From the Laboratory of Molecular Endocrinology,
Massachusetts General Hospital, Howard Hughes Medical Institute,
Harvard Medical School, Boston, Massachusetts 02114
Received for publication, October 31, 2000, and in revised form, January 23, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The expression of the paracrine signaling hormone
pituitary adenylate
cyclase-activating polypeptide
(PACAP) is regulated in a cyclical fashion during the 12-day
spermatogenic cycle of the adult rat testis. The precise functions of
PACAP in the development of germ cells are uncertain, but cycle-
and stage-specific expression may augment cAMP-regulated gene
expression in germ cells and associated Sertoli cells. Here we report
the existence of a heretofore unrecognized exon in the extracellular
domain of the PACAP type 1 receptor (PAC1R) that is alternatively
spliced during the spermatogenic cycle in the rat testis. This splice
variant encodes a full-length receptor with the insertion of an
additional 72 base pairs encoding 24 amino acids (exon 3a)
between coding exons 3 and 4. The PAC1R(3a) mRNA is preferentially
detected in seminiferous tubules and is expressed at the highest levels
in round spermatids and Sertoli cells. Analyses of ligand binding and
signaling functions in stably transfected HEK293 cells expressing the
two receptor isoforms reveals a 6-fold increase in the affinity of the
PAC1R(3a) to bind PACAP-38, and alterations in its coupling to both
cAMP and inositol phosphate signaling pathways relative to the wild
type PAC1R. These findings suggest that the extracellular region
between coding exons 3 and 6 of PAC1R may play an important role in the regulation of the relative ligand affinities and the relative coupling
to Gs (cAMP) and Gq (inositol phosphates)
signal transduction pathways during spermatogenesis.
PACAP1 is a paracrine
signaling peptide that functions as a neurotransmitter, a neurotrophic
factor, and a secretagogue for insulin, catecholamines, and pituitary
hormones (reviewed in Ref. 1). PACAP is synthesized in the form of a
precursor (proPACAP) and is processed into active peptides of either 27 (PACAP-27) or 38 (PACAP-38) amino acids, differing by an 11-amino acid
extension at the carboxyl terminus. High levels of PACAP are produced
by the germ cells of the rat testis (2-4) and are translated from a
testis-specific mRNA (5) transcribed from the PACAP gene using an
alternative promoter active in round spermatids (6, 7). PACAP regulates
gene expression in germ cells (8), increases cAMP in Sertoli cells (9),
and stimulates steroidogenesis in Leydig cells (10).
Part of the diversity in PACAP actions is due to the existence of
multiple receptors. Three distinct genes encode receptors capable of
interacting with PACAP-27 and PACAP-38 isoforms at physiological
concentrations. The PACAP-specific type 1 receptor and the PACAP
receptors type 2 and 3, which also bind vasoactive intestinal peptide (VIP), and are therefore
referred to as VIPR1 (VPAC1R) (11, 12) and VIPR2 (VPAC2R) (13, 14). The
PACAP type 1 receptor (PAC1R: identified by several laboratories and reviewed in Ref. 15) has a markedly higher affinity for PACAP and does
not function as a VIP receptor at physiological concentrations. All
three receptors are seven-transmembrane-spanning domain G protein-coupled receptors that stimulate adenylate cyclase activity. However, the PAC1R also stimulates
phospholipase C (PLC), leading to
the accumulation of inositol phosphates and diacyl glycerol. The
VPAC1R and VPAC2R may also couple to PLC in some cells (16, 17).
Multiple receptor isoforms are generated by alternate splicing of the
PAC1R mRNA. Two 81-bp exons ("hip" and "hop") are
alternatively spliced within the third intracellular loop and play a
role in modulating inositol phosphate responsiveness (18, 19).
Similarly, a variant of the receptor lacking the fourth and fifth exons
encoding sequence in the extracellular domain has been described in
hypothalamus and pituitary (20). The loss of 21 amino acids from the
extracellular ligand-binding domain results in an increased affinity
for PACAP-27. Other variants of the type I PACAP receptor have also
been described (21, 22), as well as alternative splicing of the
5'-untranslated region (23). Here we describe a novel PAC1R splice
variant highly expressed in the testis and consisting of the insertion
of a 72-bp exon encoding 24 amino acids in the extracellular
ligand-binding domain. This alternatively spliced exon substantially
alters PACAP-27 and PACAP-38 ligand binding affinities and the relative
coupling of the receptor to the cAMP-coupled and inositol
phosphate-coupled signal transduction pathways.
Oligonucleotides--
Oligonucleotides specific for the
PAC1R were (forward) PRF-7 (5'-aagcaccatggccagagtcc-3'), PRF-79
(5'-gcagcgagtggacagtggcagg-3'), PRF-70 (5'-gcatcttcaagaaggagcaagc-3'),
PRF-215 (5'-ccttgtaagctgccctgaggtct-3'), PRF-426
(5'-ggagatcaggattattactacctg-3'); (reverse) PRR-282
(5'-acctatggtttctgtcatccaga-3'), PRR-552 (5'-gcgagtgcaatgcagcttcc-3'),
PRR-1256 (5'-caccttccagctcctccatttcC-3'), PRR-1404
(5'-ctcaggtggccaagttgtcg-3'), PRR-1465
(5'-cttcctttgggaggagcccagcttcctacc-3'), DP1
(5'-ggaaacacttcttctccagcactgcaccta-3'). Oligonucleotides specific for
the follicle-stimulating hormone receptor were (forward) FRF-11 (5'-ggaatctgtggaagtttttgacg-3') and (reverse) FRR-2196
(5'-atggcctgctcttcagaagg-3'). Oligonucleotides specific for PACAP
(testis-specific isoform) were (forward) PCPTF-32
(5'-agatttccaagatacggctcaacttcatgc-3') and (reverse) PCPR-1120
(5'-cggtagctggcaactcatcg-3'). Oligonucleotides specific for SOX-17 were
(forward) SOX-17F1 (5'-atctagtgagcagcacctcc-3') and (reverse) SOX-17R1
(5'-gcttcatgcgcttcacctgc-3').
RT-PCR and Southern Blotting--
Whole cell RNA was extracted
with TriZOL reagent (Life Technologies, Inc.) in accordance with the
manufacturer's specifications. RNA, in 10 µl of H2O, was
combined with 0.5 µg of oligo(dT)16 and heated to
65 °C for 10 min, then cooled on ice. RT buffer, dNTPs (50 µM each), dithiothreitol (5 mM),
SuperScript II (Life Technologies, Inc.) enzyme (50 units), and
H2O were added for a total volume of 40 µl, and reactions
were incubated at 42 °C for 40 min. All PCRs were performed in
50-µl reactions using 2 µl of cDNA template. Reactions
contained 20 pmol each of forward and reverse primers, 0.2 mM each of dNTPs, and 2.5 units of thermostable Taq polymerase (TaKaRa Biomedical Inc., Berkeley, CA). For
nested PCR reactions, a second reaction with nested primers was carried out using 2 µl of the first reaction as template. PACAP receptor PCR
from cDNA in Figs. 1 and 3A was carried out in two
rounds with primers PRF-79 and PRR-1256 followed with primers PRF-70 and PRR-552. The first round of PCR used 10-s denaturation at 94 °C,
20-s annealing at 58 °C, and a 2-min extension at 72 °C for 20 cycles. The second round was identical except for a shorter extension
time (1 min) and 30 cycles. PAC1R(3a) PCR from cDNA in Fig.
3B was carried out using the same protocol, but with a different primer pair for the first round (PRF-79 and PRR-1465).
Cloning of the full-length PAC1R and PAC1R(3a) was done with
Pfu polymerase (Stratagene, La Jolla, CA) and the primer
pairs PRF-79 and PRR-1465, followed by PRF-7 and PRR-1404. Second round extension time was increased to 2 min, but other parameters remained as
described above. For PACAP (PCPTF-32 and PCPR-1120) and SOX-17 (SOX-17F1 and SOX-17R1): 10-s denaturation at 94 °C, 20-s annealing at 58 °C, and 1-min extension at 72 °C for 25 cycles. For
follicle-stimulating hormone receptor, FRF-11 and FRR-2196: 10-s
denaturation at 94 °C, 20-s annealing at 58 °C, and 2-min
extension at 72 °C for 30 cycles. The intron between coding exons 3 and 4 was amplified from rat genomic DNA with the primers PRF-215 and
PRR-285 and a 1:50 mixture of Pfu and standard
Taq polymerase. A "touchdown" PCR protocol was used,
with cycling parameters of 2-s denaturation at 94 °C and 3-min
extension at 72 °C for 5 cycles, followed by 2 s at 94 °C
and 3 min at 67 °C for 30 cycles.
For Southern hybridization, PCR products were transferred to MagnaGraph
membrane (Micro Separations Inc., Westboro, MA) by capillary transfer.
Hybridization with [ Cloning and Sequencing of DNAs--
Products of RT-PCR reactions
were cloned into pCR2.1 by TA cloning (Original TA cloning kit:
Invitrogen, Carlsbad, CA). To facilitate ligation, products amplified
with Pfu polymerase were purified and treated with standard
Taq polymerase for 10 min at 72 °C in the presence of
dNTPs. Sequencing was carried out with reagents from a Sequenase 2.0 kit (Amersham Pharmacia Biotech). Sequencing gels were exposed to Kodak
film, from which scanned images were generated on a computing
densitometer (Molecular Dynamics). Nucleotide sequences were
interpreted from gel images using DNA codes software (PDI Imageware
Systems, New York City).
Creation of Stable Cell Lines Expressing Recombinant PACAP
Receptor-encoding DNA--
The PAC1R and PAC1R(3a) were subcloned into
pCR 3.1 (Invitrogen). Linearized constructs were transfected into
HEK293 cells, and stably expressing cells were selected by exposure to
G418 (Life Technologies, Inc.) at 0.2-1.0 mg/ml. Cell lines used in experiments described in the legends to Figs. 4 and 5 were selected on
the basis of a similar maximal binding of 125I-PACAP-27 by
an identical number of cells. Cells were cultured in Dulbecco's
modified Eagle's medium (DMEM: Life Technologies) + 10% FCS.
Measurement of Ligand Binding Affinities--
HEK293 cells
stably expressing the receptors were harvested with trypsin and
transferred to receptor binding buffer (RBB: 138 mM NaCl,
5.6 mM KCl, 1.2 mM MgCl2, 0.4 mM CaCl2, and 10 mM HEPES, pH 7.4)
with 0.1% BSA. Approximately 20,000-50,000 cells were used per
binding reaction. For details of binding reactions, see Kieffer
et al. (24). 125I-PACAP-27 (30,000 cpm) and
competing unlabeled peptide were added followed by a 30-min incubation,
a single wash in RBB, 0.1% BSA, and determination of bound labeled
peptide in an Apex automatic Measurement of cAMP Formation--
Stable cell lines in 10-cm
dishes were transfected with reporter plasmid pCRE-luc and control
plasmid pRSV- Measurement of Inositol Phosphate Formation--
Stably
expressing cell lines were plated into 24-well plates at matching
densities and cultured for 24-48 h prior to assay. In the 24 h
preceding assay, the media were substituted with inositol-free DMEM + 10% FCS containing 3 µCi/ml
myo-[3H]inositol (Amersham Pharmacia Biotech).
At the time of assay, wells were washed twice in DMEM + 0.1% BSA and
incubated for 40 min in 0.8 ml of DMEM + 0.1% BSA containing 10 mM LiCl and a predetermined concentration of agonist
(PACAP-27 or PACAP-38; Novabiochem). Reactions were terminated by
replacing the media with 0.5 ml of ice-cold 5% trichloroacetic acid.
Wells were scraped and transferred to 1.5-ml tubes. Recovered
precipitate was extracted with 0.5 ml of chloroform and neutralized
with 10 µl of 0.1 M NaOH. Extraction of total inositol
phosphates was performed by a modified chromatographic procedure
using AG1-X8 resin (Bio-Rad) prepared in 10 mM
myo-inositol. 400 µl of the supernatant was added to
~100 µl of resin and gently mixed at room temperature for 20 min.
The resin was washed twice with 1 ml of 60 mM sodium
borate, 5 mM borax. Inositol phosphates were eluted with
200 µl of 2 M ammonium formate, 0.1 M formic acid and quantified by scintillation counting. Results shown in Fig. 4,
A and B, are from single experiments performed in quadruplicate.
Measurement of Intracellular Calcium--
Receptor effects on
intracellular calcium were investigated in the stably transfected
HEK293 cells, stimulated with PACAP-27 or PACAP-38 at 100 nM. Measurements of intracellular calcium were carried out
according to the methods described in Ref. 25.
Fractionation of Testis Cell Populations--
Separation of
testis cells was achieved by velocity sedimentation at unit gravity in
a STAPUT chamber based on the method of Bellve (26). Briefly, a single
testis from adult rat was dissociated to single cells by sequential
digestion in collagenase (5 mg/ml: Sigma) and trypsin/DNase I (5 mg/ml
and 1 µg/ml: Sigma and Roche Molecular Biochemicals). Cells
were centrifuged at 800 × g for 2 min and then
resuspended in DMEM + 10% FCS and sieved through a 100-µm mesh
strainer. Cells were collected by centrifugation once more and
resuspended in 60 ml of enriched Krebs-Ringer buffer with 0.5% BSA.
This suspension was introduced into a 23-cm diameter STAPUT chamber,
followed by a 2.1-liter gradient of 2-4% BSA in enriched Krebs-Ringer
buffer. Separation was carried out at 4 °C for 2-3 h. The apparatus
was then drained and the contents collected into 120-ml fractions.
Cells were concentrated by centrifugation, and early fractions not
containing significant amounts of cells were discarded. Samples were
taken from each remaining fraction and processed for RT-PCR as
described above. Cultures of adult Sertoli cells used for RT-PCR
analysis were prepared from rat testes by sequential digestion as
above, followed by selection of adherent cells by plating in DMEM + 10% FCS on plastic culture flasks. The cells were trypsinized and
replated after 24 h in culture, and again after 72 h, to
reduce germ cell contamination. The Sertoli cells were further purified
by one or two rounds of trypsinization and replating, and in some cases
by culture in media containing 3 µg/ml cytosine arabinofuranoside to
suppress fibroblast growth.
Data Analyses--
The hydrophobicity profile (Fig.
2B) was generated with Protean 3.14 (DNASTAR Inc., Madison,
WI). The Km and EC50 values (Table I),
and associated confidence intervals, were calculated with Prism 2.0.1 (GraphPad Software Inc., San Diego, CA).
Detection of PAC1R Variants by Nested PCR--
PCR primers were
designed to detect the existence of different isoforms of PAC1R
mRNA following a nested PCR protocol. Distal primers encompassing a
region including all of the extracellular domain and transmembrane
domains were used for initial DNA amplification, followed by proximal
primers specific for the extracellular domain (Fig.
1A). PAC1R products were then
identified by Southern hybridization with an end-labeled
oligonucleotide probe. RT-PCR analysis of RNA from 2-mm segments of
seminiferous tubules (Fig. 1B) revealed the presence of
three isoforms. Sequencing of the cloned products identified the lower
most band (arrow 1) to be identical to the published rat
PAC1R sequence (GenBankTM accession number Z23279). The
largest product (arrow 3) contained additional sequence
between coding exons 4 and 5 and is likely a consequence of the
inclusion of part of the 5'-region of the intron between exons 4 and 5 (intron slippage). The additional sequence includes two in-frame stop
codons. The product corresponding to arrow 2 contained a
novel region of 72 nucleotides inserted between coding exons 3 and 4 with no interruption of the translational reading frame. The predicted
translation products are shown (Fig. 1C). Exon 3a is an
alternatively spliced "cassetted" exon and not due to intron
slippage. The nested PCR approach was further utilized to isolate
full-length receptor clones. Three full-length versions of the PACAP
type 1 receptor were cloned (Fig. 1C): 1) PAC1R, equivalent
to GenBankTM accession number Z23279; 2) the PAC1R with the
additional 24 amino acids; and 3) the PAC1R receptor "HOP" variant.
The identities of all three clones were verified by sequencing. The
PAC1R clone 1 was identical to the published sequence (18). In addition to the extra 72 bp of coding sequence (Fig.
2A), the longer clone 2 had
one substitution (G to A) at nucleotide 798, compared with the
published sequence. This G to A substitution did not alter the
conceptual translation product and is most likely the result of
misincorporation of a nucleotide during the PCR procedure. Clone 3 (Fig. 1C) is an intron slippage splice variant resulting in
a frameshift and the formation of a truncated protein.
To determine whether the sequence coding for the insertion of an
additional 24 amino acids into the PAC1R was due to the splicing in of
a separate exon or was part of an existing exon or intron incorporated
by a splice slippage event, the region between coding exons 3 and 4 was
amplified from rat genomic DNA. Partial sequencing of this
~2.5-kilobase pair fragment revealed that the inserted sequence was a separate exon (termed 3a), separated from the preceding exon 3 by 161 bp. (Fig. 2B). The additional 24 amino acids
encoded by the alternatively spliced exon 3a predict a markedly altered hydrophobicity profile of the extracellular domain by introducing a
short stretch of hydrophilic amino acids, followed by a predominantly hydrophobic region (Fig. 2C). Furthermore, analysis of the
inserted exon 3a using PROSITE software (27, 28) identified a
potential N-linked glycosylation site (NTSS),
which may be a glycosylation-sensitive site for the regulation of the
biologic functions of exon 3a.
PAC1R(3a) mRNA Occurs in Distinct Cell Populations of the
Testis--
The tissue distribution of the novel receptor variant was
assessed by RT-PCR of several rat tissues responsive to PACAP (Fig. 3). A DNA product corresponding to PAC1R
was amplified from cerebellum, hypothalamus, pituitary, pancreatic
islets, seminiferous tubule, and the INS-1 cell line (Fig.
3A; 482-bp band in upper panel). A product
corresponding in size to that expected for PAC1R(3a) is also strongly
detected in seminiferous tubules. The identity of this product is
confirmed by Southern hybridization to an oligonucleotide probe (DP1)
complementary to the exon 3a sequence (lower panel). The strongest signal for the PAC1R(3a) occurs in the seminiferous tubule. Other tissues show only trace amounts of PAC1R(3a) PCR product.
To determine which cell populations within the testis express
PAC1R(3a), dissociated rat testis cells were fractionated by velocity
sedimentation at unit gravity. Fractions were examined by RT-PCR for
products specific for certain cell types, as well as PAC1R(3a) (Fig.
3B). A PCR product corresponding to PAC1R(3a) was detected
in highest amounts in fractions enriched for Sertoli cells (located by
the follicle-stimulating hormone receptor PCR signal) and round
spermatids (PACAP PCR signal). The product is absent in spermatocytes
(fractions 4 and 5), in which a pachytene spermatocyte marker,
transcription factor SOX-17, is expressed. PCR products corresponding
in size to PAC1R and PAC1R(3a) were also detected in cultures of adult
rat Sertoli cells substantially purified as described under
"Materials and Methods" (data now shown). However, as some germ
cells remain associated with Sertoli cells in these cultures, and in
STAPUT fractionations, the presence of the variant receptor on adult
Sertoli cells cannot be verified.
PAC1R(3a) Has an Increased Binding Affinity for
PACAP-38--
Binding studies were carried out in HEK293 cells stably
transfected with recombinant plasmids expressing either the PAC1R or
the PAC1R(3a). The receptors were compared for their relative binding
of PACAP-27 and PACAP-38 by displacement of 125I-labeled
PACAP-27 with unlabeled peptides (Fig.
4). Whereas the PAC1R and PAC1R(3a)
displayed equal affinity for PACAP-27 (Fig. 4A), the
PAC1R(3a) showed a significantly increased (6.1-fold) affinity for
PACAP-38 over the PAC1R (Fig. 4B). The amino-terminal truncated PACAP-(6-38) peptide, which lacks signaling activity and
therefore acts as an antagonist of the PACAP receptor, displayed a
1.9-fold increase in binding affinity for PAC1R(3a) compared with the
PAC1R (Fig. 4C). The Km values of the
different ligand/receptor combinations are summarized in Table
I. In other experiments, the
sequence-related peptides VIP, PHI (peptide
histidine isoleucine), and PRP did not displace
125I-PACAP-27 from either the PAC1R- or
PAC1R(3a)-expressing cell lines (data not shown).
The PAC1R(3a) Has Altered Signal Transduction Coupling Efficiencies
Compared with PAC1R--
The efficiency of signaling through the cAMP
pathway was assayed using a cAMP-responsive reporter gene. The
specificity of this method was assessed by using specific inhibitors of
protein kinase A (H89) and PLC (U73122). H89
dose-dependently inhibited the accumulation of luciferase
activity in both PAC1R- and PAC1R(3a)-expressing cell lines treated
with PACAP-27, and no inhibition was observed with U73122 (data not
shown). The cAMP responsiveness of the PAC1R(3a)-expressing cell line
was right-shifted with respect to the PAC1R-expressing line with both
PACAP-27 and PACAP-38 (Fig. 4, A and B). This
difference in ligand concentration coupling to cAMP formation between
PAC1R and PAC1R(3a) was observed in a total of four independent
experiments. Coupling to PLC-dependent signaling pathways
was assessed by measuring the accumulation of inositol phosphates in
response to PACAP-27 or PACAP-38 in the receptor-expressing stable cell
lines. The PAC1R(3a)-expressing cell line clearly has a reduced
sensitivity to both PACAP-27 and PACAP-38 compared with the
PAC1R-expressing cell line (Fig. 4B). This observation, seen
in a total of three independent experiments, suggests that the 24 amino
acids inserted in the alternatively spliced PAC1R(3a) also reduces
coupling to the PLC-dependent signaling pathway. The
EC50 values for cAMP and inositol phosphate generation are
given in Table I. No difference was detected in the ability of PAC1R
and PAC1R(3a) to elevate intracellular calcium in response to treatment
of the HEK293 with PACAP38 or PACAP27.
Alternative splicing of the PAC1R pre-mRNA has been described
previously for the 5'-untranslated region, the third intracellular loop, and the extracellular ligand-binding domain (18, 20, 23). Here we
describe a new splice variant of the PAC1R resulting from the
cassetting in of an exon that adds an additional 72 nucleotides in the
amino-terminal extracellular domain and results in alterations of both
ligand binding and signaling function. Inclusion of the additional
72-bp in-frame exon (termed exon 3a) results in the translation of an
addition 24 amino acids within the amino-terminal extracellular domain
of the mature receptor providing the unique receptor PAC1R(3a).
Although the identity of amino acids between PACAP and VIP receptors
(VPAC1R and VPAC2R) in the extracellular amino-terminal domain is 38%
for PAC1R, compared with VPAC1R and 41% for PAC1R, compared with
VPAC2R, the region encoded by coding exons 3a, 4, and 5 is unique to
the PAC1R. We speculate that the presence of exon 3a perturbs the
overall alignment of conserved residues within the extracellular
domains of the three receptors. The inclusion of coding exon 3a in the
receptor increases the affinity of the receptor for PACAP-38 by 6-fold,
but does not affect the affinity of the receptor for PACAP-27.
PAC1R(3a) also has a small increase in affinity for the truncated PACAP
peptide, PACAP-(6-38), compared with the PAC1R.
The interaction between PACAP and its receptors has been examined in
experiments with mutated and truncated receptors. The amino-terminal
extracellular domain of the PAC1R and a single membrane-anchoring
region is sufficient to mediate specific binding of PACAP, albeit with
a 20-fold reduction in affinity compared with the full-length receptor
(29). The role of the extracellular loops in stabilizing ligand binding
is apparent in experiments in which ligand binding by the VPAC1R was
found to be dependent on a conserved cysteine residue in the third
extracellular loop (30).
Three distinct regions within PACAP are believed to contribute to
receptor binding. A region within the carboxyl terminus of the 1-27
peptide comprising two adjacent The coding exons 3a, 4, and 5 in the extracellular domain of the
receptor may provide conformational alterations that affect the
relative affinity of the receptor for PACAP-27 and PACAP-38 (Fig.
5). The length of this extracellular
domain of the receptor may affect the degree to which the three regions
within the PACAP ligands can cooperate. The longer domain (exons 3a, 4, and 5) may enhance cooperation between the COOH-terminal cationic
region (region 3 of PACAP in Fig. 5) and the other receptor binding
regions of PACAP. The absence of exons 3a, 4, and 5 may enhance the
interaction of the PACAP amino-terminal region (region 1 of PACAP in
Fig. 5) while weakening the effect of the COOH-terminal region, thus bringing PACAP-27 and PACAP-38 closer together in their receptor binding affinities (20). The smaller increase in affinity of PACAP-(6-38) for binding to PAC1R(3a) compared with that of PACAP-38 suggests that the amino-terminal activation region of PACAP also plays
an important role in the binding to its receptor. The increased binding
affinity of PACAP to PAC1R(3a) compared with PAC1R suggests that the
amino-terminal activation domain of the receptor also plays an
important role in this cooperative binding of PACAP to its
receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP-labeled oligonucleotide
probes was done in a solution of 5 × SSC, 1% SDS, 10×
Denhardt's, and 100 µg/ml denatured salmon sperm DNA for 3 h at
37 °C. Blots were washed to a maximum stringency of 0.5 × SSC
at 52 °C.
counter (Micromedic Systems
Inc.).
-galactosidase (Stratagene, La Jolla, CA) 18 h
prior to assay. Immediately following transfection, the cells were
trypsinized and replated in 24-well plates. At the time of assay, cells
were transferred into DMEM + 0.1% BSA, and agonist (PACAP-27 or
PACAP-38; Novabiochem, San Diego, CA) was added in a range of
concentrations (10
11 M through
5 × 10
6 M and an
unstimulated control). Luciferase activity was quantified after a 6-h
incubation using the Promega luciferase system in accordance with the
manufacturer's instructions (Promega Corp., Madison, WI). Results
shown in Fig. 4, A and B, are from single experiments, with each treatment performed in quadruplicate. Inhibitor compounds H89 and U73122 (Calbiochem) were used to verify the predominance of the cAMP pathway in pCRE-luc activation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (32K):
[in a new window]
Fig. 1.
Alternatively spliced exon (3a) in the
extracellular domain of the PAC1 receptor (PAC1R). A,
diagram of the PAC1R mRNA denoting the sites of the oligonucleotide
amplimers (arrows) used to generate DNA products by reverse
transcription-polymerase chain reaction. PR = PAC1R,
F = forward amplimer, R = reverse
amplimer (see "Materials and Methods"). PRR-413 is the labeled
oligonucleotide used for the Southern blot in B. Numbers refer to nucleotide in the PAC1R mRNA
(GenBankTM accession number Z23279). ATG = start codon for translation. TGA = stop codon for
translation. B, reverse transcription-PCR of PAC1R mRNA
prepared from 2-mm segments of a rat seminiferous tubule. Shown are
ethidium bromide-stained PCR products hybridized with labeled
oligonucleotide probe PRR-413 (Southern blot). Nested PCR amplification
was done first with amplimers PRF-79 and PRR-1256 followed by a second
round of amplification using amplimers PRF-70 and PRR-552.
Numbers 1, 2, and 3 (arrows) refer to the segments of the PAC1R mRNA
designated in C. C, diagram of the three
alternatively spliced forms of the PAC1R mRNA detected by the PCR
analysis shown in B. Numbered boxes
(top) represent the exons of PAC1R. Dashed lines
between exons 4 and 5 indicate an alternative intron slippage splice
present in product 3, which results in the formation of a frameshifted
truncated protein. Product 2 contains the alternatively spliced exon 3a
of 72 bp. The positions of transmembrane-spanning domains are indicated
by solid bars beneath the corresponding exons.
View larger version (27K):
[in a new window]
Fig. 2.
Sequence and hydrophobicity profile of the
alternatively spliced exon 3a of the PAC1R. A,
nucleotide and amino acid sequences of exon 3a are boxed.
B, the position of exon 3a in rat genomic DNA relative to
exons 3 and 4. C, hydrophobicity profile (Kyte-Doolittle) of
the PAC1R amino-terminal extracellular domain using 9 amino acid
groupings. Exon 3a is indicated by the boxed sequence.
View larger version (59K):
[in a new window]
Fig. 3.
Tissue distribution of alternatively spliced
exon 3a of PAC1R. A, RT-PCR analysis of several
PAC1R-expressing tissues from rat, and the rat INS-1 cell line. The
PAC1R and PAC1R(3a) PCR products on an electrophoresis gel stained with
ethidium bromide are indicated by arrows at 482 and 554 bp,
respectively (top). Identity of the PAC1R(3a) product of 554 bp was confirmed by Southern blotting and hybridization with the
oligonucleotide probe DP1 (bottom). B, RT-PCR
products obtained from RNA in fractions of rat testis cells separated
by sedimentation velocity. The cell types were identified by RT-PCR
analyses of products expressed predominantly in major cell types,
follicle-stimulating hormone receptor in Sertoli cells, SOX-17 in
pachytene spermatocytes, and PACAP in round spermatids. Nested PCR for
PAC1R appears in the lower panel. The PAC1R(3a) product is
indicated by an arrowhead. The results shown are
representative of two independent experiments.
View larger version (31K):
[in a new window]
Fig. 4.
Alterations in ligand affinity and
signal transduction for PAC1R(3a) versus PAC1R.
A, luciferase activity from a CRE-luciferase reporter
plasmid (top) and accumulation of [3H]inositol
phosphates (bottom) in PAC1R- and PAC1R(3a)-expressing
stable cell lines stimulated with increasing concentrations of
PACAP-27. Binding and displacement of 125I-labeled PACAP-27
from the same cell lines in response to increasing concentrations of
PACAP-27 is overlaid on both graphs. B, luciferase activity
from a CRE-luciferase reporter plasmid (top) and
accumulation of [3H]inositol phosphates
(bottom) in PAC1R- and PAC1R(3a)-expressing stable cell
lines stimulated with increasing concentrations of PACAP-38. Binding
and displacement of 125I-labeled PACAP-27 from the same
cell lines in response to increasing concentrations of PACAP-38 is
overlaid on both graphs. C, binding and displacement of
125I-labeled PACAP-27 from PAC1R- and PAC1R(3a)-expressing
stable cell lines in response to increasing concentrations of
PACAP-(6-38).
Ligand binding affinities, cAMP formation, and inositol phosphate
production by wild type (PAC1R) and alternatively spliced variant
(PAC1R(3a)) PACAP receptors in response to PACAP isopeptides
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helices (31, 32) may provide
primary ligand-receptor recognition (33-35). The integrity of the
helices is important for high affinity binding (33). A similar
structural arrangement is also detected in VIP (36). The first 6 amino-terminal residues of PACAP are important for both binding and
signal transduction. Loss of these residues abolishes cAMP signaling
for both PACAP-(6-27) and PACAP-(6-38) (33, 37). In comparison with
PACAP-27 the binding affinity of PACAP-(6-27) is severely reduced. The
affinity of PACAP-(6-38), however, remains high rendering it an
effective antagonist. The COOH-terminal extension of PACAP-38 contains
a short
-helix and a preponderance of cationic amino acids (31). The
contribution of the COOH-terminal extension to the binding of
PACAP-(6-38) suggests that it forms a third receptor interaction domain.
View larger version (41K):
[in a new window]
Fig. 5.
Model of PACAP-38 interaction with
PAC1R. PACAP-38 contains three potential receptor interaction
domains, labeled 1 to 3 from the amino to the
carboxyl terminus. Only two of the binding domains can engage
concurrently, and only one conformation leads to receptor activation
and G protein release. In PAC1R(3a), the second, weak-activating
conformation may be favored over the first. Thus, the affinity of
PACAP-38 for the PAC1R(3a) is increased without an accompanying
increase in second messenger signaling over the entire range of
receptor occupancy.
PAC1R(3a) and PAC1R are coupled to both the cAMP and inositol phosphate signaling pathways. PAC1R(3a) exhibits increased EC50 values of coupling to both pathways in response to both PACAP-27 and PACAP-38 compared with PAC1R. Notably the increased affinity of PACAP-38 binding to the PAC1R(3a) is proportionately reflected in the accumulation of cAMP. This circumstance indicates that the stimulation of cAMP production by the PAC1R(3a) is coupled to cAMP production over the full range of receptor occupancy, whereas in contrast, the PAC1R generates near maximum levels of cAMP at only partial receptor occupancy (Fig. 4B). For example, the PAC1R generates half-maximal levels of cAMP with 10-20% receptor occupancy by PACAP-38, whereas the PAC1R(3a) receptor requires near 90% receptor occupancy to reach half-maximal cAMP production and continues to generate increasing amounts of cAMP throughout the entire range of receptor occupancy. With regard to inositol phosphate production, the wild type receptor, PAC1R, begins to generate inositol phosphate at about 80% of receptor occupancy, whereas the variant PAC1R(3a) receptor requires near 100% receptor occupancy to begin to generate inositol phosphates. These observations suggest that at low concentrations of PACAP (10-50% of receptor occupancy), the PAC1R receptor is coupled exclusively to Gs and cAMP formation. At higher concentrations of PACAP the receptor recruits Gq, resulting in the activation of phospholipase C and the formation of inositol phosphates. In contrast to PAC1R, the variant-spliced receptor PAC1R(3a) remains coupled predominantly to Gs and cAMP formation at all concentrations of PACAP and only begins to recruit Gq at very high concentrations of PACAP, equivalent or close to 100% receptor occupancy. It is worth noting that our findings support earlier studies showing that the deletion of exons 4 and 5 enhances receptor coupling to inositol phosphate production relative to cAMP formation (20).
In addition to the apparent differences in coupling to G proteins
described above, the PAC1R(3a) differs from the wild type PAC1R in
another interesting aspect. The range of PACAP concentrations involved
in the generation of both cAMP and inositol phosphate is one to 2 orders of magnitude greater for PAC1R(3a) than it is for PAC1R. For
example, for both PACAP-27 and PACAP-38 cAMP formation occurs over a
concentration range of 1011 to
10
5 M with PAC1R(3a) compared
with a range of 10
11 to
10
7 M with PAC1R. This difference
of an order of magnitude in the range of PACAP in the activation of
signal transduction between the two receptors further emphasizes the
situation that quantitative aspects of signal transduction by the PACAP
receptor are altered, or modulated, by the presence of alternatively
spliced exon 3a in the extracellular domain of the receptor. Although
several other splice variants of the PAC1R are reported to affect the relative coupling to cAMP or inositol phosphate signaling pathways (18,
19, 38), these splice variations occur in the third intracellular loop,
a region that could interact directly with G protein subunits. In
instances cited above in which variations occur in the amino-terminal
extracellular domain, the effects appear to be mediated by
conformational changes in the receptor/ligand interaction.
Several lines of evidence suggest that ligand/receptor interactions can affect coupling of receptors to signaling pathways. There are reported differences in the relative potencies of PACAP-27 and PACAP-38 in stimulating second messenger synthesis. In general, PACAP-38 is reported to be a more effective stimulator of cAMP production than is PACAP-27 (19, 20, 33), although there are exceptions (18). In two instances it has been reported that PACAP-38 is more effective than PACAP-27 in the activation of phospholipase C (18, 20). If alterations in the relative affinities of PAC1R for the two PACAP peptides, PACAP-27 and PACAP-38, indicate a change in the way regions within PACAP interact with the receptor, it is possible that these changes also affect the potency of PACAP for stimulating the activation of coupling to different G protein subunits. In at least one system, the glucagon-like peptide-1 hormone, relative activation of two different G protein subunits by one receptor has been shown to be ligand dose-dependent (39).
If our experimental model reflects the true biology of the interactions
of PACAP peptides with the different spliced forms of the PACAP
receptor in the seminiferous tubules during the cycles of
spermatogenesis, then the implications of our findings are significant
because they indicate that local ambient concentration gradients of
PACAP can regulate the recruitment of different signal transduction
pathways during the spermatogenic cycle. Ambient concentrations of
PACAP are likely to be high at certain stages of the spermatogenic
cycle due the stage-specific production of PACAP by round spermatids
(2-4, 7). The PAC1R and PAC1R(3a), expressed in round spermatids and
Sertoli cells, will therefore experience high concentrations of
autocrine and paracrine PACAP, potentially in the micromolar range.
Given that PACAP levels are higher in the testis than any other tissue,
it is tempting to speculate that these isopeptides are important
regulators of the differentiation of germ cells. Concentration
gradients established by the stage-specific expression of PACAP, and
acting through the PAC1R/PAC1R(3a), may modulate and coordinate
cellular responses in germ and Sertoli cells.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Research Centre for Developmental Medicine and Biology, Starship Hospital, Level 1, The University of Auckland, Private Bag 92019, Auckland 1, New Zealand. Tel.: 64-9-373-7599 (ext. 2568); Fax: 64-9-373-7497.
¶ Present address: 370A Heritage Medical Research Centre, University of Alberta, 87th Ave. & 13th St., Edmonton, Alberta T6G 2S2, Canada. Tel.: 780-492-7428; Fax: 780-492-6702; E-mail: tim.kieffer@ualberta.ca.
To whom correspondence should be addressed: Laboratory of
Molecular Endocrinology, Massachusetts General Hospital, 55 Fruit St.,
WEL320, Boston, MA 02114. Tel.: 617-726-5190; Fax: 617-726-6954; E-mail:
jhabener@partners.org.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M009941200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PACAP, pituitary adenylate cyclase-activating polypeptide; VIP, vasoactive intestinal peptide; PLC, phospholipase C; bp, base pair(s); RT-PCR, reverse transcription-polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; BSA, bovine serum albumin.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Vaudry, D.,
Gonzalez, B. J.,
Basille, M.,
Yon, L.,
Fournier, A.,
and Vaudry, H.
(2000)
Pharmacol. Rev.
52,
269-324 |
2. | Arimura, A., Somogyvari-Vigh, A., Miyata, A., Mizuno, K., Coy, D. H., and Kitada, C. (1991) Endocrinology 129, 2787-2789[Abstract] |
3. | Ghatei, M. A., Takahashi, K., Suzuki, Y., Gardiner, J., Jones, P. M., and Bloom, S. R. (1993) J. Endocrinol. 136, 159-166[Abstract] |
4. | Hannibal, J., and Fahrenkrug, J. (1995) Regul. Pept. 55, 111-115[CrossRef][Medline] [Order article via Infotrieve] |
5. | Hurley, J. D., Gardiner, J. V., Jones, P. M., and Bloom, S. R. (1995) Endocrinology 136, 550-557[Abstract] |
6. | Kononen, J., Paavola, M., Penttila, T. L., Parvinen, M., and Pelto-Huikko, M. (1994) Endocrinology 135, 2291-2294[Abstract] |
7. |
Daniel, P. B.,
and Habener, J. F.
(2000)
Endocrinology
141,
1218-1227 |
8. | West, A. P., McKinnell, C., Sharpe, R. M., and Saunders, P. T. (1995) J. Endocrinol. 144, 215-223[Abstract] |
9. | Heindel, J. J., Powell, C. J., Paschall, C. S., Arimura, A., and Culler, M. D. (1992) Biol. Reprod. 47, 800-806[Abstract] |
10. |
Rossato, M.,
Nogara, A.,
Gottardello, F.,
Bordon, P.,
and Foresta, C.
(1997)
Endocrinology
138,
3228-3235 |
11. | Ishihara, T., Shigemoto, R., Mori, K., Takahashi, K., and Nagata, S. (1992) Neuron 8, 811-819[Medline] [Order article via Infotrieve] |
12. | Sreedharan, S. P., Patel, D. R., Huang, J. X., and Goetzl, E. J. (1993) Biochem. Biophys. Res. Commun. 193, 546-553[CrossRef][Medline] [Order article via Infotrieve] |
13. | Lutz, E. M., Sheward, W. J., West, K. M., Morrow, J. A., Fink, G., and Harmar, A. J. (1993) FEBS Lett. 334, 3-8[CrossRef][Medline] [Order article via Infotrieve] |
14. | Inagaki, N., Yoshida, H., Mizuta, M., Mizuno, N., Fujii, Y., Gonoi, T., Miyazaki, J., and Seino, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2679-2683[Abstract] |
15. | Arimura, A., and Shioda, S. (1995) Front. Neuroendocrinol. 16, 53-88[CrossRef][Medline] [Order article via Infotrieve] |
16. | MacKenzie, C. J., Lutz, E. M., McCulloch, D. A., Mitchell, R., and Harmar, A. J. (1996) Ann. N. Y. Acad. Sci. 805, 579-584[Medline] [Order article via Infotrieve] |
17. | Van Rampelbergh, J., Poloczek, P., Francoys, I., Delporte, C., Winand, J., Robberecht, P., and Waelbroeck, M. (1997) Biochim. Biophys. Acta 1357, 249-255[Medline] [Order article via Infotrieve] |
18. | Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P. H., and Journot, L. (1993) Nature 365, 170-175[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Pisegna, J. R.,
and Wank, S. A.
(1996)
J. Biol. Chem.
271,
17267-17274 |
20. |
Pantaloni, C.,
Brabet, P.,
Bilanges, B.,
Dumuis, A.,
Houssami, S.,
Spengler, D.,
Bockaert, J.,
and Journot, L.
(1996)
J. Biol. Chem.
271,
22146-22151 |
21. | Svoboda, M., Tastenoy, M., Ciccarelli, E., Stievenart, M., and Christophe, J. (1993) Biochem. Biophys. Res. Commun. 195, 881-888[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Chatterjee, T. K.,
Sharma, R. V.,
and Fisher, R. A.
(1996)
J. Biol. Chem.
271,
32226-32232 |
23. |
Chatterjee, T. K.,
Liu, X.,
Davisson, R. L.,
and Fisher, R. A.
(1997)
J. Biol. Chem.
272,
12122-12131 |
24. | Kieffer, T. J., Heller, R. S., Unson, C. G., Weir, G. C., and Habener, J. F. (1996) Endocrinology 137, 5119-5125[Abstract] |
25. |
Holz, G. G.,
Leech, C. A.,
Heller, R. S.,
Castonguay, M.,
and Habener, J. F.
(1999)
J. Biol. Chem.
274,
14147-14156 |
26. | Bellve, A. R. (1993) Methods Enzymol. 225, 84-113[Medline] [Order article via Infotrieve] |
27. |
Hofmann, K.,
Bucher, P.,
Falquet, L.,
and Bairoch, A.
(1999)
Nucleic Acids Res.
27,
215-219 |
28. | Bucher, P., and Bairoch, A. (1994) Intell. Syst. Mol. Biol. 2, 53-61 |
29. | Cao, Y. J., Gimpl, G., and Fahrenholz, F. (1995) Biochem. Biophys. Res. Commun. 212, 673-680[CrossRef][Medline] [Order article via Infotrieve] |
30. | Gaudin, P., Couvineau, A., Maoret, J. J., Rouyer-Fessard, C., and Laburthe, M. (1995) Biochem. Biophys. Res. Commun. 211, 901-908[CrossRef][Medline] [Order article via Infotrieve] |
31. | Wray, V., Kakoschke, C., Nokihara, K., and Naruse, S. (1993) Biochemistry 32, 5832-5841[Medline] [Order article via Infotrieve] |
32. | Inooka, H., Endo, S., Kitada, C., Mizuta, E., and Fujino, M. (1992) Int. J. Pept. Protein Res. 40, 456-464[Medline] [Order article via Infotrieve] |
33. | Robberecht, P., Gourlet, P., De Neef, P., Woussen-Colle, M. C., Vandermeers-Piret, M. C., Vandermeers, A., and Christophe, J. (1992) Eur. J. Biochem. 207, 239-246[Abstract] |
34. | Cao, Y. J., Kojro, E., Gimpl, G., Jasionowski, M., Kasprzykowski, F., Lankiewicz, L., and Fahrenholz, F. (1997) Eur. J. Biochem. 244, 400-406[Abstract] |
35. |
Cao, Y. J.,
Kojro, E.,
Jasionowski, M.,
Lankiewicz, L.,
Grzonka, Z.,
and Fahrenholz, F.
(1998)
Ann. N. Y. Acad. Sci.
865,
82-91 |
36. | Fry, D. C., Madison, V. S., Bolin, D. R., Greeley, D. N., Toome, V., and Wegrzynski, B. B. (1989) Biochemistry 28, 2399-2409[Medline] [Order article via Infotrieve] |
37. | Robberecht, P., Gourlet, P., De Neef, P., Woussen-Colle, M. C., Vandermeers-Piret, M. C., Vandermeers, A., and Christophe, J. (1992) Mol. Pharmacol. 42, 347-355[Abstract] |
38. | Journot, L., Waeber, C., Pantaloni, C., Holsboer, F., Seeburg, P. H., Bockaert, J., and Spengler, D. (1995) Biochem. Soc. Trans. 23, 133-137[Medline] [Order article via Infotrieve] |
39. |
Montrose-Rafizadeh, C.,
Avdonin, P.,
Garant, M. J.,
Rodgers, B. D.,
Kole, S.,
Yang, H.,
Levine, M. A.,
Schwindinger, W.,
and Bernier, M.
(1999)
Endocrinology
140,
1132-1140 |