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Department of Psychiatry, Mental Retardation Research Center, University of California at Los Angeles, Los Angeles, California 90024-1759
Address all correspondence and requests for reprints to: James A. Waschek, 68–225 NPI, Department of Psychiatry, University of California at Los Angeles, 760 Westwood Plaza, Los Angeles, California 90024. E-mail: jwaschek{at}mednet.ucla.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unlike VPAC1 and VPAC2 receptor messenger RNAs (mRNAs), which are widely distributed in the mammalian brain and peripheral tissues (3, 7, 12, 13), PAC1 receptor mRNA is located predominantly in the brain (14). Because the molecular structures and characteristics of PACAP and PAC1 receptors are similar in mammals and nonmammals, many functions of the PACAP ligand/receptor system (15), particularly the regulation of various neuroendocrine functions, might be conserved in the lower vertebrate species. For example, early studies in the isolated frog distal lobe pituitary showed that PACAP ligand/receptor system stimulated intracellular cAMP accumulation (16) and enhanced the secretion of GH and PRL (17). The mechanism of PACAP action on these cells involved calcium mobilization or stimulation of intracellular cAMP accumulation (16, 17). In peripheral tissues, the PACAP ligand/receptor system was reported to promote corticosteroid secretion in the adrenal gland cells of the frog (18). Based on the wide distribution of PACAP in the frog brain (19), it seems likely that PACAP ligand/receptor system executes many other functions in the frog brain, although the frog PAC1 receptor has not been cloned and its distribution in the brain is still unknown.
Recently, we identified a Xenopus PACAP ligand complementary DNA (cDNA) and reported its mRNA distribution in the brain (Hu, Z., V. Lelievre, A. Chao, X. Zhou, J. A. Waschek, manuscript submitted). To further address functional significance of PACAP ligand/receptor system in the frog brain, we sought to determine the molecular structure and brain distribution of Xenopus PAC1 receptor mRNA. To identify the receptor, we screened a Xenopus laevis brain cDNA library using a homology-based approach with a rat PAC1 receptor cDNA probe. We isolated a full-length cDNA clone that encodes a protein that shows high homology with human PAC1receptor. When expressed in Xenopus oocytes, the receptor selectively bound PACAP with high affinity. This is consistent with the pharmacological characteristics of PAC1 receptor. We then examined the distribution of PAC1 receptor mRNA in the Xenopus brain by in situ hybridization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-ZAPII frog brain
cDNA library (2.5 x 106 independent clones)
was prepared using both oligo(dT) and random primers
(Stratagene, La Jolla, CA). Library phage DNA was
transferred to the nylon membranes (Amersham Pharmacia Biotech, Piscataway, NJ), exposed to denaturing buffer
(0.5 M NaOH/1.5 M NaCl),
washed in 1.5 M NaCl/1.0 M
Tris-HCl, and soaked in 2 x SSC (1 x SSC = 0.15
M NaCl/0.15 M sodium
citrate) for 5 min each. DNA was fixed on the membranes by baking the
membranes at 80 C for 4 h. A subset of library (7 x
105 plaques) was screened with a
32P-labeled, randomly primed probe corresponding
to the complete coding region of the rat PAC1
receptor cDNA. (This cDNA was kindly supplied by Dr. Wank, NIH, Ref.
5). Overnight hybridization at 35 C was carried out in 50% formamide,
5 x SSC, 50 mM phosphate buffer (pH 6.5),
5 x Denhardt’s solution (0.1% BSA, 0.1% Ficoll, and 0.1%
polyvinylpyrrolidone), 0.1% SDS, and 200 µg/ml salmon sperm DNA. The
membranes were initially washed under the condition of low stringency
(3 x 15 min wash at 35 C with 2 x SSC/0.2% SDS). Filters
were exposed, and then washed again, but with higher stringency
condition (3 x 15 min wash at 52 C with 0.1 x SSC/0.2%
SDS). One cDNA clone that hybridized only at low stringency and not at
high stringency was plaque-purified from the library, essentially
according to manufacturer’s protocol (Stratagene).
Plasmid DNA (PZL1) prepared from this positive plaque was sequenced
initially with vector-specific primers, and subsequently with the
primers corresponding to newly-sequenced portions of the cDNA.
2. Binding of 125I-labeled PACAP-27 to Xenopus oocytes
injected with in vitro synthesized PAC1 receptor mRNA
The plasmid DNA (PZL1) containing 3.3-kb complete coding
sequence of PAC1 was linearized using
XbaI endonuclease. Capped PAC1
receptor mRNA (C-mRNA) was synthesized using T7 RNA polymerase and an
in vitro transcription kit (mCAP RNA capping Kit,
Stratagene). C-mRNA was dissolved in DEPC-treated
H2O at a concentration of approximately 200 ng/µl and the
size checked on a 1.5% agarose-formaldehyde gel.
Xenopus oocytes were enzymatically defolliculated and
injected with 2.5–5.0 ng C-mRNA or an equal volume of vehicle. After
injection, the oocytes were maintained at 18 C for 5 days to allow the
expression of injected mRNA. Pharmacological experiments were performed
on intact oocytes using a modification of a procedure previously
described (11). Pharmacological parameters (Kd
and Bmax) of the expressed receptors on intact
oocytes were determined using saturation experiments. Oocytes (8/group,
n = 3) were incubated at 4 C for 2 h with increasing
concentrations (from 0.5f M to 0.2
nM) of 125I-PACAP-27 (2,200
ci/mmol, NEN Life Science Products) in the presence
(nonspecific binding) or absence (total binding) of 1
µM native rat PACAP-38. After two washes at 4 C
with 2 ml of SOS buffer (100 mM NaCl, 2
mM KCl, 1.8 mM
CaCl2, 1 mM
MgCl2, 5 mM HEPES, pH 7.0)
containing BSA at 1 mg/ml, the radioactivity bound on the oocytes was
measured in a 
counter (AutoGamma, EG\|[amp ]\|G Wallac, Turku,
Finland). Selectivity of the examined receptors was assayed by
displacement of the binding of 125I-PACAP-27 by
various native analogues. Injected and uninjected oocytes (8/group,
n = 3), were suspended in 1 ml of SOS with BSA at 1 mg/ml and
incubated for 2 h at 4 C with 35,000 cpm of
125I-PACAP-27 (2200 Ci/mmol) in the presence of
increasing concentrations (from 1 pM to 1
µM) of native analogs, PACAP-27, PACAP-38, or
VIP. Oocytes were washed twice at 4 C with 2 ml of SOS containing BSA
at 1 mg/ml. The amount of radioactivity on oocytes was then determined
with a counter.
3. Northern blot analysis of the tissue distribution of
PAC1 receptor mRNA
Total RNA was isolated from brain, lung, muscle,
intestine, and liver with the use of TriZOL reagent (Life Technologies, Inc.). Total RNA (25 µg) of each tissue was
fractionated on a 1.5% agarose-formaldehyde gel, blotted to the nylon
membrane (Magna, Micron Separations, Inc., Westboro, MA), and
fixed on the membrane by baking at 80 C for 2 h. The detailed
procedure for Northern hybridization had been described elsewhere (11).
Briefly, after overnight prehybridization, the membrane was hybridized
with the cloned 3.3 kb PAC1 receptor cDNA (25 ng)
that was radiolabeled to a specific activity (3
x 108 cpm/µg) by random
primer labeling (Life Technologies, Inc.). The
32P-labeled probe was added to the hybridization
solution (1% SDS, 1 M NaCl, 10% dextran sulfate, and 50%
formamide) with salmon sperm DNA, and overnight hybridization was
performed at 42 C. The blot was washed in 2 x
SSC/0.2% SDS and 0.2 x SCC/0.2% SDS (each for
3 x 15 min at 55 C), and exposed for 16 h
in a PhosphorImager (Molecular Dynamics).
4. Preparation of a 33P-labeled frog PAC1
receptor riboprobe
To increase the selectivity of the PAC1
receptor riboprobe for in situ hybridization studies, PCR
was employed to amplify a 300 bp cDNA fragments from PZL1. The forward
and antisense primers were 5'-GGTTGGT CTCCAGAGAAC-3' and
5'-CCGTAGAATGAATGAGAC-3', respectively, and generated a fragment
corresponding to the nucleotide sequence 589–888 (shown in Fig. 1
). This region was chosen because an
alignment of the nucleotide sequence with the published sequences of
PACAP related receptors showed that this region is not homologous to
any of published sequences except PAC1 receptor.
After an initial 2 min denaturation at 94 C, 30 PCR thermal cycles were
performed consisting of 30 sec at 94 C, 30 sec at 50 C, 30 sec at 72 C.
PCR products were checked by size in 1.5% agarose gel and then
transferred to nylon membrane. An internal oligonucleotide
hybridization probe, 5'-CCAGACCAGGATACATAC-3' (corresponding to
721–739 in Fig. 1), was end-labeled with
[-32P]ATP and hybridized overnight with the
membrane at 37 C to verify the PCR products. The resulting 300-bp DNA
fragment was ligated into PCR II TA-vector (Invitrogen)
and sequenced to confirm identity. 33P-labeled
antisense and sense probes were made by digesting the plasmid
containing the 300 bp PAC1 receptor cDNA with
Xho and BamHI, and using SP6 and T7 RNA
polymerase, respectively.
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). The open reading frame (ORF)
of 1398 bp encoded a 466-amino acid protein with a calculated molecular
mass of 53 kDa. The sequence allows for four potential
N-linked glycosylation consensus sites (-Asn-X-Ser/Thr-) in the N
terminus (Fig. 2) at
Asn47,59,115,137, similar to human
PAC1 receptor and frog VPAC receptor (5, 22). Ten
conserved cysteine residues are present in the extracellular domains of
frog PAC1 receptor, seven of which are located in
the N-terminal segment, two in the first extracellular loop, and one in
the second extracellular loop (Fig. 2). A hydropathy plot of the
deduced amino acid sequence showed eight regions of hydrophobic
residues, seven corresponding to putative transmembrane domains (data
not shown). The other region, composed of 20 N-terminal amino acids, is
a putative signal peptide (Fig. 1).
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). This
showed that the Xenopus PAC1 receptor
had homology with the human PAC1 receptor devoid
of hip/hop cassettes (null receptor, 74% identity), goldfish
PAC1 (71%), rat PAC1
(74%), frog VPAC (48%), and rat VPAC1 (48%),
VPAC2 (48%), secretin (46%), PTH-PTHrP (39%),
and porcine calcitonin (29% identity) receptors. The greatest degree
of homology is in the transmembrane domains and the lowest homology is
in the N-extracellular terminus, the latter with 55% identity to human
PAC1 receptor, 32% identity to goldfish
PAC1 receptor, and 21% identity to frog VPAC
receptor.
Partial comparison of the extracellular N terminus of
PACAP-VIP-secretin-glucagon receptor family indicated that this cloned
cDNA, like the mammalian PAC1 receptor, encodes a
21-amino acid insertion (D87-EMDFV
DRSLGWSPENIEEQQS-E109) unique to receptor
PAC1 between the third
(Cys76) and fourth (Cys116)
conserved cysteine (Fig. 2). This 21-amino acid domain is poorly
conserved across PAC1 receptors in different
species and is absent in other members of the VIP-secretin-glucagon
receptor family as well as in one splice variant of mouse
PAC1 receptor (23). The amino acid sequence of
the third intracellular loop (between V and VI transmembrane domains)
showed 100% identity with the goldfish PAC1
receptor and has only one amino acid difference from the human
PAC1 receptor (at position of 334 in Fig. 2).
2. Pharmacological characteristics of functional expressed
PAC1 receptor cDNA
Pharmacological studies showed that injection of synthesized
mRNA of cloned Xenopus PAC1 receptor
cDNA resulted in the robust expression of receptors on intact
Xenopus oocytes. Analyses of the data in the saturation
experiment revealed a single class of binding sites with an average
Kd of 40.8 ± 3.4 pM
and a Bmax of about 3.7 x
107 receptors per oocyte (Fig. 3A). Displacement of
[125I]-PACAP-27 was performed using increasing
concentrations of native peptides, PACAP-27, PACAP-38, and VIP (Fig. 3B). PACAP-38 and PACAP-27 showed slightly different capacity to
displace binding sites of [125I]-PACAP-27. The
IC50 for PACAP-38 and PACAP-27 were 27 ± 0.14
pM and 110 ± 9.01 pM,
respectively. VIP did not displace
[125I]-PACAP-27 binding, except at high
concentrations of 1 µM
(IC50 > 1 µM).
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), similar to that in
mammals (4, 5, 9). We then used in situ hybridization to
study the distribution of PAC1 receptor mRNA in
the brain. The PAC1 receptor gene had a clear
region-specific pattern of expression in the brain. Figures 5, 6, and 7 illustrate the main hybridized
structures from forebrain to medulla.
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, A–B). Throughout the anterior/posterior axis of the pallium
(corresponding to the mammalian hippocampus),
PAC1 receptor mRNA signals were dense and fairly
uniformly distributed (Fig. 5, C–F). Other regions in telencephalon,
such as the nuclei of medial septum and accumben septum only had weak
to moderate mRNA signals (Fig. 5, C–D). In the anterior diencephalon,
PAC1 receptor mRNA was strongly observed in the
hypothalamic preoptic area (Fig. 5, E–F). In frog, this area includes
both the hypothalamic paraventricular and supraoptic nuclei. On the
same sections, silver grains were weakly to moderately localized in the
medial amygdala. In the posterior diencephalon, mRNA signals were still
heavily localized in the hypothalamic preoptic area. Obvious
hybridization signals were also observed in the infundibular nucleus
and moderate mRNA signals were seen in the dorsal habenular nuclei
(Fig. 5, G–H). Along with the rostral/caudal axis of the thalamus,
PAC1 receptor mRNA was expressed at low levels in
the nuclei of posterocentral and ventrolateral thalamus (Fig. 5, G–H).
In the anterior mesencephalon, the PAC1 receptor
mRNA was weakly detected in the nucleus of oculomotor nerve and
moderate in the nuclei of posterior thalamus (Fig. 6, A–B). Weak
PAC1 receptor mRNA expression was also detected
in stratum griseum superficiale tecti. At the middle/posterior
mesencephalon (Fig. 6 C–D), expression of PAC1
mRNA was strong in stratum griseum periventricular tectum, torus
semicircularis, and the nuclei of anteroventricular tegmental
mesencephalon. Conversely, the lateral portion of stratum griseum
superficial tectum, on the same sections, contained only weak
hybridization signals. On more posterior sections (Fig. 6, E–F),
strong hybridization signals were still observed in the hypothalamic
preoptic areas. PAC1 receptor mRNA signals were
only moderately present over the distal lobe of the pituitary (Fig. 6, E–F). The posterior and intermediate pituitary lobes totally lacked
hybridized signals. At the same levels, higher levels of
PAC1 receptor gene expression were observed in
the nucleus of posteroventricular tegmental mesencephali and stratum
griseum central tecti (Fig. 6, E–F). Moderately intense
PAC1 mRNA signals were identified in the Purkinje
cell layer of the cerebellum (Fig. 6, G–H). In the medulla, high
abundance of PAC1 mRNA was detected in the
nucleus of cerebellum and the griseum centrale rhombencephali (Fig. 6, G–H). On the same sections, moderate PAC1 gene
expression was also found in the motor nucleus of trigeminal nerve.
High power photomicrographs in Fig. 7 show hybridization signals with
antisense (A–D) and sense (E and F) probes in representative brain
areas.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Structure-activity investigations have shown that the extracellular N terminus and the first transmembrane domains can confer relatively high affinity PACAP binding (27). For the human PAC1 receptor, PACAP-38 and PACAP-27 were reported to have a fairly similar EC50 in adenylate cyclase stimulation, whereas PACAP-38 was more potent than PACAP-27 in phospholipase C (PLC) activation (28). The PACAP-38 vs. PACAP-27 specificity in humans appears to be due to the presence of the 21-amino acid domain in the N-terminal extracellular region of PAC1 receptor that impairs PLC induced by PACAP-27 (23). Based on the presence of the 21 amino acid insertion in the cloned Xenopus PAC1 receptor, we speculate that the PLC stimulation on this receptor by PACAP-27 in the frog brain will not be an important pathway of the signal transduction. We are presently addressing the detailed signal transduction pathways of the frog PAC1 receptor, including the activation of adenylate cyclase, phospholipase C, and specific ion channels.
The low conservation of the amino acid sequence in the N-terminal extracellular region of VIP/PACAP and PACAP receptors of other species suggests that relatively few residues are required to impart specific binding characteristics. Mutational studies performed on the mammalian VPAC1 receptor revealed that the presumed disulfide bonds between the cysteine residues in the extracellular N terminus contribute to the active conformation of the receptor (29, 30). Seven cysteine residues were present in the sequence for the N terminus of the Xenopus PAC1 receptor. The conservation of cysteine residues suggests that they are important in the conformation of PAC1 receptor. Four potential N-glycosylation sites were found in the N-terminal part of frog PAC1 receptor. All of these sites matched those found in VPAC1 and PAC1 receptors of other species. The first two N-glycosylation sites (Asn47,59) have been shown to be necessary for correct delivery of VPAC1 receptor to the plasma membrane (31). Conservation of these two asparagine residues in frog suggests that they are also used for the transport of the Xenopus PAC1to cell membrane.
2. The distribution of PAC1 receptor mRNA in
the frog brain
In agreement with previously reported distributions of
PAC1 receptor in the mammals (32, 33), our
northern hybridization revealed abundant expression of
PAC1 receptor mRNA in the frog brain. This is
obviously different from the recently cloned frog (Rana
ridibunda) VPAC receptor, which has features of both
VPAC1 and VPAC2 receptor,
and is distributed widely in peripheral tissues. Our in situ
hybridization showed that PAC1 receptor mRNA was
widely distributed in the frog brain. Its expression overlaps with that
of the PACAP ligand in many brain areas (unpublished data), for
example, pallium, hypothalamic preoptic area, the nucleus of
cerebellum. This suggests that numerous functions of frog
PAC1 receptor would appear to be executed in the
brain.
Our hybridization results in Xenopus partially agree with the study of Jeandel et al. (34) who examined PACAP binding sites in the frog (Rana ridibunda) brain by autoradiography. They found that PACAP specific binding sites were highest in the olfactory bulb, pallium, striatum, habenular nuclei, ventromedial thalamic nucleus, corpus geniculatum, posterior tubercle, dorsal part of the magnocellular preoptic nucleus, and the molecular cell layer of the cerebellum. Our study showed that PAC1 receptor mRNA signals were very strong in the olfactory bulb, pallium, hypothalamic preoptic areas, and the nucleus of the cerebellum. No PAC1 receptor signals were obviously present in the striatum or molecular layer of the cerebellum. Conversely, we identified moderate expression of frog PAC1-R gene in the cerebellar Purkinje cell layer. These discrepancies might be due to different frog species or differences in methodologies.
In mammals, PAC1 receptor mRNA is expressed abundantly in the olfactory bulb, hypothalamus, the anterior pituitary lobe, and hippocampus (14, 33). In the frog Xenopus brain, we found marked localization of PAC1 receptor mRNA in corresponding regions including the hypothalamic preoptic area and pallium, suggesting that functions of frog PAC1 receptor in these areas might be similar to that of mammals. Although rat PAC1 receptor mRNA was detected in the rat amygdala, bed nucleus of the stria terminalis and septum (14, 33), PAC1 receptor mRNA signals were not obviously present in these areas in the Xenopus brain. Conversely, PAC1 receptor gene expression in the cerebellar Purkinje cell layer was moderately present in the Xenopus brain, and weakly in the rat brain. These differences imply some unique functions of PACAP ligand/receptor system in the brains of various species.
The localized distribution of PAC1 receptor mRNA in the frog brain is also clearly different from that of recently cloned frog VPAC receptor (22). Unlike PAC1 receptor, VPAC receptor mRNA is expressed highly in the frog bed nucleus of the pallial commissure, lateral and medial septum (22). These results imply that effects of PACAP or VIP on these structures might be executed through the VPAC receptor. However, the VPAC receptor mRNA was detected at low levels in the pallium, suggesting that both receptors function in this area. In the distal lobe of the pituitary, moderate PAC1 receptor mRNA were detected, similar to that of VPAC receptor mRNA. Thus, both PAC1 and VPAC receptor are likely to be involved in regulation of cells in the pituitary distal lobe.
In summary, a frog (Xenopus laevis) PACAP type I receptor has been identified. This receptor has the highest degree of homology with mammalian PAC1 receptors and shows pharmacological characteristics of typicalPAC1 receptor. PAC1 receptor mRNA exhibits a region-specific pattern of expression in the frog brain. The cloning of this receptor in frog (Xenopus laevis) will provide the molecular basis for studying the structural determinants for ligand binding and the potential functions of PAC1 receptor and signaling pathways in the frog brain. Further, because the PACAP ligand/receptor system may function in mammalian neurogenesis (22), it will be of interest to determine if PACAP functions similarly in lower vertebrates.
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Acknowledgments |
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Footnotes |
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2 The first two authors made equal contributions to this work.
Received September 13, 1999.
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