Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide

Neuron,

Vol. 8, 811-819,

April,

1992, Copyright

0 1992 by Cell Press

Functional Expression and Tissue Distribution of a Novel Receptor for Vasoactive Intestinal Polyp&tide Takeshi Ishihara,*+ Ryuichi Shigemoto,* Kensaku Mori,* Kenji Takahashi,+ and Shigekazu Nagata* *Osaka Bioscience institute 6-2-4 Furuedai, Suita Osaka 565 Japan +Department of Biophysics and Biochemistry Faculty of Science University of Tokyo 7-3-l Hongo, Bunkyo-ku Tokyo 113 Japan *Department of Morphological Brain Science Kyoto University Faculty of Medicine Kyoto 606 Japan

Summary Vasoactive intestinal polypeptide (VIP), a 28 amino acid peptide hormone, plays many physiological roles in the peripheral and central nerve systems. A functional cDNA clone of the VIP receptor was isolated from a rat lung cDNA library by cross-hybridization with the secretin receptor cDNA. VIP bound the cloned VIP receptor expressed in mouse COP cells and stimulated adenylate cyclase through the cloned receptor. The rat VIP receptor consists of 459 amino acids with a calculated M, of 52,054 and contains seven transmembrane segments. It is structurally related to the secretin, calcitonin, and parathyroid hormone receptors, suggesting that they constitute a new subfamily of the G, proteincoupled receptors. VIP receptor mRNA was detected in various rat tissues including liver, lung, intestines, and brain. In situ hybridization revealed that VIP receptor mRNA is widely distributed in neuronal cells of the adult rat brain, with a relatively highexpression in thecerebral cortex and hippocampus. Introduction Vasoactive intestinal polypeptide (VIP) is a 28 amino acid polypeptide hormone that belongs to the family of brain-gut peptide hormones (Said, 1986). The amino acid sequence of VIP is related to those of secretin and glucagon, indicating that it is a member of the secretinlglucagon family, which includes peptide histidine isoleucine (PHI) (Tatemoto and Mutt, 1981) or peptide histidine methionine (PHM) (Itoh et al., 1983), pituitary adenylate cyclase activating polypeptide (PACAP) (Miyata et al., 1989), and helodermin (Vandermeers et al., 1984). VIP and VIP-immunoreactive substances are found in various tissues, such as lung, liver, and brain (Cozes and Brenneman, 1989), and seem to work as a neurotransmitter or neuromod-

ulator in the central and peripheral nervous systems (Hokfelt et al., 1980). VIP induces relaxation of smooth muscle (Piper et al., 1970) and stimulates secretion of electrolytes from the gastrointestinal tract (Barbezat and Grossman, 1971). VIP also stimulates glycogenolysis in the cerebral cortex (Magistretti et al., 1981) and regulates cell death and differentiation of the retinal ganglion cells and sympathetic neuroblasts (Kaiser and Lipton, 1990; Pincus et al., 1990). In autonomic ganglia,VIP is found in neuronscontainingacetylcholine (Hokfelt et al., 1980), and the signal induced by acetylcholine seems to be modulated by VIP (Lundberg et al., 1982; Kawatani et al., 1985). These diverse effects of VIP are mediated by its interaction with the receptor for VIP (Rosellin, 1986). Since VIP stimulates the cellular accumulation of CAMP, it was postulated that the VIP receptor is coupled to the a subunit of the adenylate cyclase-stimulating G protein (C,) (Gozes and Brenneman, 1989). The VIP receptor seems to be structurally related to the receptor for other members of the secretin/glucagon family because the binding of VIP to the receptor is competed for by secretin, PACAP, and PHI (Robberecht et al., 1990; Couvineau et al., 1990) and viceversa. Recently, two apparently different polypeptides (M, 53,000 and 18,000) that bind VIP were purified to homogeneity from porcine liver (Couvineau et al., 1990) and guinea pig lung (Brugger et al., 1991), respectively. The molecular basis for these two VIP-binding proteins (receptors) remains to be determined. Furthermore, the detailed mechanism of the signal transduction mediated by the VIP receptor is also unknown. Previously, we isolated the cDNA for rat secretin receptor (Ishihara et al., 1991) and showed that the secretin receptor contains seven transmembrane domains. Meanwhile, Sreedharan et al. (1991) have reported a human cDNA clone, GPRNI, which they believe encodes the VIP receptor. Although the polypeptide coded by GPRNI cDNA contains seven transmembrane domains, it has no significant homology with the rat secretin receptor. In this study, we have isolated a novel VIP receptor from a rat lung cDNA library. The receptor has 48% identity with the secretin receptor at the amino acid sequence level and mediates the VIP-triggered cellular accumulation of CAMP. Furthermore, in situ hybridization with antisense RNA indicates that theVIP receptor is expressed in neuronal cells throughout the brain regions. Results Isolation of Rat lung VIP Receptor cDNA The human colon adenocarcinoma WiDr cell line is a derivative of human HT29, which strongly expresses the VIP receptor (Laburthe et al., 1978). To isolate VIP receptor cDNA, a cDNA library (1.5 x IO6 clones) was constructed with mRNAfrom WiDrcellsand screened

Met nrg Pro Pro ser Pro l,TG CCC CCT CCG ,,GC CC?, AL: Se= P=o Gin llis Clu GCC I~GC 02~ CAG cnc GAG Thr GLy@ Se= Lys Met AC,, GGC TGC KC h&G r\TG Gin ,,eu p,,e Ala P=o Ile Cl,G Cl-G T','T GCC CCC ATT ,,la m Gly ,,eu As" Asp GCC TGC GGC Cl-6 A,,= GAC Tvr ync

Ser ~eu Se= Lou Ala nGc we *Cc cry GCC

CACCAGCCnChGACCCCGTTGCCGCTCCCGGCCGCTTGCCGCCGGCTCAGGGCGGACC -1 -20 Ala GLy Ala Leu Ala Cys Ala Le" Arg P=o Ala Gly Se= Cl" Ala Pro IliS va1 nrg Trp Le" cys va1 Leu CCC CAT G'I'C CGC TGG CTC TGC CTG CTG GCh GGA GCC CTT GCC TGC GCC CTC AGA CCC GCG GGC AGC CRG GCA 100 20 * -GLu Tyr Leu GLn Leu Ile Glu Ile Gln Arg GLn GLn @j Le" Glu GLu Ala GLn Leu GLu As,, Glu Thr GIG CGT ChG C&G 'ICC CT% GAG GAG GCC CAG CTG GAG AAT GAA KC TGT GAG Tt~c CTG CAG TX ATT GAG nTri 200 C" V" 40 Pro Thr Thr Pro Arg Gly Gin RLa Val VaL Le" Aspm Pro Leu Ile Phe Trp Asp A% Leu Thr mT=p ncc CCG AGG CCC CAG GCG GTA GTC mu GAC wc ccc crc wrc TTT TGG GAC MC CI'C ACC 'EC TGG CCG ACA ._ 300 SO His Gly Tyr A% Ile Se= Arg Se= DThr GLu Glu Gly Trp Se= Gln Leu GLu P=o GLy P=o Tyr His Ile C,,T GGT TAT AK RTC AGC CGT ,tGC TGC ACT GM GAG GGC TGG TCA CA,, CTG GAA CCA GGC CCC TAC CAC ATT 400 120 100 Arg Ala Se= Se= Le" Asp Glu Gln GLn Gin Thr Lys Phe Tyr Asn Thr Val Lys Thf &y TY= Thr Ile GL AGG GO. TCG AGT CTG G&T GAG C,'tG CAA GIG ICC AfiG TTC TAC hhT KC! GTG AAG ACC.GGC TAC ACC ATC GG$ 500 140 Se= Leu Leu Val Ala "et Ala Ile Leu Se= Leu Phe Arg Lys Leu His Cys Thr Arg Am TV= Ile His Met j,GC C~C L"I‘G c,“r GCC ATG GCT ATC '11G AGC GIG * AGG AAG CTG CAC IS= Kc CGA 'hhc 'rAC AX c*c AT6

180 160 llis Leu Phe Met Se= Phe ILe Leu A=q Ala The ALa Val Phe 11~ Lys Asp Met Ala Leu Phe Asn Se= Gly GLu Ile Asp llis Cys Se= CA'T C+C TTC AT 'ICC TTC ATC CIY; A CC CCC ACT GCC GTC 7-l-C A'TC AAG GAC ATG CCC CTC TTC AAC AGC! GGG GAG ATA GAC CR,'.? 'KC 'ITT 200 GLu Ala Se= Val Gly Cys Lys GAG CCC TCG GTG GGG TGC MC 700

220 Le" Tyr Th= Leu Leu Ala Val Se= Pile Phe Se= Glu Arg Lys CTA TAC Kc CTG CTG GCC GTC KC 'I-K TTC TCC GAG CGG MC 800 $$

Thr Kc

ALd pro GCC

240 Tyi Phe T=p GLv Ty= ILe Le" Ile GlV T=D Gl,' ‘/al Pro Se= Val TAC '1°K TGG GGG TAC ATT CTC ATC GGC 'EC GGA GTG CCC ACT GTG $

Ile T=D Th= ATI, n;$ KG

"al "al. ,,=g ILe Tyr Phe Glu Asp Phe Gly Cys Trp Asp Th= GTC GTC AGG ATA TAT TTT GAG GhT 7-l-C GGG TGC TGG Gi,C KC 900

I,,e

se= IL=

Leu L="

LSU "~1 TG GT

200

nsn AA

260 Ile Ile A& Se= Se= ATC ATC AAC TCC 'ICC

eu Tm TTD Ile Ile LYS TG TGG TGG ATC ATA AAb 300

phe "al ~,eu Phe Ile Cvs Ile 11% Arg Ile Leu Val Gin LYS I&u A=g P=o P=o ASP ILe 1°K GTC CTG TTT hTC ‘TGC ATC AX CGG ATC CTG GTT CAG iG,h Cl-A CGG CL’, CCC GAC ATT 1000 320

Gly ,,ya hs,, Asp ser ser Pro Tyr Se= Arg La" Ala Lys ,Se= Thr Le" Le" Le" Ile GGG ,,,iG AAT GAT ,632 AGC CCA TAT TCG AGG CTG CCC AhG -Cm 'KC ACG 7"“ Cl 'C AT 1100

PKO Le"

Phe Gly

ILe

1118 Tvr

Val

Met

Phe Ala

340

Asp Am Phe Lys Ala Cln Val Lys GAC AAC Tl'C AAG CCC CAG GTG MA

360

Met Val Phe Glu ATG GTC TTC GAA 1200 380

Glu

'?a1 Gln Ala Glu Leu At-g Arg Lys Trp Arg Arg Trp His Leu Gin Gly GAG Gn; CAG GCG GAG CT‘? CCC CGG AAG 'KG CGG CGT 'IGG CAT Cl% CAG 'XC

400 His Pro Trp Gly Gly Se= Am Gly Ala Thr Cys Ser Thr Gln Val Se= Met Leu Thr CAG CAT CCC TGG GGA GGC AGC AAC GGC CCC ACA TGC AGC ACG CAG GTA WC ATG CTC ICC

Gln

Leu Gly Trp Se= Ser Lye GTC CT'2 GGC TGG MC TCC MA 1.___ ?OO

Val

Arg Val Se= Pro Se= Ala Arg CGC GTC AGC CCG AGC GCA ‘XC

Se= TCC

420 Arg Se= Se= CGC TCC TCC

1400 429 Ser Phe Gln Ala Glu Val Se= Leu Val AGC TTC CAA GCG GAG GTC TCC CTG GTC TGA CCGCCAGAGGCCGCCAGGCCCCTTCACACC~CTCCACAACGGC~~GAGCAGAGGT~TAGCGACACCGT~G~ 1500 CCAGCTTTTCATAGGGACACA~C~T~CCGCAGCCAGGACCGGGT~A~~AGC~GAGAGCGGGGAGCT~AAC~~GAG~~~AGG~GA~CAGCA~AGAC~CACCTC 1600

CCAAGGCCATTCTGCCAGTCA~CGCACAAAATC~CATACAC~GAAAGCAACCGCT~CCCTCAAAG~T~GAA~CGCGGGGCAGAAG~~CGCCCGCC~~G~C~AGA 1700 ATCCACn;GGACGCTCGTGTG~~G~TAAGCGT~CCATTGAGA~C~CTCTGAGGA~CAGCCCCCTCCC~CGT~ATC~CCAG~~TC~GG~GGTGA~T~T~AC 1000 CTCAGGCAGATGGTTATGACCTGGAGT~CAG~TGGGGATCGCAGC~~T~~GTCCTCTCTCAGGATGGAA~GTCCCAGTGTATC~CAGAC~GGGAG~CAGGGAC~AC~

1900 ~GGGGAGGGAGAAGACAACCCCAGTCAGTGCTCACCAAGTGACTT~TCCAACAAATAGGACCAGTCCCACTAGCCTTATGTACAACCAAAGCAATATCGAGC~C~G~CTGGGCG 2000 2100 CTCATCTACACACGCCAACCACTGCTGCAAGGGTCAGAGGCATGAACACACAGGCCCCT~ATCCTGAGGTCACCCTAACATCAC~GTAGTGACAC~GGGT~CGG~AGAG~GGT 2200

ATC~TTCCCGTGGCTAGTCGAACGTAAAGA~AC~T~TACAT~~A~ATC~C~G~TTCTCT~GGGTTGTA~ACCACAGA~~AT~CCCCACAGTCCCCAGTTCT~CT 2300

GC~TGGAGCGTGATTATGTGCTGACATAAAAGCCCAGGGGTCAAGGCAT~TAAG~CTC~CCACCCTTAATTCAACACGG~T~AATCCAAGCA~TGTCAGCAGGCCCC~CA 2400

GAGCTGCACCAGGCTTGTACGAC~~AAACGT~GCTCTGACAGGCTTAGTTCCCC~GCCACTGTGGGTCTC~CTTCTCC~AGAAG~GTGTCACCTCTGCAGTGGTGAGAGGTG 2500

ACCCACTAAAGCTACCACCCTTCACTCCTCGGACAGGCAGGAGCCAC~ACTTCCATGGGAGGACTTATCCCAAGAGCCTT~ATTCACATTCGAG~TGAAGAGAT~~ACAGGGGT 2600

2700

TCCGTTGTGACCCTCTCTCCCCTAAACCCATCTGGAAGGAGACAGATGCTGCCTCCGACCGAATCAAAACAGCCAACGGAGC~TCACAAACAGCAAGGGTTCACGATCAT~CTGAAA 2800

ACACAGCAAAGAGGATAn;CAGCCACCACDTn;CTGCTGTCTACGGGCCAACCAAGAGGGTCGGGTCAG~GAGGTTTCCCAG~AAGTCAGTGAGGAGAGGCCATGTGG~AAGGGA 2900 AAGGAGTAGACACAGCAATGGGGCAAAGCAGGTGACATTGGTCAGAGAGAAAGAGAGAAAGAGAGAGAGAGAGCACTCCCAAGG~TTG~CAAACACA~TGT~AAA~TGTGGAGT 3000

TGTCPTGCTGATGAAGCCAGAAGTGTATGCn;GTGTGTGAAGAAGCCAGAATGGGCTAGCAGGGGGG 3100 3129 Figure

1. Nucleotide

Sequence

and Predicted

Amino

Acid

Sequence

of the Rat VIP Receptor

cDNA

Numbers above and below each line refer to the amino acid and nucleotide positions, respectively. Numbering of the amino acids begins at Ala-l of the postulated mature receptor, with negative numbers for the signal peptide. The seven putative transmembrane segments are underlined, and the potential N-glycosylation sites are marked by stars. The cysteine residues (boxed) in the first extracellular domain are conserved between the VIP and secretin receptors.

Molecular 813

Cloning

of a Novel

VIP Receptor

under low stringency conditions using rat secretin receptor cDNA (Ishihara et al., 1991) as the probe. Nine clones (pWVl-9) gave positive results, and these were subjected to nucleotide sequence analysis. The positive clones contained sequences homologous to rat secretin cDNA, but their sequences were interrupted by putative intervening sequences, or had deletions in the regions homologous to secretin receptor cDNA. To isolate the intact cDNA clone, various rat tissues were examined by Northern hybridization to identify the presence of the corresponding mRNA. These analyses showed that rat lung most abundantly expresses the 5.5 kb mRNA hybridizing with a cDNA clone (pWV2) from the human WiDr cell line. A rat lung cDNA library was constructed with the CDM8 vector, and the library was screened using pWV2 cDNAas the probe under low stringencyconditions. Twenty-two positive clones were isolated from 5 x IO5 clones, and plasmid DNA from each clone was introduced into COS cells by the DEAE-dextran method. When these transfected COS cells were examined for the ability to bind 1251-VIP as described (Ishiharaetal.,1991),5cloneswerepositive,indicating that they encode the VIP receptor. Since restriction mapping and DNA sequence analyses of these clones have shown that they overlapped, the longest cDNA clone (pV19) was further characterized. Structure of the Rat VIP Receptor Figure 1 shows the complete nucleotide sequence of the pV19 clone together with the deduced amino acid sequence. The sequence contains an open reading frame coding for a protein of 459 amino acids. Since the first 30 amino-terminal residues seem to serve as a signal sequence (von Heijne, 1986), the mature VIP receptor may consist of 429 amino acids with a calculated M, of 48,946. This agrees reasonably well with the value (55 kd) estimated for the VIP receptor in rat lung by cross-linking studieswith 1251-VIP(Provowand Velicelebi, 1987). The small difference (6 kd) may be explained by glycosylation at some of the potential N-glycosylation sites (Asn-X-Ser/Thr) in the extracellular region of the VIP receptor (Figure 1). The VIP receptor contains seven putative transmembrane segments, and its amino acid sequence is markedly homologous to that of the rat secretin receptor (identity48%) (Ishihara et al., 1991). Furthermore, it has 33% and 39% identity with the porcine calcitonin receptor (tin et al., 1991) and theopossum receptor for parathyroid hormone and parathyroid hormone-related peptide (Juppner et al., 1991), respectively, although VIP has no apparent homology with calcitonin and parathyroid hormone (see Discussion). On theother hand, the VIP receptor has little homology with other members of the G protein-coupled receptor superfamily, including the human GPRNI cDNA encoding the putative “VIP receptor” (Sreedharan et al., 1991). Binding Characteristics of the Cloned VIP Receptor To confirm that pV19 cDNAencodes theVIP receptor,

a

0.0

1.0

Bound

2.0

(nM)

VIP Added (nM)

0 0.0

I 1 .o

0.5

Bound Figure 2. Scatchard Analysis from COP Cells Transfected from Rat Lung

1.5

(nM)

of ‘Lrl-VIP Binding to Membranes with the VIP Receptor cDNA and

Membrane fractions from COP cells transfected with the VIP receptor cDNA (a) or from rat lung (b) were incubated with various concentrations of ‘251-VIP with or without an excess of unlabeled VIP as described in Experimental Procedures. The Scatchard plots were obtained using the MacLigand Program (Munson and Rodbard, 1980). Insetsshowthesaturation bindingof ‘251-VIP to the membrane fractions. The specific binding (open circles) is shown as the difference between total (closed squares) and nonspecific (closed circles) binding.

the pV19 plasmid or CDM8vector was transfected into mouse COP cells (Tyndall et al., 1981) and a membrane fraction was prepared from the transfected cells. lz51VIP could bind to membranes from pV19-transfected COP cells in a saturating manner (Figure 2a), whereas no specific binding of 1251-VIP to the membranes from CDM8-transfected COP cells was observed. Scatchard analysis of VIP binding data to the membranes revealed two classes of the VIP-binding sites with apparent dissociation constants of 173 pM and 21.0 nM, respectively. The concentrations of the high and low affinity binding sites were 4.1 and 53 pmol per mg of protein, respectively. These dissociation constants observed with the recombinant VIP receptor are simi-

80

60

PEPTIDE

PEPTIDE

CONCENTRATION

Figure 4. Accumulation of Intracellular CAMP in COSGsl Expressing the Rat VIP Receptor in Response to VIP and lated Peptides

(M)

b

Cells Its Re-

COSCsl cells were transfected with the rat VIP receptor expression vector and incubated for45 min with various concentrations of VIP (open squares), PACAP(closed circles), PACAP-27(open circles), helodermin (closed squares), PHM (open triangles), secretin (closed triangles), and glucagon (crosses). The CAMP accumulated in the cells was quantified as described in Experimental Procedures. As a control, COSGsl cells transfected with the CDM8 vector were treated with 10 uM VIP (X). The assays were done in duplicate, and the values agreed within 5% error. The average values are plotted in the figure.

80

60

40

glucagon in the binding reaction did not inhibit the binding of 1251-VIP to the receptor. These results correlated well with the specificity observed with the native VIP receptor in the rat lung membrane (Figure 3b).

20

0

-4

PEPTIDE Figure

CONCENTRATION(M)

3. Binding

Specificity

CONCENTRATION

(M)

of the VIP Receptor

The binding of 1251-VlP to membranes was determined in the presence of the indicated concentrations of VIP (open squares), PACAP(closed circles), PACAP(open circles), helodermin (closed squares), PHM (open triangles), secretin (closed triangles), and glucagon (crosses). (a) Displacement of 1*51-VIP binding to membranes from COP cells transfected with the VIP receptor cDNA. (b) Displacement of ‘z51-VIP binding to membrane from rat lung. The binding assays were done in duplicate, and the average values are plotted. The difference in duplicate was within 10%.

lar to those obtained with membrane fractions of rat lung (Figure 2b; Leroux et al., 1984). Members of the glucagonkecretin family compete with VIP for the VIP receptor. Figure 3a shows the binding specificity of the cloned VIP receptor using the membrane fraction from pVl9-transfected COP cells. PACAP(I& = 1.0 nM) and PACAP(I& = 2.5 nM) were slightly more potent than VIP (I& = 3.0 nM) in displacing the binding of 1251-VIP to the membrane. Helodermin and PHM inhibited the binding of 1251-VIP to the membrane with an apparent I& value of 6 nM. On the other hand, secretin was about 100 times less potent than VIP in displacing the binding of 1251-VIP, and the presence of 2.5 1M unlabeled

Intracellular Accumulation of CAMP Mediated by the Cloned VIP Receptor To determine whether the cloned VIP receptor transduces the signals, the VIP expression plasmid pV19 or its vector CDM8 was introduced into COSGsl cells, which overexpress the a subunit of the rat G, protein (Ishihara et al., 1991). As shown in Figure 4, VIP stimulated the accumulation of CAMP in COSGsl cells transfected with pV19, but not in those cells transfected with CDM8. The CAMP levels reached about 400 pmol per IO5 transfected cells after a 45 min incubation with 0.1 uM VIP. This value is comparable to that obtained with the cloned secretin receptor (lshihara et al., 1991). PACAP(EC5,, = 0.28 nM) and PACAP(ECsO = 0.42 nM) were slightly more potent than VIP (E& = 0.57 nM) in stimulating the accumulation of CAMP, whereas, helodermin and PHM (E& = 1.0 nM) were less potent than VIP. Secretin also stimulated adenylate cyclase activity through the recombinant VIP receptor, although about 30 times more secretin than VIP was necessary to elicit the half-maximal response. In accordance with the inability of glucagon to bind thecloned VIP receptor (Figure 3a), it barely stimulated the accumulation of CAMP. Tissue Distribution of the VIP Receptor Figure 5a shows the tissue distribution

of the VIP re-

Molecular

Cloning

of a Novel

VIP Receptor

815

4 28s 418s

in situ using labeled antisense RNA. The prominent expression of VIP receptor mRNA was observed in the cerebral cortex, hippocampus, and mitral cell layer of the olfactory bulb, while weak hybridization signals were widely distributed in the subcortical regions and cerebellarcortex(Figure6a).These hybridizing signals seem to be specific, since no signals were detected when a IOO-fold excess of unlabeled antisense RNA was included in the hybridization buffer (Figure 6b). The bright-field photomicrograph of the emulsiondipped section of the cerebral cortex indicated that VIP receptor mRNA is concentrated in neurons (Figure 6~). However, we cannot rule out the possible expression of the VIP receptor in glia.

Discussion Sreedharan et al. (1991) recently isolated a human cDNA clone, GPRNI, from the human pre-B lymphoblastic cell line Nalm 6 and the colon carcinoma cell line HT-29 and suggested that GPRNI cDNA encodes the VIP receptor. This cDNA is the human counterpart

4 4

Figure ceptor

5. Northern

Hybridization

Analysis

28s 18s

of the

Rat VIP

Re-

Poly(A) RNA was prepared from the indicated rat tissues (a) or subregions of the rat brain (b), and 2.5 pg (a) or 2.0 pg (b) of poly(A) RNA was electrophoresed on 1.5% agarose gel. Northern hybridization was performed using the’*P-labeled 1 kb Pstl fragment of pV19 as the probe. The filters were exposed to X-ray film for 24 hr (a) or 7 days (b).

ceptor as determined by Northern hybridization using poly(A) RNA from various rat tissues and pV19 cDNA as the probe. The 5.5 kb VIP receptor mRNA was most abundant in the lung, and moderate levels were found in the liver and intestine. Weak expression of the VIP receptor was also observed in the thymus and brain, but no expression was observed in the heart, kidney, spleen, pancreas, stomach, or adrenal glands. VIP mRNA was detected in all subregions of the adult rat brain, although mRNA from the cerebral cortex and hippocampus gave relatively stronger signals (Figure 5b). It is noteworthy that a single 5.5 kb VIP mRNA was detected in the lung, liver, and colon, but mRNA from the brain and small intestine showed two additional bands of 2.4 and 1.3 kb. To analyze the localization of the VIP receptor in the brain, sections from adult male rat were hybridized

of the previously isolated dog RDCI cDNA clone (Libert et al., 1989), which encodes a protein containing seven transmembrane domains. To confirm their results, a set of oligonucleotides conserved between dog RDCI and human GPRNI cDNAclones were used as polymerase chain reaction primers to amplify the corresponding sequence from rat genomic DNA. The amplified DNA fragment was then used to screen the rat lung cDNA library to identify the rat homolog of GPRNI cDNA. One of the cDNA clones encoded a protein consisting of 362 amino acids, and 92% of the sequence was identical to the sequences encoded by the human GPRNI and dog RDCI cDNA clones. However, when this cDNA was expressed in COS cells, there was no specific binding of 1251-VIP to the transfected cells (unpublished data). Vassart et al. also observed similar negative results with dog RDCI cDNA (Vassart, personal communication). In this study, rat VIP receptor cDNA was isolated by cross-hybridization with rat secretin receptor cDNA and expressed in COS cells to determine its ability to bind labeled VIP. The mature VIP receptor encoded by the cloned cDNA consists of 429 amino acids with a calculated M, of about 49,000, which agrees well with the size of the VIP receptor (M, 53,000) purified from porcine liver (Couvineau et al., 1990), but not with that of the VIP-binding protein (M, 18,000) purified from guinea pig lung (Brugger et al., 1991).TheVIP receptor contains seven transmembrane domains and is markedly homologous to the secretin, calcitonin, and parathyroid hormone receptors (Figure 7). However, no significant homologywasfound with other G proteincoupled receptors (O’Dowd et al., 1989). These results suggest that these receptors constitute a subfamily of the G protein-coupled receptor superfamily. The receptors of other members of the secretin/glucagon peptide family, such as the glucagon and PACAP re-

Neuron RI6

Figure ceptor Adult

6. In Situ Hybridization of VIP RemRNA in Parasagittal Sections of the Rat Brain

Parasagittal sections of adult male rat brain were hybridized with %-labeled antisense RNA for the VIP receptor. (a) Negative film image of the in situ hybridization of a parasagittal section. Cb, cerebellar cortex; Cx, cerebral cortex; Hi, hippocampus; Hy, hypothalamus; OB, olfactory bulb; SC, superior colliculus; Sep, lateral septal nucleus; Th, thalamus. (b) Control hybridization carried out in an adjacent section using the same labeled probe in the presence of a IOtT-fold molar excess of the unlabeled probe RNA. (c) Bright-field photomicrographs of emulsion-dipped sections showing layer II through the cerebral cortex. Bars, 5 mm (a and b); 50 nm fc).

ceptors, are likely to be members of this subfamily, and their cDNAs may be isolated using the secretin and/or VIP receptor cDNA as the probe. Despite the high homology between the VIP and secretin receptors, they preferentially bind the respective ligand (Figure 3; lshihara et al., 1991). Previously, we have postulated that the cysteine-rich region in the first extracellular domain of the secretin

VIP R (1 49) Secret,” R (I -60, PTH-PTWP R (1.95) Calcmnm R (l-60)

receptor is responsible for binding of the ligand (Ishihara et al., 1991). This region was found to be less conserved among the VIP, secretin, calcitonin, and parathyroid hormone receptors (Figure 7). It is now possible to examine the ligand-binding domain by constructing various hybrid receptors between members of the subfamily. The VIP, secretin, calcitonin, and parathyroid hor-

ASPQHECEYLQLIEIQRQ AHTVGVPPRLCDVRRVLLEERA DADDVITKEEQIILLRNAQAQCEQRLKEVLRVPEAESAKDWMSRS~TKKEKP AHTPTLEPEPFLYILGKQMLEAQHFf$DRM-----------

VIP R (50.140) secrean R (61.147) PTH-PTHrP R (96165) Cahlonin R (61.147)

NSGEID"C-----------------SEA SSDDVTYC-----------------DA" SGVSTDEIERITEEELRAFTEPPPADKA --VpNGELVK----------------m

VIPR (141.217) Secretin R (146-224) PTH-PTHrP R (166-260) Cakitonin R (146-222)

VIP R (218310) Seuetin R (225-317) PTH-PT”,P R (281973) Wcimnin R (223312)

PDIGKNDS-S

VIPR (311.367) secdn R (318393) PTH-PTHrP R (374454) Calcitm~nR (313-366)

Figure

7. Amino

TE-VSGILWQVQMH T--PLLGKIYDYW Actd

Sequence

Alignment

of the Rat VIP Receptor

and the Secretin,

Calcitonin,

and Parathyroid

Hormone

Receptors

The amino acid sequences of the rat VIP receptor, rat secretin receptor (Ishihara et al., 1991), porcine calcitonin receptor (Lin et al., 1991), and opossum parathyroid hormone receptor (PTH-PTHrH R) (Juppner et al., 1991) are aligned. The amino acids are numbered from the putative mature protein of each receptor. Several gaps have been introduced to optimize the alignment. Amino acids identical in more than three members are boxed. The seven putative transmembrane segments are indicated by bars. The putative C,-activating region is double underlined.

Molecular 817

Cloning

of a Novel

VIP Receptor

mone receptors can mediate the ligand-dependent stimulation of theadenylatecyclase(Figure4; lshihara et al., 1991; Lin et al., 1991; Jiippner et al., 1991). Very recently, Okamoto et al. (1991) identified a G, activator region in the third inner loop of the 02-adrenergic receptor. In this region, the positions of basic amino acids are important for the interaction with the G, protein (Okamoto et al., 1991). Sequences with similar characteristics can be found in the third inner loop of the VIP, secretin, calcitonin, and parathyroid hormone receptors (Figure 7). This finding suggests that this region also plays an important role in their interactions with the G, protein. The ligand specificity of the VIP receptor has been so far determined using tissue membrane fractions (Robberecht et al., 1991). Since these tissues often contain receptors for other VIP-related molecules (Robberecht et al., 1990), the ligand specificity has not been accurately determined. The recombinant VIP receptor expressed in COP cells bound 1251-VIP with high affinity (Figure 2a). Displacement of the 1251-VIP binding to the receptor (Figure 3a) and stimulation of adenylate cyclase via the cloned receptor (Figure 4a) suggest that this receptor has slightly different affinities for VIP, PACAP, PHM, and helodermin. PACAP and helodermin have their own receptors, which bind their respective ligands with much higher affinities than VIP (Robberecht et al., 1990,199l). Other types of VIP receptors with different ligand specificities may exist. In fact, in addition to the ubiquitous 5.5 kb mRNA, we found two different mRNAs (2.4and 1.3 kb) hybridizing with the cloned cDNA in rat brain (Figure 5b). These mRNAs may represent different subtypes of the VIP receptor. Furthermore, whether the cloned VIP receptor or other subtypes of the VIP receptor are involved in the stimulation of phosphatidyl inositol turnover (Malhotra et al., 1988) remains to be studied. Northern and in situ hybridization indicated that theVIP receptor is expressed in neuronal cells throughout regions of the brain, particularly the cerebral cortex and hippocampus (Figure 5b; Figure 6). These results essentially agree with those obtained with labeled VIP (Martin et al., 1987). Baldino et al. (1989) have shown by in situ hybridization that VIP mRNA is expressed in cortical and thalamic neurons. SinceVIP stimulates glycogenolysis in cortical tissues (Magistretti et al., 1981), it is likely that it works as a neurotransmitter in the cerebral cortex to modulate energy metabolism (Magistretti, 1990). The physiological roles of the VIP receptor in other regions of the brain are currently unknown. It may bind VIP-related substances, such as PACAP, which are abundant in the brain (Kbves et al., 1990). Molecular cloning of other receptors for the secretin/glucagon familywill help us to understand the physiological functions of these hormones in the brain. Experimental

Procedures

Cells and Peptides The human WiDr

cell

line

(JCRB 0224) was obtained

from

the

JCRB cell bank and cultured in minimal Eagle’s medium supplemented with nonessential amino acids and 10% fetal calf serum Oiyclone). Mouse COP cells (Tyndall et al., 1981) were obtained from Dr. Kamen through Dr. Kishimoto and were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum. Porcine secretin was synthesized and purified as described previously (Ishihara et al., 1991). Human VIP, PHM, PACAP-38, and PACAPwere purchased from the Peptide Institute Inc. (Osaka); helodermin was from Peninsula Laboratories Inc. (Belmont, CA). 1251-labeled VIP (specific activity 2200 Ci/mmol) was purchased from DuPont/New England Nuclear. Constructions of cDNA libraries, Isolation of cDNAs, and DNA Sequence Analysis Random hexamer-primed, double-stranded cDNA was synthesizedwith mRNAfrom human WiDrcelIsaspreviouslydescribed (Ishihara et al., 1991). After addition of an EcoRl adapter, a cDNA larger than 1.4 kb was recovered from an agarosegel, phosphorylated with T4 polynucleotide kinase, and ligated with the )cgtll vector (Stratagene). A cDNA library consisting of about 1.5 x IOh recombinant clones was constructed in E. coli Y109Or. A rat lung cDNA library (a total of 5 x IO1 independent clones) carrying cDNAs larger than 2.0 kb was prepared in the expression vector CDM8 (Seed, 1987) as previously described (Ishihara et al., 1991). Plaque and colony hybridizations were performed under either high (Sambrook et al., 1989) or low stringency conditions (Fukunagaetal.,199O).TheprobeDNAwasprepared bypolymerase chain reaction using appropriate primers and was labeled with lLP by the random primer method. DNA was sequenced by the dideoxy nucleotide chain termination method usingT7 DNA polymerase (Pharmacia) and [a-‘SS]dATPaS (Amersham). Preparation of the Rat Lung Membrane Fraction The membrane fraction was prepared from the rat lung essentially as described (Provow and Velicelebi, 1987). Male Wistar rats were anesthetized with ether, decapitated, and cardiac perfused with 30 ml of ice-cold phosphate-buffered saline (PBS). The whited lungs were minced and homogenized in a Polytron homogenizer with 25 mM HEPES (pH 7.4), 250 mM sucrose, 5 mM MgCI,, 1 mM phenylmethylsulfonyl fluoride at 20 ml per g wet weighttissue.After rehomogenization inaglass-Teflon homogenizer, the homogenates were filtered through medical gauze and centrifuged at 30,000 x g for 10 min. The precipitate was suspended in a buffer containing 25 mM HEPES (pH 7.41, 5 mM MgCI,, and 1 mM phenylmethylsulfonyl fluoride. After centrifugation, the pellet was resuspended in the same buffer containing 1 mM EGTA at a concentration of 10 ml per g wet weight tissue. Binding of 9-VIP to Membranes from COP Cells and Rat Lung COP cells grown in 15 cm plates were transfected with pV19 or CDM8 plasmid DNA by the DEAE-dextran method. Crude cell membranes were prepared from the transfected COP cells as described previously (Ishihara et al., 1991). Ten micrograms of membrane fraction was incubated at 37°C for 2 hr with various concentrations of ‘*+VIP in 100 ~1 of a solution containing 25 mM HEPES (pH 7.4), 5 mM MgClz, 1 mM ECTA, 50 mM NaCI, 10 mg/ml bovine serum albumin, 2 mglml bacitracin, 0.1 mgiml leupeptin, and 0.1 mM (pamidinophenyl)methanesulfonyl fluoride hydrochloride. The reaction was terminated by centrifugation at 14,000 x g for 3 min. The resulting pellet was suspended in 1 ml of ice-cold PBS and filtered through a Whatman GF/C filter presoaked in 0.3% polyethyleneimine. To determine the nonspecific binding of ‘2rl-VIP to the membrane, a large excess of unlabeled VIP (5 PM) was included in the assay mixture and subtracted from the total binding to yield the specific binding. CAMP Assay The intracellular CAMP levels were assayed as described previously (Ishihara et al., 1991). In brief, COSCsl cells were transfected with pV19 or CDM8 by the DEAE-dextran method. At 24 hr after the treatment with glycerol, the cells were split into

NWKX 818

6 well plates and cultured at 37°C for 48 hr. Cells were washed twice with the incubation buffer (Dulbecco’s modified Eagle’s medium containing 0.5 mM I-methyl-3-isobutylxanthine and 1 mglml bovine serum albumin) and incubated at 37OC for 45 min in the same buffer containing various concentrations of peptides. After removing the buffer, 1 ml of ethanol was added to the cells, and the suspension was centrifuged at 14,000 x g for 3 min. The supernatants were dried under vacuum, and the level of CAMP was quantified using the CAMP assay system purchased from Amersham. Northern Hybridization Total cellular RNA was prepared from various rat tissues by the guanidine thiocyanate-acid phenol method (Chomczynski and Sacchi, 1987J, and poly(A) RNA was enriched using oligo(dTJ30 Latex (Takara Shuzo, Co.). About 2 pg of poly(A) RNA in each lane waselectrophoresed througha1.5% agarosegelcontaining6.6% formaldehyde, and thegel was transferred to a nylon membrane (Schleicher & Schuell). Hybridization was carried out as described (Sambrook et al., 1989), except that the hybridization temperature was 42OC and the filter was washed at 50°C in 0.1 x SSC and 0.1% SDS. For probe DNA, the Pstl fragment (nucleotides 249-1291) of pV19 was labeled with 32P by the random primer method. In Situ Hybridization In situ hybridization was carried out as described (Masu et al., 1991). Sections (10 gm thick) of adult male rat were cut on a cryostat, thaw-mounted onto poly-r-lysine-coated slides, fixed with 4% formaldehyde, and acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine buffer (pH 8.0). To prepare the ‘5S-labeled RNA, the 1.3 kb Sacl fragment of pV19 was subcloned into pBluescript KS(+), and transcribed in vitro using T7 RNA polymerase and [a-SSS]CTP. The labeled RNA product (specific activity 2 x IO9 cpm/gg) was fragmented by incubation at 60°C for 50 min in 0.1 M NaHCOl (pH 10.2) and used as a probe at a concentration of 2-3 x 105 cpmlul. Hybridization proceeded at 55OC for 5 hr in hybridization buffer (50% formamide, 10% dextran sulfate, 100 mM dithiothreitol, 1 x Denhardt’s solution, 0.2% SDS, 2x SSC, 10 mM Tris-HCI [pH 7.51, 250 pglml tRNA, 500 ug/ml herring sperm DNA). After washing twice with 2x SSC containing 10 mM B-mercaptoethanol, slides were treated at 37OC for 30 min with 20 us/ml RNAase A, washed at 60°C for 1 hr with 0.1 x SSC containing 10 mM B-mercaptoethanol, and exposed for 2 weeks to Hyperfilm-Bmax (Amersham). To monitor the nonspecific binding of the probe RNA, a IOO-fold excess of unlabeled cRNA was included in the hybridization buffer. For emulsion autoradiography, the slidesweredipped in NTBL emulsion (Kodak) diluted I:1 with distilled water and exposed for 6 weeks. After developing, the brain sections were counterstained with cresyl violet. Acknowledgments

Brugger, C. H., Stallwood, D., and Paul, S. (1991). low molecular mass vasocactive intestinal peptide tein. J. Biol. Chem. 27, 18358-18362. Chomczynski, RNA isolation form extraction.

Isolation binding

of a pro-

P., and Sacchi, N. (1987). Single-step method by acid guanidinium thiocyanate-phenol-chloroAnal. Biochem. 162, 156-159.

Couvineau, A., Voisin, T., Cuijarro, L., and Laburthe, Purification of vasoactive intestinal peptide receptor cineliver bya newlydesignedonestepaffinitychromatography. 1. Biol. Chem. 265, 13386-13390.

of

M. (1990). from por-

Fukunaga, R., Seto, Y., Mizushima, S.,and Nagata, S. (1990).Three different mRNAs encoding human granulocyte colony-stimulating factor receptor. Proc. Natl. Acad. Sci. USA 87,8702-8706. Cozes, I., and Brenneman, D. E. (1989). VIP: molecular biology and neurobiological function. Mol. Neurobiol. 3, 201-236. Hokfelt, T., Johansson, Schultzberg, M. (1980). 521.

O., Ljungdahl, A., Peptidergic neurones.

Lundberg, Nature

J., and 284, 515-

Ishihara, T., Nakamura, S., Kaziro, Y., Takahashi, T., Takahashi, K., and Nagata, S. (1991). Molecular cloning and expression of a cDNA encoding the secretin receptor. EMBO J. 7, 1635-1641. Itoh, N., Obata, K., Yanaihara, N., and Okamoto, H. (1983). Human preprovasoactive intestinal polypeptide contains a novel PHI-27-like peptide, PHM-27. Nature 304, 547-549. Juppner, H., Abou-Samra, A-B., Freeman, M., Kong, X. F., Schipani, E., Richards, J., Kolakowski, L. F., Jr., Hock, J., Potts, J. T., Jr., Kronenberg, H. M., and Segre, C. V. (1991).AG protein-linked receptor for parathyroid hormone and parathyroid hormonerelated peptide. Science 254, 1024-1026. Kaiser, P. K., and Lipton, S. A. (1990). VIP-mediated increase in CAMP prevents tetrodotoxin-induced retinal ganglion cell death in vitro. Neuron 5, 373-381. Kawatani, M., Rutigliano, M., and de Groat, ization and muscarinic excitation induced glion by vasoactive intestinal polypeptide.

W. C. (1985). Depolarin a sympathetic ganScience 229,879~881.

Koves, K., Arimura, A., Somogyvari-Vigh, A., Vigh, S., and Miller, J. (1990). lmmunohistochemical demonstration of a novel hypothalamic peptide, pituitary adenylate cyclase activating polypep tide in the ovine hypothalamus. Endocrinology 727, 264271. Laburthe, M., Rousset, M., Boissard, C., Chevalier, G., Zweibaum, A., and Rosselin, C. (1978). Vasoactive intestinal peptide: a potent stimulator of adenosine 3’5’~cyclic monophosphate accumulation in gut carcinoma cell lines in culture. Proc. Natl. Acad. Sci. USA 75, 2772-2775. Leroux, P., Vaudry, H., Fournier,A., St.-Pierre, S., and Pelletier, G. (1984). Characterization and localization of vasocactive intestinal peptide receptors in the rat lung. Endocrinology 774,1506-1512. Libert, F., Parmentier, M., Lefort, A., Dinsart, C., Van Sande, J., Maenhaut, C., Simons, M.-J., Dumont, 1. E, and Vassart, G. (1989). Selective amplification and cloning of four new members of the G protein-coupled receptor family. Science 244, 569-572.

We thank Drs. R. Yoshida, K. Katoh, and K. lmamura for help with the in situ hybridization, Dr. M. Nishizawa for discussion, and Ms. M. lkeda for secretarial assistance. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advefltisement” in accordance with 18 USC Section 1734 solely to indicate this fact.

Lin, H. Y., Harris, T. L., Flannery, M. S., Aruffo, A., Kaji, E. H., Gorn, A., Kolakowski, L. F., Jr., Lodish, H., and Goldring, 5. R. (1991). Expression cloning of an adenylate cyclase-coupled calcitonin receptor. Science 254, 1022-1024.

Received

Magistretti, P. J., Morrison, J. H., Shoemaker, W. J., Sapin,V.,and Bloom, F. E. (1981). Vasoactive intestinal polypeptide induces glycogenolysis in mouse cortical slices: a possible regulatory mechanism for the local control of energy metabolism. Proc. Natl. Acad. Sci. USA 78, 6535-6539.

December

19, 1991; revised

January

31, 1992.

References Baldino, F., Jr., Fitzpatrick-Mcelligott, S., Gozes, I., and Card, J. P. (1989). Localization of VIP and PHI-27 messenger RNA in rat thalamic and cortical neurons. J. Mol. Neurosci. 7, 199-207. Barbezat, stimulation

G. O., and Grossman, M. I. (1971). Intestinal by peptides. Science 774, 422-423.

secretion:

Lundberg, J. M., Hedlund, B., and Bartfai, T. (1982). Vasoactive intestinal polypeptideenhances muscarinic ligand binding in cat submandibular salivary gland. Nature 295, 147-149. Magistretti, Pharmacol.

P. J. (1990). VIP neurons Sci. 77, 250-254.

in thecerebral

cortex.Trends

Malhotra, R. K., Wakade, T. D., and Wakade, A. R. (1988). Vasoactive intestinal polypeptide and muscarine mobilize intracellularCa2+ through breakdown of phosphoinositides to inducecatecholamine secretion. J. Biol. Chem. 263, 2123-2126.

Molecular 819

Cloning

of a Novel

VIP Receptor

Martin, J. L., Dietl, M. M., Hof, P. R., Palacios, J. M., and Magistretti, P. J. (1987). Autoradiographic mapping of [mono[12sl]iodoTyr”‘, MetO’? vasoactive intestinal peptide binding sites in the rat brain. Neuroscience 23, 539-565. Masu, M., Tanabe, Y., Tsuchida, K., Shigemoto, R., and shi, S. (1991). Sequence and expression of a metabotropic mate receptor. Nature 349, 768765.

Nakanigluta-

Miyata, A., Arimura, A., Dahl, R. R., Minamino, N., Uehara, A., Jiang, L., Culler, M. D., and Coy, D. H. (1989). Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem. Biophys. Res. Commun. l&1,567-574. Munson, P. J., and Rodbard, D. (1980). puterized approach for characterization tems. Anal. Biochem. 707,220-239.

LIGAND: a versatile of ligand-binding

O’Dowd, B. F., Lefkowitz, R. J., and Caron, of the adrenergic and related receptors. 12, 67-83.

comsys-

M. G. (1989). Structure Annu. Rev. Neurosci.

Okamoto, T., Murayama, Y., Hayashi, Y., Inagaki, M., Ogata, E., and Nishimoto, I. (1991). Identification of a C, activator region of the 8Zadrenergic receptor that is autoregulated via protein kinase A-dependent phosphorylation. Cell 67, 723-730. Pincus, D. W., DiCicco-Bloom, E. M., and Black, I. B. (1990). Vasoactive intestinal peptide regulates mitosis, differentiation and survival of cultured sympathetic neuroblasts. Nature 343, 564 567. Piper, P. J., Said, S. I., and Vane, J. R. (1970). Effects on smooth muscle preparations of unidentified vasoactive peptides from intestine and lung. Nature 225, 11441146. Provow, S., and Velicelebi, C. (19873. Characterization lization of vasoactive intestinal peptide receptors membranes. Endocrinology 720, 2442-2452. Robberecht, Heterogeneity 66.

and solubifrom rat lung

P., Cauvin, A., Courlet, P., and Christophe, of VIP receptors. Arch. Int. Pharmacodyn.

J. (1990). 303,51-

Robberecht, P., Gourlet, P., Cauvin, A., Buscail, L., Neef, P. D., Arimura, A., and Christophe, J. (1991). PACAP and VIP receptors in rat liver membranes. Am. J. Physiol. 260, G97-Cl02 Rosellin, G. (1986). The receptors of the VIP family peptides (VIP, secretin, CRF, PHI, PHM, GIP, glucagon and oxyntomodulin). Specificities and identity. Peptides 7, (suppl. I), 89-100. Said, S. I. (1986). Vasoactive vest. 9, 191-200. Sambrook, J., Fritsch, Cloning: A Laboratory bor, New York: Cold

intestinal

peptide.

J. Endocrinol.

In-

E. F., and Maniatis, T. (1989). Molecular Manual, Second Edition (Cold Spring HarSpring Harbor Laboratory).

Seed, B. (1987). An LFA-3 cDNA membrane protein homologous 840-842.

encodes a phospholipid-linked to its receptor CD2. Nature329,

Sreedharan, S. P., Robichon, A., Peterson, K. E., and Goetzl, E. 1. (1991). Cloning and expression of the human vasoactive intestinal peptide receptor. Proc. Natl. Acad. Sci. USA 88, 4986-4990. Tatemoto, K., and Mutt, V. (1981). Isolation and characterization of the intestinal peptide porcine PHI (PHI-27), a new member of the glucagon-secretin family. Proc. Natl. Acad. Sci. USA 78,66036607. Tyndall, C., La Mantia, C., Thacker, C. M., Favaloro, J., and Kamen, R. (1981). A region of the polyoma virus genome between the replication origin and late protein coding sequence is required in cis for both early gene expression and viral DNA replication. Nucl. Acids. Res. 9, 6231-6250. Vandermeers, A., Vandermeers-Piret, M.-C., Robberecht, P., Waelbroeck, M., Dehaye, J.-P., Winand, J., and Christophe, J. (1984). Purification of a novel pancreatic secretory factor (PSFJ and novel peptide with VIP- and secretin-like properties (helodermin) from gila monster venom. FEBS Lett. 766, 273-276. von Heijne, C. (1986). A new method for predicting quence cleavage sites. Nucl. Acids Res. 74, 4683-4690.

signal

se-

CenBank

Accession

Number

The GenBank accession this paper is M86835.

number

for the sequent

:e reported

in