modifications and efficiency of prosegment cleavage of the prohormone convertases PC1 and PC2: glycosylation, sulphation and identification of the intracellular site of prosegment cleavage of PC1 and PC2, Biochem. J. 294 Ž1993. 735–743. w5x M. Benyamina, C. Delbende, S. Jegou, P. Leroux, F. Leboulenger, ´ M.C. Tonon, J. Guy, G. Pelletier, H. Vaudry, Localization and identification of a-melanocyte-stimulating hormone Ž a-MSH. in the frog brain, Brain Res. 366 Ž1986. 230–237. w6x J.A. Braks, K.C.W. Guldemond, M.C.H.M. van Riel, A.J.M. Coenen, G.J.M. Martens, Structure and expression of Xenopus prohormone convertase PC2, FEBS Lett. 305 Ž1992. 45–50. w7x A. Bruzzaniti, K. Goodge, P. Jay, S.A. Taviaux, M.H.C. Lam, P. Berta, T.J. Martin, J.M. Moseley, M.T. Gillespie, C8, a new member of the convertase family, Biochem. J. 314 Ž1996. 727–731. w8x N. Chartrel, J.M. Conlon, J.M. Danger, A. Fournier, M.C. Tonon, H. Vaudry, Characterization of melanotropin-release-inhibiting factor Žmelanostatin. from frog brain: homology with human neuropeptide Y, Proc. Natl. Acad. Sci. USA 88 Ž1991. 3862–3866. w9x N. Chartrel, Y. Wang, A. Fournier, H. Vaudry, J.M. Conlon, Frog vasoactive intestinal polypeptide and galanin: primary structures and effects on pituitary adenylate cyclase, Endocrinology 136 Ž1995. 3079–3086. w10x N. Chartrel, J.M. Conlon, F. Collin, B. Braun, D. Waugh, M. Vallarino, S.L. Lahrichi, J.E. Rivier, H. Vaudry, Urotensin II in the central nervous system of the frog Rana ridibunda: immunohisto-

12

w11x

w12x

w13x

w14x

w15x

w16x

w17x

w18x

w19x

w20x

w21x

w22x

w23x

w24x

w25x

w26x

w27x

D. Vieau et al.r Molecular Brain Research 63 (1998) 1–13 chemical localization and biochemical characterization, J. Comp. Neurol. 364 Ž1996. 324–339. P. Chomczynski, N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction, Anal. Biochem. 162 Ž1987. 156–159. F. Collin, N. Chartrel, A. Fasolo, J.M. Conlon, F. Vandesande, H. Vaudry, Distribution of two molecular forms of gonadotropin-releasing hormone ŽGnRH. in the central nervous system of the frog Rana ridibunda, Brain Res. 703 Ž1995. 111–128. J.M. Conlon, F. O’Harte, D.D. Smith, M.C. Tonon, H. Vaudry, Isolation and primary structure of urotensin II from the brain of a tetrapod, the frog Rana ridibunda, Biochem. Biophys. Res. Commun. 188 Ž1992. 578–583. J.W.M. Creemers, E.F. Usac, N.A. Bright, J.W. Van de Loo, E. Jansen, W.J.M. Van de Ven, J.C. Hutton, Identification of a transferable sorting domain for the regulated pathway in the prohormone convertase PC2, J. Biol. Chem. 41 Ž1996. 25284–25291. J.M. Danger, J. Guy, M. Benyamina, S. Jegou, F. Leboulenger, J. ´ Cote, ˆ ´ M.C. Tonon, G. Pelletier, H. Vaudry, Localization and identification of neuropeptide Y ŽNPY.-like immunoreactivity in the frog brain, Peptides 6 Ž1985. 1225–1236. R. Day, M.K.H. Schafer, S.J. Watson, M. Chretien, N.G. Seidah, ´ Distribution and regulation of the prohormone convertases PC1 and PC2 in the rat pituitary, Mol. Endocrinol. 6 Ž1992. 485–497. I. De Bie, M. Marcinkiewicz, D. Malide, C. Lazure, K. Nakayama, M. Bendayan, N.G. Seidah, The isoforms of proprotein convertase PC5 are sorted to different subcellular compartments, J. Cell Biol. 135 Ž1996. 1261–1275. L. Desrues, M.C. Tonon, J. Leprince, H. Vaudry, J.M. Conlon, Isolation, primary structure and effect on a-MSH release of frog neurotensin, Endocrinology 139 Ž1998. 4140–4146. K. Dierickx, F. Vandesande, Immuno-enzyme cytochemical demonstration of mesotocinergic nerve fibres in the pars intermedia of the amphibian hypophysis, Cell Tissue Res. 174 Ž1976. 25–33. W. Dong, M. Marcinkiewicz, D. Vieau, M. Chretien, N.G. Seidah, ´ R. Day, Distinct mRNA expression of the highly homologous convertases PC5 and PACE4 in the rat brain and pituitary, J. Neurosci. 15 Ž1995. 1778–1796. M. Furuta, H. Yano, A. Zhou, Y. Rouille, ´ J.J. Holst, R. Carroll, M. Ravazzola, L. Orci, H. Furuta, D.F. Steiner, Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2, Proc. Natl. Acad. Sci. USA 94 Ž1997. 6646–6651. D. Julius, A. Brake, L. Blair, R. Kunisawa, J. Thorner, Isolation of the putative structural gene for the lysine–arginine-cleaving endopeptidase required for processing the yeast prepro-a-factor, Cell 37 Ž1984. 1075–1089. M.C. Kiefer, J.E. Tucker, R. Joh, K.E. Landsberg, D. Saltman, P.J. Barr, Identification of a second human subtilisin-like protease gene in the fesr fps region of chromosome 15, DNA Cell Biol. 10 Ž1991. 757–769. M. Lamacz, C. Hindelang, M.C. Tonon, H. Vaudry, M.E. Stoeckel, Three distinct thyrotropin-releasing hormone-immunoreactive axonal systems project in the median eminence-pituitary complex of the frog Rana ridibunda. Immunocytochemical evidence for co-localization of thyrotropin-releasing hormone and mesotocin in fibers innervating pars intermedia cells, Neuroscience 32 Ž1989. 451–462. A. Laquerriere, ` P. Leroux, B.J. Gonzalez, C. Bodenant, R. Benoit, H. Vaudry, Distribution of somatostatin receptors in the brain of the frog Rana ridibunda: correlation with the localization of somatostatin-containing neurons, J. Comp. Neurol. 280 Ž1989. 451–467. G.Y. Lazar, ´ ´ Z.S. Liposits, P. Toth, ´ S.L. Trasti, J.L. Maderdrut, I. Merchenthaler, Distribution of galanin-like immunoreactivity in the brain of Rana esculenta and Xenopus laeÕis, J. Comp. Neurol. 310 Ž1991. 45–67. O. Lesouhaitier, M. Esneu, M.K.L. Kodjo, C. Hamel, V. Contesse, L. Yon, I. Remy-Jouet, A. Fasolo, A. Fournier, F. Vandesande, G. ´ Pelletier, J.M. Conlon, E.W. Roubos, M. Feuilloley, C. Delarue, F.

w28x

w29x

w30x

w31x

w32x

w33x

w34x

w35x

w36x

w37x w38x

w39x

w40x

w41x

w42x

w43x

w44x

w45x

Leboulenger, H. Vaudry, Neuroendocrine communication in the frog adrenal gland, Zool. Sci. 12 Ž1995. 255–264. I. Lihrmann, J.C. Plaquevent, H. Tostivint, R. Raijmakers, M.C. Tonon, J.M. Conlon, H. Vaudry, Frog diazepam-binding inhibitor: peptide sequence, cDNA cloning and expression in the brain, Proc. Natl. Acad. Sci. USA 91 Ž1994. 6899–6903. J. Lusson, D. Vieau, J. Hamelin, R. Day, M. Chretien, N.G. Seidah, ´ cDNA structure of the mouse and rat subtilisinrkexin-like PC5: a novel candidate proprotein convertase expressed in endocrine and nonendocrine cells, Proc. Natl. Acad. Sci. USA 90 Ž1993. 6691– 6695. J. Lusson, S. Benjannet, J. Hamelin, D. Savaria, M. Chretien, N.G. ´ Seidah, The integrity of the RRGDL sequence of the proprotein convertase PC1 is critical for its zymogen and C-terminal processing and for its cellular trafficking, Biochem. J. 326 Ž1997. 737–744. J. Meerabux, M.L. Yaspo, A.J. Roebroek, W.J.M. Van de Ven, T.A. Lister, B.D. Young, A new member of the proprotein convertase gene family ŽLPC. is located at a chromosome translocation breakpoint in lymphomas, Cancer Res. 56 Ž1996. 448–451. I. Merchenthaler, G. Lazar, ´ ´ J.L. Maderdrut, Distribution of proenkephalin-derived peptides in the brain of Rana esculenta, J. Comp. Neurol. 281 Ž1989. 23–39. A. Mitra, L. Song, L.D. Fricker, The C-terminal region of carboxypeptidase E is involved in membrane binding and intracellular routing in AtT-20 cells, J. Biol. Chem. 269 Ž1994. 19876–19881. B. Mulatero, A. Fasolo, Calcitonin gene-related peptide immunoreactivity in the hypothalamo-hypophysial system of the green frog, Rana esculenta, Gen. Comp. Endocrinol. 81 Ž1991. 349–356. T. Nakagawa, M. Hosaka, S. Torii, T. Watanabe, K. Murakami, K. Nakayama, Identification and functional expression of a new member of the mammalian Kex2-like processing endoprotease family: its striking structural similarity to PACE4, Biochem. J. 113 Ž1993. 132–135. K. Nakayama, W.S. Kim, S. Torii, M. Hosaka, T. Nakagawa, J. Ikemizu, T. Baba, K. Murakami, Identification of the fourth member of the mammalian endoprotease family homologous to the yeast Kex2 protease: its testis-specific expression, J. Biol. Chem. 267 Ž1992. 5897–5900. T.J. Neary, R.G. Northcutt, Nuclear organization of the bullfrog diencephalon, J. Comp. Neurol. 213 Ž1983. 262–278. P. Netchitaılo, ¨ M. Feuilloley, G. Pelletier, F. Leboulenger, M. Cantin, J. Gutkowska, H. Vaudry, Atrial natriuretic factor-like immunoreactivity in the central nervous system of the frog, Neuroscience 22 Ž1987. 341–359. R.G. Northcutt, E. Kicliter, Organization of the amphibian telencephalon, in: S.O.E. Ebbeson ŽEd.., Comparative Neurology of the Telencephalon, Plenum, New York, 1980, pp. 203–255. M. Olivereau, F. Vandesande, E. Boucique, F. Ollevier, J.M. Olivereau, Immunocytochemical localization and spatial relation to the adenohypophysis of a somatostatin-like and a corticotropin-releasing factor-like peptide in the brain of four amphibian species, Cell Tissue Res. 247 Ž1987. 317–324. T. Ouimet, A. Mammarbachi, T. Cloutier, N.G. Seidah, V.F. Castellucci, cDNA structure and in situ localization of the Aplysia californica pro-hormone convertase PC2, FEBS Lett. 330 Ž1993. 343–346. P. Panzanelli, B. Mulatero, L.H. Lazarus, A. Fasolo, Neuromedin B-like immunoreactivity in the brain of the green frog Rana esculenta, Eur. J. Basic Appl. Histochem. 35 Ž1991. 359–370. A.J.M. Roebroek, J.A. Schalken, M.J.G. Bussemakers, H. van Heerinkhuizen, C. Onnekink, F.M.J. de Bruyne, H.P.J. Bloemers, W.J.M. van de Ven, Characterization of human fesr fps reveals a new transcription unit Ž fur . in the immediate upstream region of the proto-oncogene, Mol. Biol. Rep. 11 Ž1986. 117–126. A.K. Salm, G.I. Hatton, G. Nilaver, Immunoreactive glial fibrillary acidic protein in pituicytes of the rat neurohypophysis, Brain Res. 236 Ž1982. 471–476. M.K.H. Schafer, R. Day, W.E. Cullinan, M. Chretien, N.G. Seidah, ¨ ´

D. Vieau et al.r Molecular Brain Research 63 (1998) 1–13

w46x

w47x

w48x

w49x

w50x

w51x

w52x

S.J. Watson, Gene expression of prohormone and proprotein convertases in the rat CNS: a comparative in situ hybridization analysis, J. Neurosci. 13 Ž1993. 1258–1279. N.G. Seidah, L. Gaspar, P. Mion, M. Marcinkiewicz, M. Mbikay, M. Chretien, cDNA sequence of two distinct pituitary proteins ´ homologous to Kex2 and furin gene products: tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases, DNA Cell Biol. 6 Ž1990. 415–424. N.G. Seidah, M. Marcinkiewicz, S. Benjannet, L. Gaspar, G. Beaubien, M.G. Mattei, C. Lazure, M. Mbikay, M. Chretien, Cloning ´ and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, furin and Kex2: distinct chromosomal localization and messenger RNA distribution in brain and pituitary as compared to PC2, Mol. Endocrinol. 5 Ž1991. 111–122. N.G. Seidah, R. Day, J. Hamelin, A. Gaspar, M.W. Collard, M. Chretien, Testicular expression of PC4 in the rat: molecular diversity ´ of a novel germ cell-specific Kex2rsubtilisin-like proprotein convertase, Mol. Endocrinol. 6 Ž1992. 1559–1570. N.G. Seidah, M. Chretien, R. Day, The family of subtilisinrkexin ´ like pro-protein and pro-hormone convertases: divergent or shared functions, Biochimie 76 Ž1994. 197–209. N.G. Seidah, J. Hamelin, M. Mamarbachi, W. Dong, H. Tadros, M. Mbikay, M. Chretien, R. Day, cDNA structure, tissue distribution, ´ and chromosomal localization of rat PC7, a novel mammalian proprotein convertase closest to yeast kexin-like proteinases, Proc. Natl. Acad. Sci. USA 93 Ž1996. 3388–3393. S.P. Smeekens, D.F. Steiner, Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing protease Kex2, J. Biol. Chem. 265 Ž1990. 2997–3000. S.P. Smeekens, A.S. Avruch, J. LaMendola, S.J. Chan, D.F. Steiner, Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans, Proc. Natl. Acad. Sci. USA 88 Ž1991. 340–344.

13

w53x A.B. Smit, S. Spijker, W.P.M. Geraerts, Molluscan putative prohormone convertases: structural diversity in the central nervous system of Lymnaea stagnalis, FEBS Lett. 312 Ž1992. 213–218. w54x D. Taverna, B. Mulatero, A. Fasolo, Co-occurrence of substance P and enkephalin-like immunoreactivities in the proencephalon of two amphibians, Eur. J. Histochem. 37 Ž1993. 33–42. w55x M.C. Tonon, A. Burlet, M. Lauber, P. Cuet, S. Jegou, L. Gouteux, ´ H. Vaudry, Immunohistochemical localization and radioimmunoassay of corticotropin-releasing factor in the forebrain and hypophysis of the frog Rana ridibunda, Neuroendocrinology 40 Ž1985. 109–119. w56x M.C. Tonon, L. Desrues, M. Lamacz, N. Chartrel, B. Jenks, H. Vaudry, Multihormonal regulation of pituitary melanotrophs, Ann. N.Y. Acad. Sci. 680 Ž1993. 175–187. w57x H. Tostivint, I. Lihrmann, C. Bucharles, D. Vieau, Y. Coulouarn, A. Fournier, J.M. Conlon, H. Vaudry, Occurrence of two somatostatin variants in the frog brain: characterization of the cDNAs, distribution of the mRNAs, and receptor-binding affinities of the peptides, Proc. Natl. Acad. Sci. USA 93 Ž1996. 12605–12610. w58x H. Vaudry, J.M. Conlon, Identification of a peptide arising from the specific post-translation processing of secretogranin II, FEBS Lett. 284 Ž1991. 31–33. w59x H. Vaudry, N. Chartrel, J.M. Conlon, Isolation of wPro 2 , Met 13 xsomatostatin-14 and somatostatin-14 from the frog brain reveals the existence of a somatostatin gene family in a tetrapod, Biochem. Biophys. Res. Commun. 188 Ž1992. 477–482. w60x L. Yon, M. Feuilloley, Y. Charnay, H. Vaudry, Immunohistochemical localization of delta sleep-inducing peptide-like immunoreactivity in the central nervous system and pituitary of the frog Rana ridibunda, Neuroscience 47 Ž1992. 221–240. w61x L. Yon, M. Feuilloley, N. Chartrel, A. Arimura, J.M. Conlon, A. Fournier, H. Vaudry, Immunohistochemical distribution and biological activity of pituitary adenylate cyclase-activating polypeptide ŽPACAP. in the central nervous system of the frog Rana ridibunda, J. Comp. Neurol. 324 Ž1992. 485–499.