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Endocrinology Vol. 143, No. 4 1327-1336
Copyright © 2002 by The Endocrine Society

RECEPTORS

Identification of a Potential Receptor for Both Peptide Histidine Isoleucine and Peptide Histidine Valine

Dicky Lai-Yin Tse, Ronald Ting-Kai Pang, Anderson On-Lam Wong, Siu-Ming Chan, Hubert Vaudry and Billy Kwok-Chong Chow

Department of Zoology (D.L.Y.T., R.T.K.P., A.O.L.W., S.M.C., B.K.C.C.), University of Hong Kong, Hong Kong, People’s Republic of China; and European Institute for Peptide Research (H.V.), INSERM Unité 413, UA Centre National de la Recherche Scientifique, University of Rouen,76821 Mont-Saint-Aignan, France

Address all correspondence and requests for reprints to: Dr. Billy K. C. Chow, Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong, SAR, PRC. E-mail: . bkcc{at}hkusua.hku.hk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peptide histidine isoleucine (PHI), peptide histidine valine (PHV), and vasoactive intestinal polypeptide (VIP) are cosynthesized from the same precursor and share high levels of structural similarities with overlapping biological functions. In this study, the first PHI/PHV receptor was isolated and characterized in goldfish. To study this receptor using homologous peptides, we have also characterized the goldfish prepro-PHI/VIP, and, surprisingly, a shorter transcript lacking the VIP coding region was isolated. A PHI/VIP precursor without the VIP coding sequence has never before been reported. Initial functional expression of the PHI/PHV receptor in Chinese hamster ovary cells revealed that it could be activated by human PHV [50% effective concentration (EC50): 43 nM] and to a lesser extent human PHI (EC50: 133 nM) and helodermin (EC50: 166 nM) but not fish and mammalian pituitary adenylate cyclase-activating polypeptides and VIPs. Subsequent studies indicated that, similar to the pituitary adenylate cyclase-activating polypeptide receptors (PAC1-R, VPAC1-R, and VPAC2-R), the receptor isolated in this study is able to interact with goldfish PHI and its C-terminally extended form, PHV with EC50 values 93 and 43 nM, respectively. Northern blot and RT-PCR/Southern blot analyses revealed that the PHI/VIP gene is expressed in the intestine, brain, and gall bladder and the PHI/PHV receptor gene is primarily expressed in the pituitary and to a lesser extend in the intestine and gall bladder, suggesting that PHI/PHV may play a role, notably in the regulation of pituitary function. In conclusion, our results demonstrate for the first time the existence of a PHI/PHV receptor, indicating that the functions of PHI and PHV could be mediated by their own receptor in addition to VIP receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VASOACTIVE INTESTINAL POLYPEPTIDE (VIP) and peptide histidine isoleucine (PHI) are members of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon family. These peptides share many structural and biological similarities. They are cosynthesized from a common precursor; colocalized in the central nervous, gastrointestinal, reproductive, and respiratory systems; and coreleased in response to stimulation (1, 2, 3, 4, 5, 6, 7). It is interesting to note that similar to PACAP-27 and PACAP-38, a C-terminally extended form of PHI, known as peptide histidine valine (PHV), is also present in high concentrations in the circulation (8), although the function of this peptide is still unclear.

VIP and PHI mediate a number of similar physiological actions in many systems, but the potencies of PHI are one to three orders of magnitude lower than those of VIP in various studies (5, 9, 10, 11, 12, 13). Thus, PHI has been considered a weak agonist for VIP. In the gastrointestinal system, they both augment water and electrolyte transport in the jejunum (9, 14) and glucagon release from the pancreas (15). In the neuroendocrine system, they stimulate the secretion of PRL in the pituitary (10) and increase melatonin synthesis in the pineal gland (12). In the reproductive system, both peptides inhibit fallopian smooth muscle activity (5). In the vascular system, they produce an increase in vertebral artery blood flow (13).

The biological effects of VIP are mediated through at least two receptor subtypes, termed VPAC1-R (16) and VPAC2-R (17), that exhibit high affinity for both VIP and PACAP. A third receptor, termed PAC1-R, that possesses high affinity for PACAP but a much lower affinity for VIP has been characterized (18). Up to now, a selective PHI receptor has not been identified, and it was generally accepted that the effects of PHI were mediated through VIP receptors. As a matter of fact, there is evidence that PHI shares common receptors with VIP: 1) PHI has been reported to bind VIP-preferring sites in many preparations (19, 20, 21); 2) maximal doses of PHI and VIP do not produce additive effects (5, 10, 11, 12, 13, 16); and 3) cross-desensitization of PHI and VIP has been demonstrated (11, 12, 21). Thus, it is likely that at least some of the actions of PHI are mediated by binding to VIP-preferring sites. However, there are examples indicating that the biological responses to PHI are distinct from those evoked by VIP: 1) PHI injection in the preoptic area of rats facilitates LH secretion, whereas VIP injection abolishes LH secretion (22); 2) VIP and PHI produce different effects on the secretion of hormones in mouse pancreatic islets (11); and 3) VIP antagonists do not affect PHI-stimulated responses (23, 24). These latter studies suggest the existence of PHI receptor distinct from VIP receptor mechanisms. Finally, the strongest piece of evidence supporting the occurrence of authentic PHI receptors come from radioligand binding studies: using radiolabeled PHI and VIP, receptors with greater or equal affinities for PHI, compared with VIP, have been demonstrated in rat liver (25), rat pineal cells (26), rat pituitary tumor cells (27), rat insulinoma cells (28), rabbit gastric smooth muscle cells (29), and mouse neuroblastoma cells (30).

Here we report the cloning and identification of a PHI/PHV-preferring receptor in the goldfish, which is the first PHI receptor characterized in vertebrates. We also describe the cDNA sequence of the goldfish PHI/VIP precursor so that homologous peptides could be used to functionally characterize the PHI/PHV receptor. The observation that evolutionary pressure has acted to conserve the primary structure of human peptide histidine methionine (PHM), rat PHI, and the goldfish orthologs strongly suggests that these peptides play vital functions throughout the vertebrate phylum.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of goldfish PHI receptor cDNA
Goldfish, Carassius auratus, 25–40 g, of both sexes at a sexually regressed stage were purchased from a local dealer and maintained in a 200-liter aquarium at 18 C under a 12-h light/12-h dark photocycle for at least 1 wk before experimentation. The fish were fed to satiation daily with commercial fish food. Total and poly(A)+ RNA were isolated from the brain and pituitary of freshly killed goldfish using the guanidium thiocyanate-acid phenol method (31), and poly(A)+ RNA was enriched by the PolyATtract mRNA isolation system (Promega Corp., Madison, WI). Reverse transcription and nested-PCR was performed as described previously (32). PCR products between 500 and 600 bp corresponding to the transmembrane domains (TMD) 2–6 of this family of receptors were subcloned and sequenced. A partial cDNA clone, which showed high sequence identities with mammalian VIP/PACAP receptors but was distinct from goldfish VPAC1-R (32), PAC1-R (33), and GHRH receptor (34) was obtained and used as a probe to screen a goldfish brain and pituitary cDNA library. The library was constructed using the ZAP Express cDNA synthesis kit and Gigapack II gold packaging extract (Stratagene, Cambridge, UK). The probe was radiolabeled with [{alpha}-32P]-dATP (3000 Ci/mmol) (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) using the RadPrime DNA labeling system (Life Technologies, Inc., Gaithersburg, MD). After library screening, in vivo excision of the positive clones was performed according to the manufacturer’s instructions. A cDNA clone of the receptor in pBK-CMV vector (Stratagene) was produced and analyzed subsequently by restriction mapping and DNA sequence analysis. DNA sequences were analyzed using MacDNASIS Pro v3.6 (Hitachi, Tokyo, Japan) and GeneWorks (Intelligenetics, Inc., Mountain View, CA) software.

Cloning of the goldfish prepro-PHI/VIP cDNA
Total RNA was isolated from intestines of freshly killed goldfish (31), and poly(A)+ RNA was enriched by the PolyATtract mRNA isolation system (Promega Corp.). Rapid amplification of cDNA ends (RACE) was then performed using a Marathon cDNA amplification kit (CLONTECH Laboratories, Inc., Palo Alto, CA) as previously described (35). Two degenerate primers, DVIP1: 5'-GAYGCAGTNT TYACNGAYAA YTA and DVIP2: 5'-TAYTTYTTNG CNGCCATYTG YTT, designed on the basis of the conserved regions of known VIP sequences, were used for 5' and 3' RACE, respectively. The conditions of PCR were 1 min each at 94 C, 55 C, and 72 C for 30 cycles. The amplified products were blunt ended, subcloned into pBluescript KS+ (Stratagene), and sequenced using the Autoread sequencing kit (Amersham Pharmacia Biotech). A probe was obtained by PCR amplification using primer pairs VIPF: 5'-CTTCTGCAGG ACTCTCGCAT TGCC and VIPR: 5'-CAGTTATCAC GAATGACGGC AGTC, designed from the nucleotide sequences of the RACE products using goldfish brain first-strand cDNA as the template. The PCR contained 50-pmol primers and PCR SuperMix (Life Technologies, Inc.). The conditions of PCR were 1 min each at 94 C, 59 C, and 72 C for 30 cycles. The probe was used to screen the goldfish brain and pituitary cDNA library. After library screening, in vivo excision of the positive clones to the pBK-CMV vector was performed and DNA sequences were analyzed using MacDNASIS Pro v3.6 (Hitachi) and GeneWorks software (Intelligenetics, Inc.).

Functional expression of the cloned receptor
Chinese hamster ovary (CHO)-K1 cells were cultured in MEM supplemented with 10% FBS and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). The cells were incubated at 37 C under 5% carbon dioxide and saturated humidity. The construct containing the cDNA (pBK-CMV/VR7) was transfected into CHO cells using LipofectAMINE reagent (Life Technologies, Inc.). A permanent cell line with stable expression of the cDNA was obtained by selection in the presence of 500 µg/ml G418 (Life Technologies, Inc.) for 3 wk. For the functional study of the receptor, cAMP assays were performed as described previously (32), and cAMP levels were quantified by a cAMP [125I] RIA kit (NEN Life Science Products, Boston, MA). For each cAMP induction experiment, receptor-transfected (with no peptide stimulation), vector transfected, and nontransfected CHO cells were used as controls, and there were no significant differences among these samples. Human PHM, PHV, VIP, human PACAP38, PACAP27, secretin, GHRH, and glucagon were purchased from Bachem (Bubendorf, Switzerland). Cod VIP and helodermin were obtained from Peninsula Laboratories, Inc. (Belmont, CA). Goldfish PACAP38 and PHV (predicted amino acid sequences from the clone 2A) were synthesized by the Department of Biochemistry, University of Toronto (Toronto, Canada) and the European Institute for Peptide Research, University of Rouen (Rouen, France), respectively. Goldfish PACAP38, GHRHcarp-like, and GHRHcatfish-like peptides were synthesized by the Laboratory of Cellular Physiology and Immunology, Rockefeller University (New York, NY). Carp GHRH was a generous gift from Dr. J. Rivier (The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, San Diego, CA).

Tissue distribution studies by Northern blot analysis and RT-PCR
Poly(A)+ RNAs from various goldfish tissues were isolated as described above and separated by a 1% denaturing agarose gel with 0.72% formaldehyde (wt/vol), transferred, and cross-linked to Hybond-N nylon membrane (Amersham Pharmacia Biotech). After overnight hybridization in the Rapid-hyb Buffer (Amersham Pharmacia Biotech) supplemented with 100 µg/ml denatured calf thymus DNA (Life Technologies, Inc.) at 65 C, the membrane was washed to a final stringency of 0.1x standard saline citrate/0.1% SDS at 65 C before autoradiography. For RT-PCR, first-strand cDNA was prepared from 0.5 µg poly(A)+ RNA using random hexanucleotide and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). The primer pairs, VIPF and VIPR, were used for the detection of goldfish PHI/VIP transcript in PCR. The conditions of PCR were 1 min each at 94 C, 59 C, and 72 C for 28 cycles. For the amplification of the goldfish VPAC1-R transcripts, the PCR conditions used were 1 min each at 94 C, 64 C, and 72 C for 34 cycles, using primers gfVIPRF: 5'-TTCGAGGCTG GCAAAATCCA CACT and gfVIPRR2: 5'-CTCCAGAAGA TCACTTTCGC CTTA. Primers B1F: 5'-CCCACGGGAA TGTAGAATGT TC and B1R: 5'-GCCAGGGAAT CGGGGCATCA TTCC were used to amplify the PHI/PHV receptor, and the condition was 1 min each at 94 C, 60 C, and 72 C for 33 cycles. For the detection of the goldfish PAC1-R, the primers used were 27C1: 5'-AGTGTCGGCA AGGTCGTGGA GGTC and 27C4: 5'-CGCAGGTAGA TGCTGGACTC GTTC. The PCR conditions used were 1 min each at 94 C, 68 C, and 72 C for 27 cycles. The products were separated on an agarose gel (1%) and transferred to the Hybond-N membrane (Amersham Pharmacia Biotech). Probe labeling and blotting procedures were the same as described (32, 33). To demonstrate the quality of the first-stranded cDNA, an actin PCR was performed in the following conditions: 1 min each at 94 C, 60 C, and 72 C for 28 cycles using primers ß-actin F: 5'-CACTGTGCCC ATCTACGAG and ß-actin R: 5'-CCATCTCCTG CTCGAAGTC.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of goldfish PHI receptor cDNA
We have previously characterized the VPAC1-R (32), PAC1-R (33), and GHRH receptor (34) from goldfish. Using degenerate oligonucleotides designed based on the amino acid sequence alignment of these PACAP/VIP receptors, we looked for novel receptor clones from the goldfish brain. We isolated a partial cDNA clone (534 bp) that shared high-sequence homologies with the TMD2 to TMD6 of VIP/PACAP receptors but was distinct from the goldfish GHRH, VPAC1, and the PAC1 receptors. This clone was then used as a probe to screen a goldfish brain and pituitary cDNA library. Two clones, VR6 and VR7, were isolated and found to contain inserts of different sizes: 2.6 kb and 3.2 kb, respectively. DNA sequence analysis of these clones indicated that VR7 contained an entire opening reading frame of a G protein-coupled receptor and VR6 was a 5' truncated cDNA clone of VR7 (from nucleotide 684 to 3200, data not shown).

Nucleotide sequence analysis of the goldfish PHI receptor cDNA
VR7 is 3200 bp in length and contains a single opening reading frame of 1338 bp (from nucleotides 121 to 1458) that encodes a 446-amino-acid-long polypeptide, with a predicted molecular mass of 51.8 kDa (Fig. 1Go). At the 3' end, a consensus polyadenylation signal, AATAAA, is present. A Kyte-Doolittle hydrophobicity analysis of the receptor revealed seven hydrophobic domains presumably forming the transmembrane spanning regions (data not shown). The protein contains a putative signal peptide with 16 hydrophobic residues followed by a large extracellular hydrophilic domain (113 amino acids) and a C-terminal hydrophilic cytoplasmic tail (53 amino acids). Amino acid sequence comparison of this clone with other members of the secretin/VIP/glucagon receptor family showed that VR7 shares the highest sequence identities with frog (36) and human (37) VPAC2-Rs (55.8% and 53.9%, respectively). This is consistent with the phylogenetic studies, which indicate that the goldfish PHI/PHV receptor is grouped with other VPAC2-Rs to form a subbranch within the phylogenetic tree of the secretin/glucagon receptor family (data not shown). There are seven conserved cysteine residues (positions 55, 64, 78, 96, 158, 213, and 389), compared with VPAC1, VPAC2, PAC1, GHRH, and secretin receptors, and three additional conserved cysteine residues (positions 40, 151, and 282), compared only with goldfish VPAC1, PAC1, and frog VPAC2 receptors. Three putative N-linked glycosylation sites (Asn-X-Ser/Thr, where X is any amino acid except proline) were identified in the N-terminal extracellular domain (positions 61 and 95) and the first exoloop (position 201). The consensus signature (FQGBBVAXBY CFXNXEVQ, where X and B represent any or hydrophobic amino acid residue, respectively) for the secretin receptor family is located at the seventh TMD (position 379–396) with a glutamine to lysine substitution at the last position. Interestingly, the first exoloop of the goldfish PHI receptor contains 34 amino acid residues and it is unusually long, compared with other members of the family (21–23 residues).



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  		Figure 1. Comparison of amino acid sequences of goldfish PHI receptor (top) with goldfish PAC1 (33 ), goldfish VPAC1 (32 ), and frog VPAC2 (36 ) receptors. The gfPHI receptor cDNA is 3200 bp in length and contains a single opening reading frame (1338 bp, from nucleotides 121 to 1458), which encodes a 446-amino-acid-long polypeptide. The seven TMDs are labeled with horizontal arrows. The amino acids that are identical or conserved among these receptors are boxed. The conserved cysteine residues are labeled (*).

 
Isolation of goldfish prepro-PHI/VIP cDNA
Initial functional studies of the cloned VR7 showed that it was responsive to human PHV but not fish PACAP and VIP (see below), indicating it was probably a goldfish PHI/PHV receptor. To use homologous peptides to study the characteristics of this putative PHI/PHV receptor, the goldfish prepro-PHI/VIP cDNA was isolated by RACE. The degenerate primers for RACE were designed according to the most conserved amino acid sequences of vertebrate VIPs. Using goldfish intestinal cDNAs as templates, two cDNA fragments sized 480 and 348 bp were isolated. Two primers, VIPF and VIPR designed on the basis of nucleotide sequences of the RACE products, were used for PCR to obtain a cDNA fragment of 637 bp from a goldfish brain and pituitary cDNA library (34). The DNA sequences of the PCR fragment, which shared a high level of homology with the human prepro-PHI/VIP, were used as a probe to screen the same library. Three putative cDNA clones designated 2A, 11A, and 13A were obtained and were found to contain inserts of different sizes: 815 bp (2A), 831 bp (11A), and 701 bp (13A). Clone 2A contained a poly(A)+ tail with two potential polyadenylation signals, AATAAA. These signals are located 154 bp and 21 bp upstream to the poly(A)+ tail, respectively. This clone contains an open reading frame (from nucleotide 68 to 520) coding a 151-amino-acid-long protein with a predicted molecular mass of 17,167 (Fig. 2Go). The first 30 amino acid residues of the precursor protein are predicted to be the signal peptide by virtue of their hydrophobic nature. The amino acid sequences between residues 120 and 147 are identical with the amino acid sequence of goldfish VIP reported previously (38), and the sequences between 76 and 102 are highly homologous to human PHM (78% sequence identity). The goldfish VIP and PHI are flanked by potential proteolytic cleavage sites. The C terminus of the goldfish VIP is followed by a carboxyl-terminal amidation signal motif, i.e. a glycine residue followed by two basic amino acid residues (39), indicating that the goldfish VIP is amidated at the C terminus. However, similar to the case in chicken (40), the C terminus of the goldfish PHI is not amidated because it lacks a glycine residue to act as an amide donor.



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  		Figure 2. Comparison of the nucleotide and deduced amino acid sequences of the clones (2A, 11A, and 13A) coding for goldfish preproPHI/VIP. The predicted amino acid sequences of 2A are shown above the cDNA sequences. Nucleotide residues are numbered in the 5' to 3' direction, and amino acid residues are numbered from the methionine initiation residue. Putative PHI and VIP sequences are underlined and the C-terminal residues of the putative PHV are labeled with an asterisk (*). Amino acid substitutions among the two clones are shaded. The potential polyadenylation signals (AATAAA) for clone 2A (638–643 and 771–776) are underlined. The splicing consensus "AG" flanking the VIP coding exon is typed in bold and shaded. Dashes are gaps that were introduced to yield maximal alignment. The positions of the primers used in RT-PCR are indicated in the figure.

 
Clones 11A and 13A do not possess the consensus polyadenylation signal (AATAAA), although poly(A)+ tails were found in these clones. Clone 11A is highly homologous to 2A; they share 80.1% and 88.7% DNA and amino acid sequence identities, respectively (Fig. 2Go). The amino acid substitutions of these two precursors are essentially located in the signal peptide and the N-terminal flanking peptide. Interestingly, the nucleotide sequences of clone 13A is almost identical with those of 11A except that there is a deletion of 132 bp in the former. Translation of this clone leads to the synthesis of prepro-PHI with the VIP encoding region (44 amino acids) being deleted. It is highly possible that 13A is a splice variant of 11A that contains only the goldfish PHI but not VIP. The predicted amino acid sequences of goldfish PHIs from clones 2A and 11A revealed that there are two amino acid substitutions among these peptides. The C-terminal residues of the two PHI homologues are alanine (11A) and threonine (2A), respectively. The occurrence of two distinct types of PHI/VIP transcripts is consistent with the fact that goldfish has a tetraploid genome. The VIP C-terminal flanking peptide (prepro-PHI/VIP 156–170 in human) was absent (40, 41, 42, 43) in goldfish, there were stop codons immediately behind the amidation, and cleavage-processing signals (GKRR in 2A or GKKR in 11A). Fig. 3Go shows the amino acid comparison of the PHVs in goldfish and other vertebrates.



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  		Figure 3. Amino acid sequence alignment of PHI/PHV in vertebrates. Shaded areas represent identical amino acid residues. The sequences of rat, mouse, human, chicken, and turkey PHI/PHVs were adapted from Refs. 55 , 42 , 46 , 40 , and 43 , respectively. The C-terminal residue of PHI was labeled with an asterisk (*).

 
Functional expression of VR7 in transfected CHO cells
To identify the ligand(s) for this novel receptor, the cDNA clone was stably transfected into CHO cells, and cAMP responses in the presence of various peptides were measured. Pilot experiments showed that PACAP27 (mammalian/salmon sequence), carp GHRH, and goldfish GHRHs had no significant effect on cAMP formation. The cAMP responses to a series of other peptides including human PHI and PHV, human and goldfish PACAP38, cod VIP, helodermin, human secretin, VIP, PACAP38, PHM, and PHV are shown in Fig. 4Go. Cod VIP was used in this study because of the structural similarities of goldfish and cod VIP, i.e. only one conservative amino acid substitution at position 13 (Phe replacing Tyr). It was found that only human PHV was able to activate this receptor in a dose-dependent manner. The 50% effective concentration (EC50) values of this peptide in stimulating cAMP production were 93 ± 40 nM. PHM and helodermin could activate the receptor with lower potencies. Although the receptor showed high degrees of sequence homologies with other vertebrate VPAC2-Rs, functional studies indicated that it is not a goldfish VPAC2-R: VIP (cod and human) and PACAP38 (human and goldfish) were weak agonists of the receptor and could augment cAMP synthesis only at micromolar concentrations. Secretin, generally used as a pharmacological agent to distinguish mammalian VPAC1-R and VPAC2-R, was unable to activate this receptor. The fact that the receptor could be stimulated by human PHV prompted us to clone the PHI/VIP precursor and then synthesize goldfish PHI and PHV. These peptides exhibited nearly the same potencies as human PHV and the EC50 values of these peptides in stimulating cAMP production were 93 ± 40 nM and 42.6 ± 16 nM, respectively.



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  		Figure 4. Stimulation of cAMP production in goldfish PHI receptor-transfected CHO cells. The stably transfected CHO cells were incubated with various concentrations of peptides including goldfish PHT (ortholog of mammalian PHI; C-terminal residue is threonine), goldfish PHV, PACAP38, cod VIP, helodermin, human PHM and PHV, VIP, PACAP38, and secretin. PHI is the generic abbreviation used to designate mammalian PHI and PHM as well as goldfish peptide histidine alanine (PHA) and peptide histidine threonine (PHT). Cyclic AMP levels are expressed as fold stimulation, compared with that of the control (no peptide). Data presented here were from six independent peptide stimulations, and the values are the means ± SD. The average values of basal cAMP were 2.0 ± 0.2 pmol/well.

 
Tissue distribution of goldfish prepro-VIP and PHI/PHV receptor mRNAs
By Northern blot analysis, the tissue distribution of PHI/VIP transcripts was investigated (Fig. 5DGo). Strong hybridization signal (1.1 kb) was detected in the intestine, but weaker signals were found in the brain and gall bladder. With a more sensitive approach using RT-PCR coupled with Southern blotting (Fig. 5Go, A and B), consistently, PHI/VIP transcripts were found to be abundant in the intestine, brain, and gall bladder. In addition, lower levels of expression were also observed in the heart and ovary. All the tissues examined expressed ß-actin control mRNA in about the same levels (Fig. 5CGo), indicating the integrity of the first strand cDNAs used in this study.



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  		Figure 5. Tissue distribution studies of goldfish prepro-PHI/VIP by (A) RT-PCR, (B) Southern blot, and (D) Northern blot analyses. The probe used for (B) and (D) was a PCR fragment (617 bp, using the same primers VIPF and VIPR) previously subcloned and analyzed by DNA sequencing. Amplification of goldfish ß-actin was used as a control (C) to indicate the quality of the first-strand cDNAs used in the RT-PCR. For the Northern blot analysis, 4 µg poly(A)+ RNA prepared from various tissues were used. Lane 1, Brain; lane 2, pituitary; lane 3, gall bladder; lane 4, male gonad; lane 5, female gonad; lane 6, intestine; lane 7, heart; and lane 8, liver.

 
The expression levels of PHI/PHV receptor mRNA in the brain and pituitary of goldfish was examined by Northern blot analysis. High levels of PHI receptor transcripts (3.2 kb) were detected in the pituitary, but low levels were found in the brain (Fig. 6Go). This observation suggests a physiological role of goldfish PHI in regulating pituitary functions. To investigate the seasonal changes of PHI receptor, a Northern blot analysis was performed using sexually mature and regressed fish. After normalization of the hybridization signals with actin mRNA, little seasonal variations in the expression of PHI receptor corrected for the actin mRNA signal were observed in goldfish brain and pituitary (Fig. 6Go). By RT-PCR coupled with Southern blot analysis, PHI receptor mRNA was found to be abundant in the pituitary, gall bladder, and intestine (data not shown), an observation that was consistent with the tissue-specific expression of the prepro-PHI/VIP. In addition, lower levels of expression were detected in liver, brain, male gonad, and heart.



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  		Figure 6. Northern blot analysis of goldfish PHI/PHV receptor in the brain and pituitary of sexually mature (A) and regressed (B) goldfish. About 3 µg poly(A)+ RNA was loaded in each lane. The probe used in this study was the full-length cDNA (3.2 kb) for the goldfish PHI receptor. Transcripts of 3.2 kb were detected at a high level in the pituitary and at a low level in the brain. The blot was rehybridized with ß-actin cDNA fragment as a control.

 
To investigate the possible functions of VIP and PHI in fish, we studied the distribution of goldfish PHI/VIP, VPAC1-R, PAC1-R, and PHI receptor in eight different regions of the brain (Fig. 7Go). The PHI/VIP transcripts were detected in the posterior part of the nervous system including medullar and spinal cord after 28 cycles of PCR. When we increased the PCR amplification cycles to 35, PHI/VIP transcripts were also found in the hypothalamus, optic tectum-thalamus, and olfactory bulbs/tracts (data not shown). VPAC1-R was present at higher levels in the anterior parts (olfactory bulbs-tracts, telencephalon, hypothalamus, and optic tectum-thalamus) and, interestingly, the pituitary showed a much lower level of expression. The expression pattern of PAC1-R was similar to that of VPAC1-R. The transcripts of the PHI/PHV receptor were again found in all parts of the brain, but the receptor was most abundant in the pituitary. These findings strongly suggest that PHI and/or PHV are important in regulating pituitary functions in goldfish.



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  		Figure 7. Distribution of goldfish prepro-PHI/VIP, VPAC1-R, PAC1-R, and PHI/PHV receptor transcripts in eight different regions of the goldfish brain by a combined RT-PCR and Southern blot approach. The PCR and hybridization conditions as well as probe preparation for VPAC1-R and PAC1-R were essentially the same as previously described (32 33 ). The probes for prepro-PHI/VIP and PHI/PHV receptor are described in Figs. 5Go and 6Go, respectively. The PCR product for the PHI/PHV receptor from the pituitary was subcloned and sequenced to confirm its identity. In each sample, a 200-bp DNA fragment specific for the goldfish ß-actin was amplified as a control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Structural characterization of goldfish prepro-VIP/PHI and PHI/PHV receptor mRNA
The primary structure of goldfish VIP has been previously determined (38), and it was found that there are six amino acid substitutions between the sequences of goldfish and human VIPs. The present study now reveals that the primary structures of PHI-related peptides has also been reasonably conserved from fish to mammals with only five and six amino acid substitutions between the sequences of goldfish PHIs and human PHM. Interestingly, the structures of goldfish PHIs appear to be more closely related to those of mammalian PHI/PHM (74–78% identity) than those of avian PHI (59% identity). In chicken and turkey, the major form of transcripts is prepro-VIP (98%), which lacks the PHI-encoding region (40, 43). A longer transcript, prepro-PHI/VIP, also exists but at much lower levels (about 2%). It is thus possible that avian PHIs do not exert any important function, and hence mutations of the PHI sequences were tolerated during avian evolution. Because of that, in the past, it was generally accepted that PHI was not conserved and was unlikely to be important in nonmammalian vertebrates. The observation that goldfish PHIs exhibit high-sequence similarity with mammalian PHI/PHM reveals that this view was not correct and that PHI-related peptides may also play important physiological roles.

The present report provides the first evidence for a prepro-PHI transcript that lacks the exon encoding for VIP. Studies on the structure and organization of prepro-PHI/VIP genes in mammals showed that VIP and PHI are encoded by two distinct exons (41, 42). The arrangement of the splice junctions of PHI and VIP exons allows the reading frame of the remaining coding sequences to be unchanged after either one of the exons is differentially removed. As yet, there is no evidence for the presence of these processing mechanisms in mammals. In contrast, differential splicing occurs in chicken and turkey, in which the majority of the PHI exon is spliced away from the transcript (40, 43). In this study, a novel splice variant (clone 13A) was isolated from goldfish, which revealed an in-frame 132-bp deletion resulting in the loss of the VIP-encoding exon containing also part of the bridging peptide (Fig. 2Go). The exon part of the 5' donor junctions obeyed the consensus sequences (C/AAG gt) of splicing (44); the sequences of both sites flanking the VIP encoding exons are "AG" (Fig. 2Go). Using Northern blot analysis, we were unable to detect transcripts of smaller size, suggesting that alternative splicing of the PHI/VIP mRNA is present at relatively low levels in goldfish.

By degenerate PCR and cDNA library screening techniques, we isolated a putative G protein-coupled receptor that shared the highest amino acid identities with the frog [56% (36)] and human [54%, (37)] VPAC2-Rs. Phylogenetic analysis also grouped this receptor with the VPAC2-R to form a distinct subbranch within the receptor superfamily (data not shown). We also used secretin and helodermin as pharmacological agents to distinguish VPAC1 and VPAC2 receptors (45). It was found that, at a 0.1-µM concentration, helodermin could increase cAMP production, but secretin could not. These data suggested that the novel receptor is pharmacologically more related to VPAC2-R. However, all the ligands for VPAC2-R, including human and goldfish PACAP38 and human and cod VIP, were unable to activate the receptor significantly at a concentration of 1 µM (Fig. 4Go). Instead of being a VIP/PACAP receptor, functionally, this receptor is specific for goldfish PHI and PHV. The relatively high EC50 values of goldfish PHI and PHV are indeed similar to those obtained in the mammalian studies (46).

Another interesting aspect of the goldfish PHI receptor is that human PHV exhibited higher potency and maximal activity than PHM. This observation suggested that the importance of PHV has been overlooked in the past. In fact, PHV is the predominant form of PHI in human plasma (63%) (8). The half-life of PHV (36 and 37 min), reported by Bloom et al. (47) and Gill et al. (48), respectively, are much longer than that of PHM (3.0 and 6.5 min) and VIP (0.6 and 0.8 min). PHV is also the most abundant circulating prepro-PHI/VIP-derived product in patients with VIP-producing tumors, and it appears to play a greater role than VIP in causing the watery diarrhea syndrome (49). Other than in the circulation, PHV is also found in high concentrations in tissues including the vagina and stomach (5, 8). It is more potent than PHM and VIP in reducing both the force and frequency of spontaneous contractions of isolated rat uterus and relaxing bronchial smooth muscle in guinea pig (46). Taken together, it is possible that the PHI/PHM specific activities are mediated by PHV receptors, and this could be one of the reasons for the lack of PHI/PHM receptor information in mammals in the past.

In summary, we have functionally characterized the first PHI/PHV receptor from goldfish. The discovery of this receptor provides answers to some of the controversial issues related to the PHI/VIP system. First, the isolation of PHI receptor confirms the existence of PHI-binding sites (25, 26, 27, 28, 29, 30). Second, a PHI receptor explains the PHI-specific effects detected in several experimental systems (15, 22, 30). Finally, it also explains the differential desensitization of VIP and PHI (29) as well as the lack of effect of VIP antagonists on PHI-specific activities in some systems (23, 24).

Tissue distribution of goldfish prepro-VIP and PHI/PHV receptor mRNAs
The study of tissue distribution is a key step toward the understanding of the functions of the gene products. The prepro-PHI/VIP transcripts were abundant in the gastrointestinal system (intestine, gall bladder) and brain indicating that, in the goldfish, VIP and PHI are brain-gut peptides. The pattern of distribution of PHI/VIP precursors is similar to that reported in humans (50). In the central nervous system, VIP and PHI stimulate somatostatin release (10) and increase melatonin synthesis in the rat pineal (12). In fish, VIP inhibits gastric acid secretion and reduces gastric mucosal blood flow in cod (51). The presence of the transcripts in the heart and female gonad suggests VIP and/or PHI/PHV may play roles in the vascular and reproductive systems similar to the mammalian models. For the PHI/PHV receptor, a widespread distribution of the receptor transcripts was found in peripheral tissues, and this observation is similar to that of rat VPAC2-R (52).

To obtain more information about the functions of VIP and PHI/PHV in the brain, the expression patterns of the PHI/VIP precursor and their receptors (PHI receptor, VPAC1-R and PAC1-R) were studied in different regions of the goldfish brain. High levels of PHI/VIP precursor expression were detected in the medulla and spinal cord, and receptors for VIP and PHI/PHV were found in all regions of the brain. This suggests that although PHI/VIP cell bodies are mostly present in lower brain regions, these neurons project their fibers to various regions of the brain. The expression of PHI/VIP precursors in the hypothalamus is unexpectedly low because PHI and VIP are presumably hypothalamic-releasing factors. A possible explanation is that there are only a small number of cell bodies expressing PHI/VIP in the diencephalon, and these neurons are highly diffused or only localized to discrete areas of the hypothalamus.

In contrast to goldfish VPAC1-R and PAC1-R, transcripts of goldfish PHI/PHV receptor were detected in much higher levels in the pituitary (Fig. 7Go), compared with various brain regions. This observation strongly suggests that PHI/PHV is essential to the control of pituitary functions, consistent with ideas that PHM is a hypophysiotropic factor regulating anterior pituitary hormone secretion (53). Previously, we have demonstrated that PACAP analogs interact with PAC1-R to regulate GH release in goldfish (33). Furthermore, nerve fibers with PACAP immunoreactivity are found in close proximity with somatotropes. These findings support the idea that, similar to mammals, peptides from the VIP/PACAP family also function as hypophysiotropic factors in fish. However, in our preliminary studies, goldfish PHI, cod VIP, and human VIP (10 pM–1 µM) were not effective in altering basal GH and Gonadotropin II release from goldfish pituitary cells (unpublished data). Previous evidence indicates the presence of different levels of PHI, PHV, and VIP immunoreactivities in the anterior pituitary of female and male rats (2, 54). It is possible that PHI/PHV in goldfish is also involved in sexual dimorphism and/or reproductive functions. Although we were unable to detect seasonal changes of PHI/PHV receptor expression (Fig. 6Go) or variations in transcript levels of PHI/VIP, PHI/PHV receptor, VPAC1-R, and PAC1-R in male and female goldfish brains (eight regions of goldfish brain by RT-PCR, data not shown), we cannot exclude the possibility that regulation of these proteins occurs at the posttranscriptional level by differential processing in male and female goldfish.

In summary, in the periphery, the expression patterns of peptide ligand(s) derived from goldfish PHI/VIP transcripts overlap with that of their receptors including PHI/PHV receptor and VPAC1-R. The presence of PHI/PHV receptor and VPAC1-R in all regions of the brain further substantiates the functions of PHI/PHV and VIP as neuromodulator in goldfish brain and pituitary. The high levels of PHI/PHV receptor expression in the pituitary strongly suggest a functional role of goldfish PHI/PHV in regulating pituitary functions.


   Acknowledgments
 
The authors would like to thank Dr. Svetlana Mosjov from the Laboratory of Cellular Physiology and Immunology, Rockefeller University, for her critical comments and useful discussions.


   Footnotes
 
This work was supported by the Hong Kong government Research Grants Council Grant HKU7181/99M (to B.K.C.C.), INSERM Grant U 413 (to H.V.), and a Procore exchange program (F-HK 35/01T) (to B.K.C.C. and H.V.).

Abbreviations: CHO, Chinese hamster ovary; EC50, 50% effective concentration; PACAP, pituitary adenylate cyclase-activating polypeptide; PHI, peptide histidine isoleucine; PHM, peptide histidine methionine; PHV, peptide histidine valine; RACE, rapid amplification of cDNA ends; TMD, transmembrane domain; VIP, vasoactive intestinal polypeptide.

Received September 7, 2001.

Accepted for publication December 24, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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