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Characterization of murine PACAP mRNA
Peptides, Vol. 16, No. 7, pp. 1295-1299, 1995 Copyright 0 1995 Elsevier Science Inc. Printed in the USA. All tights reserved Ol96-9781/95 $9.50 + .OO
Pergamon Ol%-9781( 95)02018-R
Characterization KIMITAKE
OKAZAKI,
Discovery
Research
YASUAKI
ITOH,
of Murine PACAP mRNA1 KAZUHIRO
OGI,
SHOICHI
OHKUBO
Laboratories Z, Discovery Research Division, Takeda Chemical 10 Wadai, Tsukuba, Zbaraki 300-42, Japan Received
AND
HARUO
Industries,
0NDA2
Ltd.,
10 April 1995
OKAZAKI, K., Y. ITOH, K. OGI, S. OHKUBO AND H. ONDA. Characrerizarion ofmurine PACAP mRNA. PEPTIDES 16(7) 1295- 1299, 1995.--.4 murine PACAP precursor cDNA was isolated by screening a brain cDNA library. The amino acid sequence of the precursor was highly similar (from 8 1% to 93% similarity) to its rat, human, and ovine counterparts. The primary structure of murine PACAP is identical with those from sheep, humans, and rats, indicating that the mature PACAP is well conserved among mammals. Northern blot analysis revealed that the approximately 2.4 kb transcript for the PACAP precursor is expressed
in murine brain. The verification that murine PACAP is identical to its human counterpart provides a rationale for physiological and pathophysiological studies of PACAP in mice. PACAP
Mouse
Precursor
cDNA
mRNA
Peptides
PITUITARY adenylate cyclase activating polypeptide (PACAP) is a newly recognized polypeptide belonging to the secretin/glucagon/vasoactive intestinal peptide (VIP) family. Two amidated forms, a 38-amino acid peptide (PACAP-38) and a truncated form corresponding to its N-terminal 27 amino acid residues (PACAP-27), were originally isolated from ovine hypothalami by Miyata et al. ( 17,18 ) . They showed that the adenylate cyclasestimulating potency of both PACAPand PACAPwere 1000 times greater than that of VIP in cultured rat pituitary cells ( 17,18). Subsequent studies showed that PACAP provokes the secretion of pituitary hormclnes from pituitary cells (9,17 ) , amylase (20), and insulin ( 13 ) from the pancreas, and adrenaline from adrenal chromaffin cells (38). PACAP also evokes endothelium-independent vasodi lation (37) and interleukin-6 synthesis in the pituitary (33). The distribution of PACAP was wide spread in rat brain and testes ( 1,16). Immunohistochemical and ligand binding studies have revealed that PACAP has a neuronal localization in both the central and peripheral nervous systems, suggesting that PACAP acts as a neurotransmitter and/or neuromodulator ( 2). PACAPand PACAPare synthesized from a common precursor, and their amino acid sequences are identical in humans ( 14), sheep ( 14)) and rats I(21) . The human PACAP gene was also sequenced and assigned to a locus on chromosome 18~11 (22). High-affinity PACAP receptors have been identified in several tissues and cell lines (28). Recently, cDNAs encoding PACAP specific receptors (type I, which binds PACAP with a much higher affinity than VIP) were isolated from a rat brain cDNA library ( 11.30). The type I PACAP receptor was also purified in a fully active form from bovine brain membranes (24). Up to the present, wide-ranging biological and physiological studies of PACAP were performed using mice (3,4,7,10), mu-
Brain
Prohormone
FIG. I. Restriction enzyme map and sequencing strategy for the murine PACAP cDNA. Lines and the whole box indicate the noncoding and coding regions, respectively. The mature PACAP and PRP regions are indicated with filled and dotted boxes, respectively. The arrows indicate the direction and extent of the sequenced regions.
tine tissues (31,32), and murine cell lines (6,12,15,26,28,34) because well-established information on the biology and genetics of mice exists. In contrast, little is known about the molecular organization of murine PACAP. We report here the molecular properties of PACAP for this laboratory animal. METHOD Puri$cation
of Murine Brain Poly(A)+
RNA
Total RNA was extracted from the brains of four adult 8week-old ICR mice as described previously (22). Poly (A)’ RNA was purified from the total RNA with an oligo( dT)-cellulose spun column (Pharmacia LKB, Uppsala, Sweden). Construction
of a Murine Brain cDNA Library
Murine brain cDNA was synthesized from the poly(A)+ RNA with AMV RNA reverse transcriptase (Seikagaku Kogyo,
’ Sequence data from this article have been deposited with the DDBJ, EMBL, and GenBank ’ Requests for reprints should be addressed to Dr. Haruo Onda.
1295
Data Libraries
under the accession
number D14716.
1296
OKAZAKI
1
GAGAGTTTCTTCGGGTTAGAAGGTAGAAAGCCAGGAGCGATTTCACTCACTGCATACCCTCTTTCCTCTCCTGCGCAGA
79
80 1
ATG ACC ATG TGT AGC CGA GCA AGG CTG CCC CTG CTG GTG TAT CCC ATA ATA ATC CAT AGC Met Thr Met Cys Ser GIY Ala Arg Leu Ala Leu Leu Val TYT GIY Ile Ile Met His Ser
139 20
140 21
AGT GTC ICC TGT + TCA CCT CCC CCC CGA CTC AGC TTC CCT GGG ATC AGA CCA GAA GAC GAG Ser Val Ser Cys Ser Pro Ala Ala GIY Leu Scr Phe Pro Gly lie Arg Pro Glu Asp Glu
199 40
200 41
GCT TAC GAC CAG GAC CGA AAC CCC CTG CAA GAC TTC TAT GAC TGG GAC CCT CCC GGC GTG Ala Tyr Asp Gln Asp CIY Asn Pro Leu Gln ASP Phe Tyr Asp Trp Asp Pro Pi-o Gly Val
259 60
260 61
GGG AGC CCC GCC TCC CCC CTG CGT GAC GCT TAC GCC CTT TAC TAT CCA GCG GAC AGG ACA GIY Ser Pro Ala Ser Ala Leu Arg ASP Ala TYT Ala Leu TYT TYT Pro Ala Asp Arg Arg
319 80
320 81
CAT GTC CCC CAC GAA ATC CTT AAC GAA CCC TAT CGA AAA GTC TTG GAC CAG CTG TCC CCC ASD Val Ala His Glu Ile Leu Asn Glu Ala Tyr Arg LYS Val Leu ASP Gln Leu Ser Ala
379 100
380 101
AGG AAG TAC CTG CAG TCG GTC GTG GCC AGG CGC GCG GGG GAC GAA CCT AGG CCG CAC CCC Arg Lys Tyr Leu Gin Ser Val Val Ala Arg GIY Ala Gly Asp Glu Pro Arg Arg His Ala
439 120
440 121
GTG GAC GAC CCC CCC CCC CTT ACC AAA CCC CAC TCG GAC GGC ATC TTC ACA CAT ACC TAC VaI Asp Asp Pro Ala Pro Leu Thr LYS A.-g His Ser Asp CIY Ile Phe Thr Asp Ser Tyr
499 140
500 141
AGC CCC TAC CGA AAA CAA ATG GCT GTC AAG AAA TAC TTG CCC GCC GTG CTA GGG AAA AGG Ser Arx Tyr Arg LYS Gin Met Ala Val LYS LYS TYT Leu Ala Ala Val Leu Gly Lys Arg
559 160
560 161
TAT AAA CAG AGG GTT AAA AA~ AAA GGA ccc CGA ATA GCA TAC TTG TAG CAGATCAGCTGCCGG Tyr LYS Gin Arg Val LYS Asn LYS GIY Arg Arg lie Ala Tyr Leu c*c
622 175
623
CTACCTTGTGTATAAAATGAAAAGTCGTTTTCCAAATTGACTGACCAGTCATCACTCGTGTTCTTTCCAAACATGTATT
701
702
TATGTATCAAGTAAAGCCATTAAATGACTATTTTGATAATAATATTGTTTTTCTTTTTATGAAGCACTAGAGAATGCAC
780
781
AGATATACTTTGTGGACCAATTATTGATATATATTATAAGTATATATATTAAGAATATATATAAGTATAACAGAGAGCA
859
860
ATTAAGATGGGTGCACAAGGATTGAAAATTCGCCTGAGCTGCTTTATGTTTTTATATAAAGTAAATAGAGAAAATAGAC
938
939
AACCATTGTTTTGAATATTACTCCTATTTTTGTAAACTGGAATTAAAGGATAGTATTTTTATCCACAACCGTCTTGAAG
1017
1018
ATACCAATAATGGCCATTTGTACAAAAAACAATGATGCCCTGCTCCAGGGGAATTCTGAGGTAATGACTTGGGGAATTG
1096
1097
CTCAAGGGCTTTCTTTCCCTCTGAGTCTGGGGCAGGCTGCTTGAACCCCAGCCTAACTAACTCAAGTGGGCATTGTCCC
1175
1176
ACTGGTTGCAGGGGCAATTCCAACAATTTCAGTTTCTTTGATTATGTGTATTTGTCTCTCCTCAGACTCTCAGCCCAGA
1254
1255
AGGAAATTCTAATTAAACAACAGCTCTATCCAAATTGTGCTTCTCCCAGAACTCATGTCATTCCCTGATAGAAGAGTTG
1333
1334
AGGAACTGTACAGAAGAGACAGGCTTGGAGAGAGAGCTCTCTTTTCTGTACTTCCTGATTCTCCAGGGAACAGACTATT
1412
1413
CTAAGGCTAGGGCAATTGGAACAAAGTGAAAGATATATAAGGGATTGGTAAAGGCA~AACATGGGGATTTGAGATTTGA
1491
1492
GAGTTGCCTCAGGTCTGAGAATCTGGGGGCAAGTCTAGCTCCTCTGTAGGTTCCACTGCCTGACAGATCAGGTGCTGGT
1570
1571
GTTGGAATGAATGCAAAGTACAATGTGTTTTTCTCCAGTGCTGTTCATGCTTTTCATGTTGTGAAATGGCCAGGATCCT
1649
1650
CCCTTTGAACACTGTTCTGCAGAAGCCAGCTCTGTTCTTTGTGGATTTTCTGGAGACCCTCCTTCCTACCCTTGCCCTC
1728
1729
ATGCATTGTTTTAGAGTCATTTGCCATTTTCCACTCACTTATCTTAAATTTGTGAATGCTAGTTATTTTTTGTTGTTGT
1807
1808
TTGATGCAAGCAGTTACTGTGAAGTTTAAGAACCCCTGTGTAGCTTCCACAGAGAAATTATGCACTAAATATGAACCTT
1886
1887
TTGTTTCTTGTTTATTGACTTTGTAGGTAAAAATGTATTTTTCTATATTATGGCTTATTGCTT~G~~~~ATTTATTTCA
1965
1966
TAAAACCAATCTTTGTCATATTAGAATGTGTAGTGTTCCAATGCTGCTCAGTTTGACTGATAAATCATTTAAACCCCAT A
2045
ET AL.
2044 2123
2124
2202
2203
2249
FIG. 2. Nucleotide sequence of the cDNA and the predicted amino acid sequence of the murine PACAP precursor protein. The probable cleavage site of the signal peptide is shown by an arrow. The box and the solid underline indicate the mature PACAP and PRP, respectively. The polyadenylation signal sequences are indicated by broken underlines. An arrowhead indicates the polyadenylation position of clones No. 12 and No. 14.
Japan), E. coli RNase H, E. coli DNA polymerase I, and T4 DNA polymerase (Amersham International plc., Buckinghamshire, UK). A murine brain cDNA library was then constructed in Agtll with the cDNA Cloning System Adaptor Method ( Amersham International plc.). Tokyo,
Cloning of the cDNA Encoding for the Precursor of Murine PACAP An Act I-Eco RI 1.17 kb fragment of rat PACAP precursor cDNA (21) was labeled with [a-32P]dCTP (110 TBq/mmol;
Amersham International plc.) using Prime-ItTM Random Primer Kit (Stratagene, La Jolla, CA) and used to screen the murine brain cDNA library. Approximately 3 X IO6 plaques on nitrocellulose filters (Schleicher & Schuell, Dassel, Germany) were screened by plaque hybridization with the DNA probe at 60°C in a hybridization buffer (7% SDS, 0.5% BSA, 1 mM EDTA, and 0.5 M sodium phosphate buffer, pH 7.2) for 16h (8). The filters were washed in 0.3 M sodium chloride and 0.03 M sodium citrate (2 X SSC) at 65°C then dried and autoradiographed using Kodak X-OMATTM AR films (Eastman Kodak Company, Rochester, NY) with an intensifying screen at
MURINE
PACAP
mRNA
Mouse Rat
1297
CHARACTERIZATION
PACAP
PACAP
1 MTNCSGARLA
LLVYGIIMHS
SVSCSPAA-G
1 ****tt****
***"****N
**tt****_-* ***'*****R
EAYDQDGNPL
QDFYDWDPPG
59
*********
et********
*t**t*****
**********
*+YS****A*
.jg
lR*******R
+++GH*****
P**GGSR***
***t******
*****tL***
SO
**yc****s*
*R*******N
***q****o
*****SE***
ijo
60 VGSPASALRD
RYALYYPADR
I_---_? DVAHEILNE
SO A*********
**********
*t********
AYRKVLDQLS ARKYLQSW ******,c*** *******Me*
61 A****f*P*A
*A*w****G*
61 ****t*+***
**‘*.r*f*~~
Human
PACAP
1
Ovine
PACAP
1
LSFPGIRPED
l
PRP
****G****
*(+***G**DK
RGAGDEPRRH **MkHNL-
****eke***
tG*H***L**
**V*GSLGGG
**********
**R*‘*TLM*(
K*L*GT*GGG
if%
120
********* ********** ********** ******** ****t** 1 1 PACAP
HSDGIFTDS
120 AVDDPAPLTK 120 ***+R***"*
YSRYRKQMAV
KKYLAAVLGK
RYKQRVKN
G RRIAYL
175
175
**********
121 *G*'AE**S*
l ********
121 *D**SE**S*
*et*******
**********
*t********
*t********
**********
********
*
******
176
***p**
176
FIG. 3. Comparison of the deduced amino acid sequences of the murine, rat, human, and ovine PACAP precursor proteins. The numbers indicate the position of the amino acid from the N-terminus of the precursor protein. Asterisks in the sequence of the rat. human, and ovine peptides indicate the same amino acid residues as in the murine PACAP precursor protein. The gaps shown by a dash in the sequence of the murine and rat precursor proteins were inserted to maximize tbz alignment of the seouences. Boxes indicate the mature PACAP and PRP regions. An arrowhead shows the probable cleavage site of the signal peptide. 0
1
-80°C for 3 days. Kpn I 2.2 kb fragment from No. 93 clone, which contained the largest cDNA fragment in the hybridization-positive clones, was subcloned into pUCl18, named pMP93K1, and used for further analysis.
Sequencing PACAP
of cDNA Encoding the Precursor of Murine
Several plasmids containing 3 ‘- or 5 ‘-deleted region of the cDNA were constructed with exonuclease III and VII from the plasmid pMP93Kl and used to prepare single-stranded DNAs for DNA sequencing (36). The DNA sequence was determined by a dideoxynucleotide chain termination method (27) with the Klenow fragment (Takara Shuzo, Kyoto, Japan) or Taq DNA polymerase (Promega Corporation, Madison, WI). Southern Blot Analysis of Murine Genomic DNA Genomic DNA samples 1:10 pg) were digested with Eco RI, Bam HI, Bgl II, and Hin dIII, electrophoresed on a 0.8% agarose Rockland, gel ( SeakemTM GTG Agarose, FMC Bioproducts, ME), and transferred to a nylon membrane filter (Biodyne A, Pall Biosupport, Glen Cove, NY) by the method of Southern (29). The Pst I-Eco RI 0.56 kb fragment of murine PACAP cDNA was labeled as described above and used as a probe. Hybridization was carried out at 65°C in a hybridization buffer (7% SDS, 5% dextran sulfate sodium salt, and 0.5 M sodium phosphate buffer, pH 7.2) (5). The filter was washed at 65°C in 0.03 M sodium chloride and 0.003 M sodium citrate (0.2 X SSC) containing 0.1% SDS and analyzed using Imaging PlateTM and Image Analyzer BAS-2000 (Fuji Film, Tokyo, Japan).
RESULTS
Phage clones with hybridization-positive signals were isolated from the murine brain cDNA library. The 2249 bp cDNA insert of clone No. 93, which was the largest cDNA insert obtained, was subcloned into plasmid pUC 118 and sequenced to deduce a coding region for the entire murine PACAP precursor (Figs. 1 and 2). The first initiation codon ( ATG) was located at position 80 in the sequence and was preceded by an in-frame upper termination codon at position 17. The sequence displayed an open reading frame of 525 bases encoding the murine PACAP precursor protein of 175 amino acid residues (Fig. 2). These numbers are identical to those of the rat PACAP precursor, whereas the human and ovine PACAP precursors have one extra amino acid residue at position 29 from the N-terminus of the precursor protein (Fig. 3). The deduced amino acid sequence of murine PACAP is exactly identical to the rat, human, and ovine sequences. The 3’-noncoding sequence of the No. 93 clone contained no poly( A) tail but ended with the putative polyadenylation signal ATTAAA. During the course of this study, two shorter cDNA clones were isolated, which had the poly (A) tail at position 1974. This case suggests that AGTAAA at position 1950- 1955 functioned as an alternative polyadenylation signal. Southern blot analysis of murine genomic DNA was performed with a Pst I-Eco RI fragment (565 bp) of the murine PACAP precursor cDNA as a probe. As in the cases of sheep, humans, and rats ( 14,21,22), a single gene for the PACAP precursor was present in the murine haploid genome (Fig. 4). Northern blot analysis showed that ca. 2.4-kb-long mRNA, which was slightly shorter than that of rat, was transcribed in the murine brain (Fig. 5 ) . DISCUSSION
Northern Blot Analysis of &brine Poly(A)+
RNA
Approximately 10 pg of poly( A) + RNA, purified from whole murine brain, was denatured with glyoxal and dimethyl sulfoxide at 50°C for 1 h. The denatured poly (A)+ RNA was electrophoresed on a 1.2% agarose gel and transferred to a nylon membrane filter ( 35 ) . Hybridization, filter washing, and autoradiography were performed the same as for Southern blot analysis.
Although numerous studies have demonstrated that PACAP is active in both in vivo and in vitro murine systems, neither the amino acid sequence of the peptide nor the nucleotide sequence of its mRNA has been determined. In this study, we report the cloning of a cDNA from mouse brain that encodes the PACAP precursor protein. The cloned cDNA indicated that the murine PACAP precursor consists of 175 amino acid residues. The mu-
1298
OKAZAKI
rine precursor shared 93% amino acid sequence identity with that of the rats, 83% with sheep, and 81% with humans. The nucleotide sequences in the coding region and 3’-noncoding region were very similar among these animals. In contrast, the 5 ‘-noncoding region of the No. 93 clone had no similarity with its rat and ovine counterparts, but resembled the 5’-flanking sequence of the human PACAP gene (23). The most likely explanation for this sequence identity is that the clone No. 93 may be derived from an alternative or insufficiently spliced mRNA because the 5’-noncoding sequence (57-78) is (T/C),,-CAG, which agrees with the consensus sequence of the acceptor site for splicing (19). The 3 ‘-noncoding sequence of the No. 93 clone ended with a putative polyadenylation signal A’ITAAA at positions 22442249. The nucleotide sequences of the other two clones showed that AGTAAA at position 1950- 1955 functioned as an altemative polyadenylation signal. Similar heterologous polyadenylation sites were observed in the PACAP cDNA from a human neuroblastoma cell line IMR-32 (23). This suggests that alternative polyadenylation may be a common feature of the PACAP precursor mRNA. Murine PACAP is identical to the rat, human, and ovine peptides, suggesting that its amino acid sequence is highly conserved among placental mammals. The dibasic amino acid residues
A
ET AL.
B
_L-
:
. 3.3
- 4.4
- 2.4 -
1.4
- 0.24 FIG. 5. Northern blot analysis of whole brain poly(A)’ RNA. Poly(A)’ RNA (IO pg) was used in each lane. Numbers on the right indicate the mobility of the size markers that are specified in kb. Lane A = mouse and lane B = rat.
W
(Lys ‘2’_Arg 17”, Arg “‘) -Arg “’ ) that are considered important in the posttranscriptional processing of mature PACAP were also conserved in the mouse. In the murine PACAP precursor, as in the previously sequenced PACAP precursors, the 29-amino acid peptide provisionally called PACAP-related peptide (PRP) was flanked by basic amino acids (Fig. 3 ) . In our previous experiments, immunoreactive PRP was detected in the culture medium of recombinant CHO cells (25 ) Therefore, PRP or an extended peptide PRP could be another bioactive peptide cleaved from the PACAP precursor as we have already discussed ( 14,21,22). The availability of cDNA and molecular information for murine PACAP can open avenues for further analysis of its role in mice-one of the most valuable laboratory mammals for physiological and pathophysiological studies.
-10.0
-4.8 73.8 3.7
ACKNOWLEDGEMENTS
FIG. 4. Southern blot analysis of murine genomic DNA. Murine genomic DNA was digested by the indicated restriction enzymes and probed with a 0.56 kb Pst I-Eco RI fragment derived from clone No. 93. Numbers
We are especially grateful to Dr. M. Fujino (Takeda Chemical Industries, Ltd.) for his encouragement throughout this work. The authors
on the right indicate in kb.
would like to thank Dr. A. Arimura suggestions.
the molecular
weights
of the restriction
fragments
(Tulane
University)
for valuable
REFERENCES
A.; Miyata, A.; Mizuno, K.; Coy, D. H.; Kitada, C. Tissue distribution of PACAP as determined by
2. Arimura, A. Pituitary adenylate cyclase activating polypeptide (PACAP): Discovery and current status of research. Regul. Pept.
RIA: Highly abundant in the rat brain and testes. Endocrinology 129:2787-2789; 1991.
37:283-303; 1992. 3. Banks, W. A.; Kastin, A. J.; Komaki,
1. Arimura, A.; Somogyvti-Vigh,
G.; Arimura,
A. Passage
of
MURINE
4.
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6.
I.
8. 9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19. 20.
21.
PACAP
mRNA CHARACTERIZATION
pituitary adenylate cyclase activating polypcptide 1-27 and pituitary adenylate cyclase activating polypeptide l-38 across the bloodbrain barrier. J. Pharmacol. Exp. Ther. 267:690-696; 1993. Banks, W. A.; Kastin, A. J ; Komaki, G.; Arimura, A. Pituitary adenylate cyclase activating polypeptide (PACAP) can cross the vascular component of the blood-testis barrier in the mouse. J. Androl. 14:170-173; 1993. Blumberg, B.; Wright, C. V. E.; De Robertis, E. M.; Cho, K. W. Y. Organizer-specific homeobox genes in Xenopus luevis embryos. Science 253:194-196; 1991. Braas, K. M.; Brandenburg, C. A.; May, V. Pituitary adenylate cyclase-activating polypepticle regulation of AtT-20/D16v corticotrope cell proopiomelanocortin expression and secretion. Endocrinology 134:1X6- 195; 1994. Chen, W.; Inui, T.; Hachiya, T.; Ochi, Y.; Nakajima, Y.; Kajita, Y. Stimulatory action of pituitary adenylate cyclase-activating polypeptide (PACAP) on thyroid gland. Biochem. Biophys. Res. Commun. 194:923-929; 1993. Church, G. M.; Gilbert, W Genomic sequencing. Proc. Natl. Acad. Sci. USA 81:1991-1995; 1984. Culler, M. D.; Paschall, C. S. Pituitary adenylate cyclase-activating polypeptide (PACAP) potcntiates the gonadotropin-releasing activity of luteinizing hormone-releasing hormone. Endocrinology 129:2260-2262; 1991. Fridolf, T.; Sundler, F.; Ahren, B. Pituitary adenylate cyclase-activating polypeptide (PACAP): Occurrence in rodent pancreas and effects on insulin and glucagon secretion in the mouse. Cell Tissue Res. 269~275-219; 1992. Hosoya, M.; Onda, H.; Ogi, K.; et al. Molecular cloning and functional expression of rat cDNAs encoding the receptor for pituitary adenylate cyclase activating polypeptide (PACAP). Biocbem. Biophys. Res. Commun. 194:133- 143; 1993. Inagaki, N.; Yoshida, H.; Mizuta, M.; et al. Cloning and functional characterization of a thind pituitary adenylate cyclase-activating polypeptide receptor subtype expressed in insulin-secreting cells. Proc. Natl. Acad. Sci. USA 91:2679-2683; 1994. Kawai, K.; Obse, C.; Watanabe, Y.; Suzuki, S.; Yamashita, K.; Ohashi, S. Pituitary adenylate cyclase activating polypeptide stimulates insulin release from the isolated perfused rat pancreas. Life Sci. 50:257-261; 1992. Kimura, C.; Ohkubo, S.; Ggi, K.; et al. A novel peptide which stimulates adenylate cyclase: Molecular cloning and characterization of the ovine and human cDNAs. Biochem. Biophys. Res. Commun. 166:81-89; 1990. Lemer, U. H.; Lundberg, I’.; Ransjo, M.; Persson, P.; Hakanson, R. Helodermin, helospectin, and PACAP stimulate cyclic AMP formation in intact bone. isolated osteoblasts, and osteoblastic cell lines. Calcif. Tissue Int. 54:284--289; 1994. Masuo, Y.; Suzuki, N.; M,stsumoto, H.; et al. Regional distribution of pituitary adenylate cyclase activating polypeptide (PACAP) in the rat central nervous syz.tem as determined by sandwich-enzyme immunoassay. Brain Res. 60257-63; 1993. Miyata, A.; Arimura, A.; Dahl, R. R.; et al. Isolation of a novel 38 residue-hypothalamic polypsptide which stimulates adenylate cyclase in pituitary cells. B&hem. Biophys. Res. Commun. 164:567-574; 1989. Miyata, A.; Jiang, L.; Dahl, R. R.; et al. Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate-cyclask activating polypeptide with 38 residues (PACAP). Biochem. Bioohvs. Res. Commun. 170643-648: 1990. Mount, S. M: A catalogue of splice junction sequences. Nucleic Acids Res. 10:459-472; 1982. Mungan, Z.; Ertan, A.; Hammer, R. A.; Arimura, A. Effect of pituitary adenylate cyclase activating polypeptide on rat pancreatic exocrine secretion. Peptidcs 12:559-562; 1991. Ogi, K.; Kimura, C.; Onda, H.; Arimura, A.; Fujino, M. Molecular cloning and characterization of cDNA for the precursor of rat pitu-
1299
itary adenylate cyclase activating polypeptide (PACAP). Biochem. Biophys. Res. Commun. 173:1271- 1279; 1990. 22. Ohkubo, S.; Kimura, C.; Ogi, K.; et al. Primary structure and characterization of the precursor to human pituitary adenylate cyclase activating polypeptide. DNA Cell Biol. 11:21-30; 1992. __ 23. Ohkubo, S.; Ogi, K.; Kimura. C.; Okazaki, K.; Onda, H.; Fujino, M. Expression of the PACAP gene in a human neuroblastoma cell line: cDNA cloning and analyses of the upstream regulatory region. Endoer. J. 2:135-145; 1994. 24. Ohtaki, T.; Masuda, Y.; Ishibashi, Y.; Kitada, C.; Arimura, A.; Fujino, M. Purification and characterization of the receptor for pituitary adenylate cyclase-activating polypeptide. J. Biol. Chem. 268:26650-26657; 1993. 25. Okazaki, K.; Kimura, C.; Kosaka, T.; et al. Expression of human pituitary adenylate cyclase activating polypeptide (PACAP) cDNAs in CHO cells and characterization of the products. FEBS Lett. 298:49-56; 1992. 26. Propato-Mussafiri. R.; Kanse. S. M.; Ghatei, M. A.; Bloom, S. R. Pituitary adenylate cyclase-activating polypeptide release 7B2, adrenocorticotrophin, growth hormone and prolactin from the mouse and rat clonal pituitary cell lines AtT-20 and GH3. J. Endocrinol. 132:107-113; 1992. 27. Sanger, F.; Nicklen, S.; Coulson, A. R. DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467; 1977. 28. Shivers, B. D.; Gores, T. J.; Gottschall, P. E.; Arimura, A. Two high affinity binding sites for pituitary adenylate cyclase-activating polypeptide have different tissue distributions. Endocrinology 128:3055-3065; 1991. 29. Southern. E. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503517; 1975. 30. Spengler, D.; Waeber, C.; Pantaloni, C.; et al. Differential signal transduction by five splice variants of the PACAP receptor. Nature 365:170-175; 1993. 3 1. Sundler. F.; Ekblad, E.; Absood. A.; H%anson, R.; Koves, K.; Arimura, A. Pituitary adenylate cyclase activating peptide: a novel vasoactive intestinal peptide-like neuropeptide in the gut. Neuroscience 46:439-454; 1992. 32. Tabarin, A.; Chen, D.; H&anson. R.; Sundler, F. Pituitary adenylate cyclase-activating peptide in the adrenal gland of mammals: Distribution, characterization and responses to drugs. Neuroendocrinology 59:113-119; 1994. 33. Tatsuno, I.; Somogyvar-Vigh. A.; Mizuno, K.; Gottschall, P. E.; Hidaka, H.; Arimura, A. Neuropeptide regulation of interleukin-6 production from the pituitary: Stimulation by pituitary adenylate cyclase activating polypeptide and calcitonin gene-related peptide. Endocrinology 129: 1797- 1804; 199 1. 34. Tatsuno, I.-; Gottschall, P. E.; Arimura, A. Inhibition of mitogenstimulated proliferation of murine splenocytes by a novel neuropeptide, pituitary adenylate cyclase activating polypeptide: A comparative study with vasoactive intestinal peptide. Endocrinology 128:728-734; 1991. 35. Thomas, P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77:5201-5204; 1980. 36. Vieira, J.; Messing, J. Production of single-stranded plasmid DNA. Methods Enzymol. 153:3-l 1; 1987. 37. Warren, J. B.; Donnelly, L. E.; Cullen. S.; et al. Pituitary adenylate cyclase-activating polypeptide: A novel, long-lasting, endothehum-independent vasorelaxant. Eur. J. Pharmacol. 197: 13 1- 134; 1991. 38. Watanabe, T.; Masuo, Y.; Matsumoto, H.; et al. Pituitary adenylate cyclase activating polypeptide provokes cultured rat chromaffin cells to secrete adrenaline. Biochem. Biophys. Res. Commun. 182:403411; 1992.
Pergamon Ol%-9781( 95)02018-R
Characterization KIMITAKE
OKAZAKI,
Discovery
Research
YASUAKI
ITOH,
of Murine PACAP mRNA1 KAZUHIRO
OGI,
SHOICHI
OHKUBO
Laboratories Z, Discovery Research Division, Takeda Chemical 10 Wadai, Tsukuba, Zbaraki 300-42, Japan Received
AND
HARUO
Industries,
0NDA2
Ltd.,
10 April 1995
OKAZAKI, K., Y. ITOH, K. OGI, S. OHKUBO AND H. ONDA. Characrerizarion ofmurine PACAP mRNA. PEPTIDES 16(7) 1295- 1299, 1995.--.4 murine PACAP precursor cDNA was isolated by screening a brain cDNA library. The amino acid sequence of the precursor was highly similar (from 8 1% to 93% similarity) to its rat, human, and ovine counterparts. The primary structure of murine PACAP is identical with those from sheep, humans, and rats, indicating that the mature PACAP is well conserved among mammals. Northern blot analysis revealed that the approximately 2.4 kb transcript for the PACAP precursor is expressed
in murine brain. The verification that murine PACAP is identical to its human counterpart provides a rationale for physiological and pathophysiological studies of PACAP in mice. PACAP
Mouse
Precursor
cDNA
mRNA
Peptides
PITUITARY adenylate cyclase activating polypeptide (PACAP) is a newly recognized polypeptide belonging to the secretin/glucagon/vasoactive intestinal peptide (VIP) family. Two amidated forms, a 38-amino acid peptide (PACAP-38) and a truncated form corresponding to its N-terminal 27 amino acid residues (PACAP-27), were originally isolated from ovine hypothalami by Miyata et al. ( 17,18 ) . They showed that the adenylate cyclasestimulating potency of both PACAPand PACAPwere 1000 times greater than that of VIP in cultured rat pituitary cells ( 17,18). Subsequent studies showed that PACAP provokes the secretion of pituitary hormclnes from pituitary cells (9,17 ) , amylase (20), and insulin ( 13 ) from the pancreas, and adrenaline from adrenal chromaffin cells (38). PACAP also evokes endothelium-independent vasodi lation (37) and interleukin-6 synthesis in the pituitary (33). The distribution of PACAP was wide spread in rat brain and testes ( 1,16). Immunohistochemical and ligand binding studies have revealed that PACAP has a neuronal localization in both the central and peripheral nervous systems, suggesting that PACAP acts as a neurotransmitter and/or neuromodulator ( 2). PACAPand PACAPare synthesized from a common precursor, and their amino acid sequences are identical in humans ( 14), sheep ( 14)) and rats I(21) . The human PACAP gene was also sequenced and assigned to a locus on chromosome 18~11 (22). High-affinity PACAP receptors have been identified in several tissues and cell lines (28). Recently, cDNAs encoding PACAP specific receptors (type I, which binds PACAP with a much higher affinity than VIP) were isolated from a rat brain cDNA library ( 11.30). The type I PACAP receptor was also purified in a fully active form from bovine brain membranes (24). Up to the present, wide-ranging biological and physiological studies of PACAP were performed using mice (3,4,7,10), mu-
Brain
Prohormone
FIG. I. Restriction enzyme map and sequencing strategy for the murine PACAP cDNA. Lines and the whole box indicate the noncoding and coding regions, respectively. The mature PACAP and PRP regions are indicated with filled and dotted boxes, respectively. The arrows indicate the direction and extent of the sequenced regions.
tine tissues (31,32), and murine cell lines (6,12,15,26,28,34) because well-established information on the biology and genetics of mice exists. In contrast, little is known about the molecular organization of murine PACAP. We report here the molecular properties of PACAP for this laboratory animal. METHOD Puri$cation
of Murine Brain Poly(A)+
RNA
Total RNA was extracted from the brains of four adult 8week-old ICR mice as described previously (22). Poly (A)’ RNA was purified from the total RNA with an oligo( dT)-cellulose spun column (Pharmacia LKB, Uppsala, Sweden). Construction
of a Murine Brain cDNA Library
Murine brain cDNA was synthesized from the poly(A)+ RNA with AMV RNA reverse transcriptase (Seikagaku Kogyo,
’ Sequence data from this article have been deposited with the DDBJ, EMBL, and GenBank ’ Requests for reprints should be addressed to Dr. Haruo Onda.
1295
Data Libraries
under the accession
number D14716.
1296
OKAZAKI
1
GAGAGTTTCTTCGGGTTAGAAGGTAGAAAGCCAGGAGCGATTTCACTCACTGCATACCCTCTTTCCTCTCCTGCGCAGA
79
80 1
ATG ACC ATG TGT AGC CGA GCA AGG CTG CCC CTG CTG GTG TAT CCC ATA ATA ATC CAT AGC Met Thr Met Cys Ser GIY Ala Arg Leu Ala Leu Leu Val TYT GIY Ile Ile Met His Ser
139 20
140 21
AGT GTC ICC TGT + TCA CCT CCC CCC CGA CTC AGC TTC CCT GGG ATC AGA CCA GAA GAC GAG Ser Val Ser Cys Ser Pro Ala Ala GIY Leu Scr Phe Pro Gly lie Arg Pro Glu Asp Glu
199 40
200 41
GCT TAC GAC CAG GAC CGA AAC CCC CTG CAA GAC TTC TAT GAC TGG GAC CCT CCC GGC GTG Ala Tyr Asp Gln Asp CIY Asn Pro Leu Gln ASP Phe Tyr Asp Trp Asp Pro Pi-o Gly Val
259 60
260 61
GGG AGC CCC GCC TCC CCC CTG CGT GAC GCT TAC GCC CTT TAC TAT CCA GCG GAC AGG ACA GIY Ser Pro Ala Ser Ala Leu Arg ASP Ala TYT Ala Leu TYT TYT Pro Ala Asp Arg Arg
319 80
320 81
CAT GTC CCC CAC GAA ATC CTT AAC GAA CCC TAT CGA AAA GTC TTG GAC CAG CTG TCC CCC ASD Val Ala His Glu Ile Leu Asn Glu Ala Tyr Arg LYS Val Leu ASP Gln Leu Ser Ala
379 100
380 101
AGG AAG TAC CTG CAG TCG GTC GTG GCC AGG CGC GCG GGG GAC GAA CCT AGG CCG CAC CCC Arg Lys Tyr Leu Gin Ser Val Val Ala Arg GIY Ala Gly Asp Glu Pro Arg Arg His Ala
439 120
440 121
GTG GAC GAC CCC CCC CCC CTT ACC AAA CCC CAC TCG GAC GGC ATC TTC ACA CAT ACC TAC VaI Asp Asp Pro Ala Pro Leu Thr LYS A.-g His Ser Asp CIY Ile Phe Thr Asp Ser Tyr
499 140
500 141
AGC CCC TAC CGA AAA CAA ATG GCT GTC AAG AAA TAC TTG CCC GCC GTG CTA GGG AAA AGG Ser Arx Tyr Arg LYS Gin Met Ala Val LYS LYS TYT Leu Ala Ala Val Leu Gly Lys Arg
559 160
560 161
TAT AAA CAG AGG GTT AAA AA~ AAA GGA ccc CGA ATA GCA TAC TTG TAG CAGATCAGCTGCCGG Tyr LYS Gin Arg Val LYS Asn LYS GIY Arg Arg lie Ala Tyr Leu c*c
622 175
623
CTACCTTGTGTATAAAATGAAAAGTCGTTTTCCAAATTGACTGACCAGTCATCACTCGTGTTCTTTCCAAACATGTATT
701
702
TATGTATCAAGTAAAGCCATTAAATGACTATTTTGATAATAATATTGTTTTTCTTTTTATGAAGCACTAGAGAATGCAC
780
781
AGATATACTTTGTGGACCAATTATTGATATATATTATAAGTATATATATTAAGAATATATATAAGTATAACAGAGAGCA
859
860
ATTAAGATGGGTGCACAAGGATTGAAAATTCGCCTGAGCTGCTTTATGTTTTTATATAAAGTAAATAGAGAAAATAGAC
938
939
AACCATTGTTTTGAATATTACTCCTATTTTTGTAAACTGGAATTAAAGGATAGTATTTTTATCCACAACCGTCTTGAAG
1017
1018
ATACCAATAATGGCCATTTGTACAAAAAACAATGATGCCCTGCTCCAGGGGAATTCTGAGGTAATGACTTGGGGAATTG
1096
1097
CTCAAGGGCTTTCTTTCCCTCTGAGTCTGGGGCAGGCTGCTTGAACCCCAGCCTAACTAACTCAAGTGGGCATTGTCCC
1175
1176
ACTGGTTGCAGGGGCAATTCCAACAATTTCAGTTTCTTTGATTATGTGTATTTGTCTCTCCTCAGACTCTCAGCCCAGA
1254
1255
AGGAAATTCTAATTAAACAACAGCTCTATCCAAATTGTGCTTCTCCCAGAACTCATGTCATTCCCTGATAGAAGAGTTG
1333
1334
AGGAACTGTACAGAAGAGACAGGCTTGGAGAGAGAGCTCTCTTTTCTGTACTTCCTGATTCTCCAGGGAACAGACTATT
1412
1413
CTAAGGCTAGGGCAATTGGAACAAAGTGAAAGATATATAAGGGATTGGTAAAGGCA~AACATGGGGATTTGAGATTTGA
1491
1492
GAGTTGCCTCAGGTCTGAGAATCTGGGGGCAAGTCTAGCTCCTCTGTAGGTTCCACTGCCTGACAGATCAGGTGCTGGT
1570
1571
GTTGGAATGAATGCAAAGTACAATGTGTTTTTCTCCAGTGCTGTTCATGCTTTTCATGTTGTGAAATGGCCAGGATCCT
1649
1650
CCCTTTGAACACTGTTCTGCAGAAGCCAGCTCTGTTCTTTGTGGATTTTCTGGAGACCCTCCTTCCTACCCTTGCCCTC
1728
1729
ATGCATTGTTTTAGAGTCATTTGCCATTTTCCACTCACTTATCTTAAATTTGTGAATGCTAGTTATTTTTTGTTGTTGT
1807
1808
TTGATGCAAGCAGTTACTGTGAAGTTTAAGAACCCCTGTGTAGCTTCCACAGAGAAATTATGCACTAAATATGAACCTT
1886
1887
TTGTTTCTTGTTTATTGACTTTGTAGGTAAAAATGTATTTTTCTATATTATGGCTTATTGCTT~G~~~~ATTTATTTCA
1965
1966
TAAAACCAATCTTTGTCATATTAGAATGTGTAGTGTTCCAATGCTGCTCAGTTTGACTGATAAATCATTTAAACCCCAT A
2045
ET AL.
2044 2123
2124
2202
2203
2249
FIG. 2. Nucleotide sequence of the cDNA and the predicted amino acid sequence of the murine PACAP precursor protein. The probable cleavage site of the signal peptide is shown by an arrow. The box and the solid underline indicate the mature PACAP and PRP, respectively. The polyadenylation signal sequences are indicated by broken underlines. An arrowhead indicates the polyadenylation position of clones No. 12 and No. 14.
Japan), E. coli RNase H, E. coli DNA polymerase I, and T4 DNA polymerase (Amersham International plc., Buckinghamshire, UK). A murine brain cDNA library was then constructed in Agtll with the cDNA Cloning System Adaptor Method ( Amersham International plc.). Tokyo,
Cloning of the cDNA Encoding for the Precursor of Murine PACAP An Act I-Eco RI 1.17 kb fragment of rat PACAP precursor cDNA (21) was labeled with [a-32P]dCTP (110 TBq/mmol;
Amersham International plc.) using Prime-ItTM Random Primer Kit (Stratagene, La Jolla, CA) and used to screen the murine brain cDNA library. Approximately 3 X IO6 plaques on nitrocellulose filters (Schleicher & Schuell, Dassel, Germany) were screened by plaque hybridization with the DNA probe at 60°C in a hybridization buffer (7% SDS, 0.5% BSA, 1 mM EDTA, and 0.5 M sodium phosphate buffer, pH 7.2) for 16h (8). The filters were washed in 0.3 M sodium chloride and 0.03 M sodium citrate (2 X SSC) at 65°C then dried and autoradiographed using Kodak X-OMATTM AR films (Eastman Kodak Company, Rochester, NY) with an intensifying screen at
MURINE
PACAP
mRNA
Mouse Rat
1297
CHARACTERIZATION
PACAP
PACAP
1 MTNCSGARLA
LLVYGIIMHS
SVSCSPAA-G
1 ****tt****
***"****N
**tt****_-* ***'*****R
EAYDQDGNPL
QDFYDWDPPG
59
*********
et********
*t**t*****
**********
*+YS****A*
.jg
lR*******R
+++GH*****
P**GGSR***
***t******
*****tL***
SO
**yc****s*
*R*******N
***q****o
*****SE***
ijo
60 VGSPASALRD
RYALYYPADR
I_---_? DVAHEILNE
SO A*********
**********
*t********
AYRKVLDQLS ARKYLQSW ******,c*** *******Me*
61 A****f*P*A
*A*w****G*
61 ****t*+***
**‘*.r*f*~~
Human
PACAP
1
Ovine
PACAP
1
LSFPGIRPED
l
PRP
****G****
*(+***G**DK
RGAGDEPRRH **MkHNL-
****eke***
tG*H***L**
**V*GSLGGG
**********
**R*‘*TLM*(
K*L*GT*GGG
if%
120
********* ********** ********** ******** ****t** 1 1 PACAP
HSDGIFTDS
120 AVDDPAPLTK 120 ***+R***"*
YSRYRKQMAV
KKYLAAVLGK
RYKQRVKN
G RRIAYL
175
175
**********
121 *G*'AE**S*
l ********
121 *D**SE**S*
*et*******
**********
*t********
*t********
**********
********
*
******
176
***p**
176
FIG. 3. Comparison of the deduced amino acid sequences of the murine, rat, human, and ovine PACAP precursor proteins. The numbers indicate the position of the amino acid from the N-terminus of the precursor protein. Asterisks in the sequence of the rat. human, and ovine peptides indicate the same amino acid residues as in the murine PACAP precursor protein. The gaps shown by a dash in the sequence of the murine and rat precursor proteins were inserted to maximize tbz alignment of the seouences. Boxes indicate the mature PACAP and PRP regions. An arrowhead shows the probable cleavage site of the signal peptide. 0
1
-80°C for 3 days. Kpn I 2.2 kb fragment from No. 93 clone, which contained the largest cDNA fragment in the hybridization-positive clones, was subcloned into pUCl18, named pMP93K1, and used for further analysis.
Sequencing PACAP
of cDNA Encoding the Precursor of Murine
Several plasmids containing 3 ‘- or 5 ‘-deleted region of the cDNA were constructed with exonuclease III and VII from the plasmid pMP93Kl and used to prepare single-stranded DNAs for DNA sequencing (36). The DNA sequence was determined by a dideoxynucleotide chain termination method (27) with the Klenow fragment (Takara Shuzo, Kyoto, Japan) or Taq DNA polymerase (Promega Corporation, Madison, WI). Southern Blot Analysis of Murine Genomic DNA Genomic DNA samples 1:10 pg) were digested with Eco RI, Bam HI, Bgl II, and Hin dIII, electrophoresed on a 0.8% agarose Rockland, gel ( SeakemTM GTG Agarose, FMC Bioproducts, ME), and transferred to a nylon membrane filter (Biodyne A, Pall Biosupport, Glen Cove, NY) by the method of Southern (29). The Pst I-Eco RI 0.56 kb fragment of murine PACAP cDNA was labeled as described above and used as a probe. Hybridization was carried out at 65°C in a hybridization buffer (7% SDS, 5% dextran sulfate sodium salt, and 0.5 M sodium phosphate buffer, pH 7.2) (5). The filter was washed at 65°C in 0.03 M sodium chloride and 0.003 M sodium citrate (0.2 X SSC) containing 0.1% SDS and analyzed using Imaging PlateTM and Image Analyzer BAS-2000 (Fuji Film, Tokyo, Japan).
RESULTS
Phage clones with hybridization-positive signals were isolated from the murine brain cDNA library. The 2249 bp cDNA insert of clone No. 93, which was the largest cDNA insert obtained, was subcloned into plasmid pUC 118 and sequenced to deduce a coding region for the entire murine PACAP precursor (Figs. 1 and 2). The first initiation codon ( ATG) was located at position 80 in the sequence and was preceded by an in-frame upper termination codon at position 17. The sequence displayed an open reading frame of 525 bases encoding the murine PACAP precursor protein of 175 amino acid residues (Fig. 2). These numbers are identical to those of the rat PACAP precursor, whereas the human and ovine PACAP precursors have one extra amino acid residue at position 29 from the N-terminus of the precursor protein (Fig. 3). The deduced amino acid sequence of murine PACAP is exactly identical to the rat, human, and ovine sequences. The 3’-noncoding sequence of the No. 93 clone contained no poly( A) tail but ended with the putative polyadenylation signal ATTAAA. During the course of this study, two shorter cDNA clones were isolated, which had the poly (A) tail at position 1974. This case suggests that AGTAAA at position 1950- 1955 functioned as an alternative polyadenylation signal. Southern blot analysis of murine genomic DNA was performed with a Pst I-Eco RI fragment (565 bp) of the murine PACAP precursor cDNA as a probe. As in the cases of sheep, humans, and rats ( 14,21,22), a single gene for the PACAP precursor was present in the murine haploid genome (Fig. 4). Northern blot analysis showed that ca. 2.4-kb-long mRNA, which was slightly shorter than that of rat, was transcribed in the murine brain (Fig. 5 ) . DISCUSSION
Northern Blot Analysis of &brine Poly(A)+
RNA
Approximately 10 pg of poly( A) + RNA, purified from whole murine brain, was denatured with glyoxal and dimethyl sulfoxide at 50°C for 1 h. The denatured poly (A)+ RNA was electrophoresed on a 1.2% agarose gel and transferred to a nylon membrane filter ( 35 ) . Hybridization, filter washing, and autoradiography were performed the same as for Southern blot analysis.
Although numerous studies have demonstrated that PACAP is active in both in vivo and in vitro murine systems, neither the amino acid sequence of the peptide nor the nucleotide sequence of its mRNA has been determined. In this study, we report the cloning of a cDNA from mouse brain that encodes the PACAP precursor protein. The cloned cDNA indicated that the murine PACAP precursor consists of 175 amino acid residues. The mu-
1298
OKAZAKI
rine precursor shared 93% amino acid sequence identity with that of the rats, 83% with sheep, and 81% with humans. The nucleotide sequences in the coding region and 3’-noncoding region were very similar among these animals. In contrast, the 5 ‘-noncoding region of the No. 93 clone had no similarity with its rat and ovine counterparts, but resembled the 5’-flanking sequence of the human PACAP gene (23). The most likely explanation for this sequence identity is that the clone No. 93 may be derived from an alternative or insufficiently spliced mRNA because the 5’-noncoding sequence (57-78) is (T/C),,-CAG, which agrees with the consensus sequence of the acceptor site for splicing (19). The 3 ‘-noncoding sequence of the No. 93 clone ended with a putative polyadenylation signal A’ITAAA at positions 22442249. The nucleotide sequences of the other two clones showed that AGTAAA at position 1950- 1955 functioned as an altemative polyadenylation signal. Similar heterologous polyadenylation sites were observed in the PACAP cDNA from a human neuroblastoma cell line IMR-32 (23). This suggests that alternative polyadenylation may be a common feature of the PACAP precursor mRNA. Murine PACAP is identical to the rat, human, and ovine peptides, suggesting that its amino acid sequence is highly conserved among placental mammals. The dibasic amino acid residues
A
ET AL.
B
_L-
:
. 3.3
- 4.4
- 2.4 -
1.4
- 0.24 FIG. 5. Northern blot analysis of whole brain poly(A)’ RNA. Poly(A)’ RNA (IO pg) was used in each lane. Numbers on the right indicate the mobility of the size markers that are specified in kb. Lane A = mouse and lane B = rat.
W
(Lys ‘2’_Arg 17”, Arg “‘) -Arg “’ ) that are considered important in the posttranscriptional processing of mature PACAP were also conserved in the mouse. In the murine PACAP precursor, as in the previously sequenced PACAP precursors, the 29-amino acid peptide provisionally called PACAP-related peptide (PRP) was flanked by basic amino acids (Fig. 3 ) . In our previous experiments, immunoreactive PRP was detected in the culture medium of recombinant CHO cells (25 ) Therefore, PRP or an extended peptide PRP could be another bioactive peptide cleaved from the PACAP precursor as we have already discussed ( 14,21,22). The availability of cDNA and molecular information for murine PACAP can open avenues for further analysis of its role in mice-one of the most valuable laboratory mammals for physiological and pathophysiological studies.
-10.0
-4.8 73.8 3.7
ACKNOWLEDGEMENTS
FIG. 4. Southern blot analysis of murine genomic DNA. Murine genomic DNA was digested by the indicated restriction enzymes and probed with a 0.56 kb Pst I-Eco RI fragment derived from clone No. 93. Numbers
We are especially grateful to Dr. M. Fujino (Takeda Chemical Industries, Ltd.) for his encouragement throughout this work. The authors
on the right indicate in kb.
would like to thank Dr. A. Arimura suggestions.
the molecular
weights
of the restriction
fragments
(Tulane
University)
for valuable
REFERENCES
A.; Miyata, A.; Mizuno, K.; Coy, D. H.; Kitada, C. Tissue distribution of PACAP as determined by
2. Arimura, A. Pituitary adenylate cyclase activating polypeptide (PACAP): Discovery and current status of research. Regul. Pept.
RIA: Highly abundant in the rat brain and testes. Endocrinology 129:2787-2789; 1991.
37:283-303; 1992. 3. Banks, W. A.; Kastin, A. J.; Komaki,
1. Arimura, A.; Somogyvti-Vigh,
G.; Arimura,
A. Passage
of
MURINE
4.
5.
6.
I.
8. 9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19. 20.
21.
PACAP
mRNA CHARACTERIZATION
pituitary adenylate cyclase activating polypcptide 1-27 and pituitary adenylate cyclase activating polypeptide l-38 across the bloodbrain barrier. J. Pharmacol. Exp. Ther. 267:690-696; 1993. Banks, W. A.; Kastin, A. J ; Komaki, G.; Arimura, A. Pituitary adenylate cyclase activating polypeptide (PACAP) can cross the vascular component of the blood-testis barrier in the mouse. J. Androl. 14:170-173; 1993. Blumberg, B.; Wright, C. V. E.; De Robertis, E. M.; Cho, K. W. Y. Organizer-specific homeobox genes in Xenopus luevis embryos. Science 253:194-196; 1991. Braas, K. M.; Brandenburg, C. A.; May, V. Pituitary adenylate cyclase-activating polypepticle regulation of AtT-20/D16v corticotrope cell proopiomelanocortin expression and secretion. Endocrinology 134:1X6- 195; 1994. Chen, W.; Inui, T.; Hachiya, T.; Ochi, Y.; Nakajima, Y.; Kajita, Y. Stimulatory action of pituitary adenylate cyclase-activating polypeptide (PACAP) on thyroid gland. Biochem. Biophys. Res. Commun. 194:923-929; 1993. Church, G. M.; Gilbert, W Genomic sequencing. Proc. Natl. Acad. Sci. USA 81:1991-1995; 1984. Culler, M. D.; Paschall, C. S. Pituitary adenylate cyclase-activating polypeptide (PACAP) potcntiates the gonadotropin-releasing activity of luteinizing hormone-releasing hormone. Endocrinology 129:2260-2262; 1991. Fridolf, T.; Sundler, F.; Ahren, B. Pituitary adenylate cyclase-activating polypeptide (PACAP): Occurrence in rodent pancreas and effects on insulin and glucagon secretion in the mouse. Cell Tissue Res. 269~275-219; 1992. Hosoya, M.; Onda, H.; Ogi, K.; et al. Molecular cloning and functional expression of rat cDNAs encoding the receptor for pituitary adenylate cyclase activating polypeptide (PACAP). Biocbem. Biophys. Res. Commun. 194:133- 143; 1993. Inagaki, N.; Yoshida, H.; Mizuta, M.; et al. Cloning and functional characterization of a thind pituitary adenylate cyclase-activating polypeptide receptor subtype expressed in insulin-secreting cells. Proc. Natl. Acad. Sci. USA 91:2679-2683; 1994. Kawai, K.; Obse, C.; Watanabe, Y.; Suzuki, S.; Yamashita, K.; Ohashi, S. Pituitary adenylate cyclase activating polypeptide stimulates insulin release from the isolated perfused rat pancreas. Life Sci. 50:257-261; 1992. Kimura, C.; Ohkubo, S.; Ggi, K.; et al. A novel peptide which stimulates adenylate cyclase: Molecular cloning and characterization of the ovine and human cDNAs. Biochem. Biophys. Res. Commun. 166:81-89; 1990. Lemer, U. H.; Lundberg, I’.; Ransjo, M.; Persson, P.; Hakanson, R. Helodermin, helospectin, and PACAP stimulate cyclic AMP formation in intact bone. isolated osteoblasts, and osteoblastic cell lines. Calcif. Tissue Int. 54:284--289; 1994. Masuo, Y.; Suzuki, N.; M,stsumoto, H.; et al. Regional distribution of pituitary adenylate cyclase activating polypeptide (PACAP) in the rat central nervous syz.tem as determined by sandwich-enzyme immunoassay. Brain Res. 60257-63; 1993. Miyata, A.; Arimura, A.; Dahl, R. R.; et al. Isolation of a novel 38 residue-hypothalamic polypsptide which stimulates adenylate cyclase in pituitary cells. B&hem. Biophys. Res. Commun. 164:567-574; 1989. Miyata, A.; Jiang, L.; Dahl, R. R.; et al. Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate-cyclask activating polypeptide with 38 residues (PACAP). Biochem. Bioohvs. Res. Commun. 170643-648: 1990. Mount, S. M: A catalogue of splice junction sequences. Nucleic Acids Res. 10:459-472; 1982. Mungan, Z.; Ertan, A.; Hammer, R. A.; Arimura, A. Effect of pituitary adenylate cyclase activating polypeptide on rat pancreatic exocrine secretion. Peptidcs 12:559-562; 1991. Ogi, K.; Kimura, C.; Onda, H.; Arimura, A.; Fujino, M. Molecular cloning and characterization of cDNA for the precursor of rat pitu-
1299
itary adenylate cyclase activating polypeptide (PACAP). Biochem. Biophys. Res. Commun. 173:1271- 1279; 1990. 22. Ohkubo, S.; Kimura, C.; Ogi, K.; et al. Primary structure and characterization of the precursor to human pituitary adenylate cyclase activating polypeptide. DNA Cell Biol. 11:21-30; 1992. __ 23. Ohkubo, S.; Ogi, K.; Kimura. C.; Okazaki, K.; Onda, H.; Fujino, M. Expression of the PACAP gene in a human neuroblastoma cell line: cDNA cloning and analyses of the upstream regulatory region. Endoer. J. 2:135-145; 1994. 24. Ohtaki, T.; Masuda, Y.; Ishibashi, Y.; Kitada, C.; Arimura, A.; Fujino, M. Purification and characterization of the receptor for pituitary adenylate cyclase-activating polypeptide. J. Biol. Chem. 268:26650-26657; 1993. 25. Okazaki, K.; Kimura, C.; Kosaka, T.; et al. Expression of human pituitary adenylate cyclase activating polypeptide (PACAP) cDNAs in CHO cells and characterization of the products. FEBS Lett. 298:49-56; 1992. 26. Propato-Mussafiri. R.; Kanse. S. M.; Ghatei, M. A.; Bloom, S. R. Pituitary adenylate cyclase-activating polypeptide release 7B2, adrenocorticotrophin, growth hormone and prolactin from the mouse and rat clonal pituitary cell lines AtT-20 and GH3. J. Endocrinol. 132:107-113; 1992. 27. Sanger, F.; Nicklen, S.; Coulson, A. R. DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467; 1977. 28. Shivers, B. D.; Gores, T. J.; Gottschall, P. E.; Arimura, A. Two high affinity binding sites for pituitary adenylate cyclase-activating polypeptide have different tissue distributions. Endocrinology 128:3055-3065; 1991. 29. Southern. E. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503517; 1975. 30. Spengler, D.; Waeber, C.; Pantaloni, C.; et al. Differential signal transduction by five splice variants of the PACAP receptor. Nature 365:170-175; 1993. 3 1. Sundler. F.; Ekblad, E.; Absood. A.; H%anson, R.; Koves, K.; Arimura, A. Pituitary adenylate cyclase activating peptide: a novel vasoactive intestinal peptide-like neuropeptide in the gut. Neuroscience 46:439-454; 1992. 32. Tabarin, A.; Chen, D.; H&anson. R.; Sundler, F. Pituitary adenylate cyclase-activating peptide in the adrenal gland of mammals: Distribution, characterization and responses to drugs. Neuroendocrinology 59:113-119; 1994. 33. Tatsuno, I.; Somogyvar-Vigh. A.; Mizuno, K.; Gottschall, P. E.; Hidaka, H.; Arimura, A. Neuropeptide regulation of interleukin-6 production from the pituitary: Stimulation by pituitary adenylate cyclase activating polypeptide and calcitonin gene-related peptide. Endocrinology 129: 1797- 1804; 199 1. 34. Tatsuno, I.-; Gottschall, P. E.; Arimura, A. Inhibition of mitogenstimulated proliferation of murine splenocytes by a novel neuropeptide, pituitary adenylate cyclase activating polypeptide: A comparative study with vasoactive intestinal peptide. Endocrinology 128:728-734; 1991. 35. Thomas, P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77:5201-5204; 1980. 36. Vieira, J.; Messing, J. Production of single-stranded plasmid DNA. Methods Enzymol. 153:3-l 1; 1987. 37. Warren, J. B.; Donnelly, L. E.; Cullen. S.; et al. Pituitary adenylate cyclase-activating polypeptide: A novel, long-lasting, endothehum-independent vasorelaxant. Eur. J. Pharmacol. 197: 13 1- 134; 1991. 38. Watanabe, T.; Masuo, Y.; Matsumoto, H.; et al. Pituitary adenylate cyclase activating polypeptide provokes cultured rat chromaffin cells to secrete adrenaline. Biochem. Biophys. Res. Commun. 182:403411; 1992.