From the Discovery Research Laboratories I, Pharmaceutical Discovery Research Division, Takeda Chemical Industries, Ltd., Wadai 10, Tsukuba, Ibaraki 300-4293, Japan, the § Department of Biophysics, Faculty of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan, and ¶ Biotechnology Laboratories, Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., Juso Hon-machi, Yodogawa-ku, Osaka 532-8686, Japan
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ABSTRACT |
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Human pituitary adenylate cyclase-activating
polypeptide (PACAP) receptor was expressed in Sf9 insect cells
and Chinese hamster ovary (CHO) cells. The recombinant receptor in
Sf9 cell membranes had low affinity for
125I-PACAP27 (Kd = 155.3 pM) and was insensitive to guanosine 5'-O-3-thiotriphosphate (GTPS), whereas the receptor in
CHO membranes had a high affinity (Kd = 44.4 pM) and was GTP
S sensitive. The receptor in Sf9
membranes was converted to a high affinity state
(Kd = 20-40 pM) following
solubilization with digitonin. A large quantity (2 mg from 8 liters of
insect cells) of the purified PACAP receptors
(Bmax = 23.9 nmol/mg of protein) were obtained in a digitonin-induced high affinity state (Kd = 17.3 pM) using biotinylated ligand affinity chromatography.
The apparent molecular weight of the purified receptor
(Mr = 48,000) was smaller than that of the
receptor from CHO cells (Mr = 58,000) due to differences in asparagine-linked sugar chains. The purified receptor reverted to a low affinity state (Kd = 182.6 pM) upon reconstitution into lipid vesicles, however, the
receptor reconstituted with Gs protein had a high affinity
(Kd = 40.2 pM) and was GTP
S
sensitive. [35S]GTP
S binding to the reconstituted
Gs protein was enhanced by PACAP27 and PACAP38
(EC50 = 42.5 and 9.4 pM, respectively) but not
by antagonist PACAP(6-38), indicating that the purified receptor was
functionally active.
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INTRODUCTION |
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Pituitary adenylate cyclase-activating polypeptide (PACAP)1 was first discovered in 1989 as a novel hypothalamic hormone that increases adenylate cyclase activity in pituitary cells (1). PACAP exists in two carboxyl-terminal-amidated forms: PACAP38 with 38 amino acid residues (1) and PACAP27 with the same amino-terminal 27 residues (2). Molecular cloning studies revealed that the structure of PACAP is highly conserved among rat, sheep, and humans (3, 4). PACAP has structural similarity to peptides in the secretin/glucagon peptide family, especially to vasoactive intestinal polypeptide (VIP) (1). PACAP is distributed in the central nervous system and various peripheral organs (5) and elicits a wide variety of biological functions, such as neuroprotective action against gp120-induced cell death (6), protection of cerebellar granule neurons from apoptosis (7), secretion of pituitary hormones (1, 8), secretion of interleukin-6 from astrocytes or folliculo-stellate cells (9, 10), secretion of catecholamines from chromaffin cells (11) or adrenal glands (12), and insulin release (13). The biological actions of PACAP are mediated by a PACAP-specific receptor (type I receptor) and a PACAP/VIP-nonselective receptor (type II receptor). The type I PACAP receptor includes the PACAP1 receptor (14-21) and a novel variant PACAPR-TM4 (22). There are two alternatively spliced exons, rat hip and hop (20) or human SV-1 and SV-2 (21), in the PACAP1 receptor gene, resulting in the possible existence of five splicing variants in the PACAP1 receptor (20, 21). All of these receptors belong to the G protein-coupled receptor superfamily and are subdivided structurally into the secretin/glucagon receptor family (23) that is distinguished from rhodopsin-type receptors.
All G protein-coupled receptors have seven hydrophobic segments that
probably form transmembrane -helices. Direct evidence for the
arrangement of transmembrane domains was obtained from the
two-dimensional crystallography of rhodopsin (24), providing valuable
information for molecular modeling of other G protein-coupled receptors. More precise modeling requires elucidating the structures of
another receptors. In particular, receptors in the secretin/glucagon receptor family are predicted to have a different arrangement in the
transmembrane domains (25). On the other hand, structural biology
directly clarifying the three-dimensional structure of a G
protein-coupled receptor has been hindered by several difficulties in
the purification and crystallization of the receptor protein. Most G
protein-coupled receptors exist at very low level in tissue membranes.
Thus, it is essential to develop an expression system that can produce
a large amount of the recombinant receptor. Parker et al.
(26) first described that a baculovirus expression system was
beneficial for the expression of
-adrenergic and muscarinic receptors at high levels (5-30 pmol/mg). Recombinant
-adrenergic receptors purified from the baculovirus-infected insect cells were
functionally active as were the
-adrenergic receptors from turkey
erythrocytes (26). The expression system was also used to produce
various G protein-coupled receptors, however, only a few reports (27)
succeeded in providing a practical amount of purified receptor for
further biochemical or structural studies. This is probably due to
difficulty in the solubilization and purification of G protein-coupled
receptors.
We previously described successful purification of the PACAP1 receptor from bovine brain membranes in a high affinity state (28). In the present study we conducted large-scale purification of the recombinant PACAP1 receptor by combining the previously described purification procedures and the baculovirus expression system. The recombinant PACAP1 receptor purified in a digitonin-solubilized form retained high affinity for PACAP and was functionally active when reconstituted with Gs protein in lipid vesicles. The purified receptor will likely contribute to the understanding of the regulatory mechanisms and structure of G protein-coupled receptors.
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EXPERIMENTAL PROCEDURES |
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Materials--
PACAP27, PACAP38, PACAP(6-38), VIP, leupeptin,
pepstatin, and E-64 were obtained from the Peptide Institute (Osaka,
Japan). GTP, GDP, and ATP were from Yamasa (Tokyo, Japan). Guanosine
5'-O-3-thiotriphosphate (GTPS) was obtained from
Boehringer Mannheim GmbH (Mannheim, Germany). Bovine serum albumin
(BSA), GMP, Nonidet P-40, and brain extract type VII were from Sigma.
Flavobacterium menigosepticum peptide-N4-(N-acetyl-
-glucosaminyl)
asparagine amidase (N-glycanase, recombinant), F. menigosepticum endo-
-N-acetylglucosaminidase
F2 (endoglycosidase F2), Streptomyces
plicatus endo-
-N-acetylglucosaminidase
(endoglycosidase H, recombinant), and Streptococcus sp.
sialidase (neuraminidase) were from Genzyme (Cambridge, MA).
Xanthomonas manihotis
-N-acetylglucosaminidase and X. manihotis
1-2,3-mannosidase were from New England
Biolabs, Inc. (Beverly, MA). SDS, digitonin, hydroxyapatite, and
phenylmethylsulfonyl fluoride were from Wako Pure Chemicals (Osaka,
Japan). Digitonin was dissolved in water at 80-90 °C, cooled, and
ultracentrifuged for removal of insoluble materials. BIGCHAP, CHAPS,
and 5-[5-(N-succinimidyloxycarbonyl)penthylamido]hexyl D-biotinamide (Biotin-(AC5)2-OSu)
were obtained from Dojindo Laboratories (Kumamoto, Japan).
Avidin-Affi-Gel 10 was prepared by immobilizing avidin (Wako Pure
Chemicals, Osaka, Japan) to Affi-Gel 10 (Bio-Rad) following the
manufacturer's instructions. Lentil lectin-Sepharose 4B was obtained
from Pharmacia Biotech (Uppsala, Sweden). 125I-PACAP27 was
prepared by the previously described method (29). [35S]GTP
S was obtained from NEN Life Science Products
(Boston, MA).
Expression in Sf9 Insect Cells--
The null variant
lacking the SV-1 and SV-2 insertion sequences (19, 21) was expressed in
Sf9 insect cells as described previously (32). The human PACAP
receptor cDNA fragment (nucleotides 1-1664) was excised from
pTS847 plasmid (19) by EcoRI digestion and cloned in
pcDNAI/Amp (Invitrogen). An EcoRV fragment was excised from the resulting plasmid, ligated with the Sse8387I
linker, digested with BamHI and Sse8387I, and
cloned in the transfer vector pBlueBacIII (Invitrogen). The resultant
transfer vector (pHPR-7) and Autographa californica nuclear
polyhedrosis virus genomic DNA were co-transfected to Sf9 cells
to generate recombinant baculovirus. The recombinant virus producing
the highest amount of the PACAP receptor was selected. The Sf9
cells (2 × 108 cells) were cultured with 200 ml of
Grace's insect cell culture medium (Life Technologies, Inc., Grand
Island, NY) containing 0.1% Pluronic F-68 (Life Technologies, Inc.,
Grand Island, NY), 10% fetal calf serum, and 20 µg/ml gentamicin in
a 1-liter spinner flask at 27 °C for 25 h, infected with the
recombinant virus at a multiplicity of infection of 3-5, and cultured
at 27 °C for 4 days. The cells were harvested, washed with
phosphate-buffered saline containing 2.7 mM EDTA, and
stored at 70 °C until used.
Stable Expression in Chinese Hamster Ovary Cells--
The PACAP
receptor cDNA fragment (nucleotides 245-1652) was obtained by
polymerase chain reactions using pTS847 plasmid as a template (19) and
cloned at SalI site in a pAKKO1.11 expression vector (33)
containing SR promoter and mouse dihydrofolate reductase gene as a
selective marker. The resulting plasmid was transfected to Chinese
hamster ovary cells deficient in dihydrofolate reductase
(CHO/dhfr
cells) by the calcium
phosphate-coprecipitation method. A clonal cell line expressing the
maximum level of the PACAP receptor (PACR19 clone) was obtained by
selection in Dulbecco's modified Eagle's medium containing 10%
dialyzed fetal calf serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin. The PACR19 cells were grown with Dulbecco's modified
Eagle's medium containing 10% fetal calf serum, 100 units/ml
penicillin, and 100 µg/ml streptomycin in Nunc Cell Factories (Nunc
A/S, Roskilde, Denmark). The cells at 70-80% confluency were
harvested by washing with phosphate-buffered saline containing 2.7 mM EDTA, and stored at
70 °C until used.
Preparation of Biotinylated PACAP38-- PACAP38 (2.2 µmol, 10 mg) was reacted with a 1.4 mol equivalent (3.0 µmol, 1.7 mg) of biotinylating reagent, biotin-(AC5)2-Osu in dimethyl sulfoxide (5 ml) containing a 10 mol equivalent of triethylamine (21 µmol, 3 µl) at room temperature for 2 h. An aliquot (1 ml) of the reaction mixture was diluted with 0.05% trifluoroacetic acid and injected into a reversed phase high performance liquid chromatography column (7.8 mm × 30 cm, ODS80TM, Tosoh, Tokyo, Japan) equilibrated with 0.05% trifluoroacetic acid. The biotinylated PACAP38 was eluted with a linear gradient of acetonitrile from 20 to 40% for 60 min at a flow rate of 2 ml/min. Several peaks of biotinylated ligands eluted behind the peak of unbiotinylated PACAP38 were collected, lyophilized, and dissolved in 0.05% CHAPS.
Preparation of the Membrane Fraction-- The infected Sf9 cells were homogenized with HOM buffer (10 mM NaHCO3, 5 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 10 µg/ml pepstatin, and 8 µg/ml E-64, pH 7.3) using a Polytron homogenizer (Kinematica GmbH, Littau, Switzerland) and centrifuged at 700 × g for 10 min (32, 33). The pellet was subjected twice to a repeated cycle of homogenization and centrifugation. The final pellet was suspended in the HOM buffer (P0 membranes). The supernatant fractions were combined and ultracentrifuged at 100,000 × g for 60 min. The resultant membrane pellet was suspended in the HOM buffer (P1+2+3 membranes). The P1+2+3 membranes from the transformed CHO cells (PACR19 clone) were prepared as above.
Solubilization and Purification of the PACAP Receptor-- The membrane protein was solubilized with 1% digitonin at a protein concentration of 2 mg/ml for 16-18 h. The clear solubilized protein fraction was obtained after ultracentrifugation at 100,000 × g for 60 min. The solubilized membrane protein was mixed with a 4-fold equivalent of biotinylated PACAP38 and avidin-Affi-Gel 10 (1 ml/10 nmol of biotinylated ligand). The mixture was gently agitated on a rotary shaker for 2 days at 4 °C. The gel was packed in a glass column and washed with the HOM buffer containing 1 M NaCl and 0.2% digitonin. The PACAP receptor was eluted with 20 mM magnesium acetate buffer (pH 4.0) including 0.2% digitonin, 1 M NaCl, and 10% glycerol.
The affinity-purified receptor was loaded onto a lentil lectin-Sepharose 4B column at a flow rate of 1 ml/min. After washing the column with 0.2% digitonin, 20 mM Tris, 0.5 M NaCl buffer (pH 7.4), the receptor was eluted with the same buffer including 0.5 MReconstitution of the Purified Receptor with G
Protein--
Reconstitution of the purified receptor was performed as
follows. Bovine brain crude lipid (brain extract type VII) was
dissolved in REC buffer (20 mM Tris, 1 mM EDTA,
3 mM MgCl2, and 160 mM NaCl, pH
7.4) containing 17% CHAPS at 40 mg/ml and stored at 70 °C. The
lipid solution was diluted 8-fold with the REC buffer before use. The
diluted lipid solution (60 µg/12 µl), purified PACAP receptor (20 pmol/4.5 µl), purified Gs
(80 pmol/8 µl), and
purified brain G
(160 pmol/25 µl) were mixed and dialyzed at
4 °C for 24-36 h in a dialyzing apparatus (Microdialysis system,
Life Technologies, Inc., Gaithersburg, MD) equipped with dialysis
membranes (Spectra/Por 2, MWCO:12-14,000, Spectrum Medical Industries,
Inc., Houston, TX) against the REC buffer supplied at a flow rate of 15 ml/h. The reconstituted receptor was used in 2-3 days.
Receptor Binding Experiments-- Receptor binding experiments were performed by the previously described method (28) in DG-BSA/TED buffer (0.05% DG, 0.1% BSA, 20 mM Tris, and 1 mM EDTA, pH 7.4), BSA/TED buffer (1% BSA, 20 mM Tris, and 1 mM EDTA, pH 7.4), and BSA/TED-Mg buffer (1% BSA, 20 mM Tris, 1 mM EDTA, and 5 mM MgCl2, pH 7.4). BSA concentration was increased in the BSA/TED and BSA/TED-Mg buffer to avoid severe sticking of 125I-PACAP27 on test tubes. Every binding reaction mixture contained 0.005% CHAPS that was derived from the vehicle of 125I-PACAP27.
GTPS Binding Experiments--
The reconstituted receptor
diluted 200-fold with the BSA/TED-Mg buffer (10 µl), PACAP, or a
related ligand dissolved in the BSA/TED buffer supplemented with 0.05%
CHAPS (1 µl), and 0.5 nM [35S]GTP
S
diluted with the BSA/TED-Mg buffer (100 µl) were mixed and incubated
at 25 °C for 1 h. The reaction mixture was diluted with 1.5 ml
of chilled TEM buffer (0.05% CHAPS, 0.1% BSA, 5 mM MgCl2, 1 mM EDTA, and 50 mM Tris,
pH 7.4) and filtered through a pre-wetted GF/F glass fiber filter
(Whatman). The filter was washed with 1.5 ml of the TEM buffer, dried,
and subjected to liquid scintillation counting. The experiment with the
membrane fraction was performed in the same manner but in the
BSA/TED-Mg buffer supplemented with 1 µM GDP and 150 mM NaCl.
Glycosidase Digestion--
Purified PACAP receptor (0.5 mg/ml,
10 µl) was mixed with 1% SDS (10 µl), denatured at 100 °C for 3 min and then diluted with 10% Nonidet P-40 (10 µl) and distilled
water (10 µl). An aliquot (4 µl) of the denatured receptor was
digested with N-glycanase (250 milliunits) in 0.1 M Tris (pH 7.6), endoglycosidase F2 (0.2 milliunits) in 0.2 M sodium acetate (pH 4.75),
endoglycosidase H (2 milliunits) in 50 mM sodium citrate
(pH 6.0), Streptococcus sp. sialidase (5 milliunits) in 50 mM sodium citrate (pH 6.0), X. manihotis
1-2,3-mannosidase (1 units) in 50 mM sodium citrate (pH
6.0), or X. manihotis
-N-acetylglucosaminidase
(1 units) in 50 mM sodium citrate (pH 4.5) at 37 °C for
18 h. The reaction mixture was diluted with a sample buffer for
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
(34), boiled at 100 °C for 3 min, and analyzed by SDS-PAGE (34).
Protein bands were visualized using a silver staining kit, 2D-Silver
StainII (Daiichi Pure Chemicals, Tokyo, Japan).
Miscellaneous Methods-- Protein determination was carried out by the method described by Schaffner and Weissman (35). Protein sequencing was performed as described previously (28).
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RESULTS |
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Low Affinity PACAP Receptor in Sf9 Cell Membranes--
The
predominant splicing variant (null variant) of the human
PACAP1 receptor, that lacks SV-1 and SV-2 insertion
sequences in the third intracellular loop (19, 21), was overexpressed in Sf9 insect cells under the control of the baculovirus
polyhedrin promoter and was stably expressed in CHO cell transformants
under the control of SR promoter. Saturation receptor binding
experiments performed in the absence of digitonin (in BSA/TED buffer)
followed by Scatchard plot analysis demonstrated that membranes from
the baculovirus-infected Sf9 cells (Sf9
P1+2+3 membranes) contained a single class of binding sites
having low affinity for 125I-PACAP27 (Kd = 155.3 ± 16.7 pM, Fig.
1A). In contrast, membranes
from the transformed CHO cells (CHO P1+2+3 membranes)
contained high affinity binding sites (Kd = 44.4 ± 2.2 pM, Fig. 1B). The maximum
receptor binding (Bmax) to the Sf9
P1+2+3 membranes was 82.6 ± 3.8 pmol/mg of protein
(Fig. 1A) and that to the CHO P1+2+3 membranes was 24.1 ± 1.5 pmol/mg (Fig. 1B).
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Lack of Functional Coupling to G Protein in Sf9 Cell
Membranes--
Agonist-dependent stimulation of
[35S]GTPS binding to the P1+2+3 membranes
was determined in BSA/TED-Mg buffer supplemented with 1 µM GDP and 150 mM NaCl. These additives were
required to decrease the basal [35S]GTP
S binding
occurring in the absence of the agonist. The binding of
[35S]GTP
S to the CHO P1+2+3 membranes
increased markedly in the presence of 1 µM PACAP27 (Table
II). The increase in
[35S]GTP
S binding to the CHO membranes was dependent
on the agonist concentration and was specific to PACAP (Fig.
2). The EC50 values were
581 ± 43 pM for PACAP27 and 107 ± 4.4 pM for PACAP38 (Fig. 2). PACAP did not increase
[35S]GTP
S binding to the membranes of mock
transfectants (data not shown). On the other hand, the Sf9
P1+2+3 membranes did not exhibit a significant increase in
[35S]GTP
S binding in response to PACAP27 stimulation
(Table II). This result further demonstrates that the PACAP receptor is
functionally coupled to G protein in the CHO cell membranes but not
in the Sf9 cell membranes.
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The PACAP Receptor in a Digitonin-induced High Affinity
State--
Saturation receptor binding experiments were further
performed in the presence of 0.05% digitonin (in DG-BSA/TED buffer). In contrast with the experiments in the absence of digitonin, both
PACAP receptors in the Sf9 and CHO P1+2+3 membranes had high affinity for 125I-PACAP27. The
Kd values were 44.6 ± 2.1 pM (Fig.
1A) and 39.3 ± 2.3 pM (Fig.
1B), respectively. The Bmax values,
146 ± 7.1 pmol/mg for Sf9 P1+2+3 membranes
(Fig. 1A) and 54.2 ± 0.5 pmol/mg for CHO
P1+2+3 membranes (Fig. 1B), were two times
higher than the respective values determined in the absence of
digitonin. Neither PACAP receptor was sensitive to GTPS in the
DG-BSA/TED buffer (Table I).
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Solubilization and Purification of the Recombinant PACAP Receptor-- The combined Sf9 membranes were subjected to solubilization with digitonin. The solubilized protein contained a single class of the PACAP receptor with high affinity (Kd = 20-40 pM) at a 3-fold higher concentration than the membranes (Table III). Digitonin worked best in that it solubilized the receptor in the high affinity state and did not destroy the receptor activity even at higher concentrations as shown for the bovine brain PACAP receptor (28, 36).
The solubilized receptor was further purified by biotinylated ligand/avidin Affi-Gel 10 affinity chromatography. Biotinylated ligand was prepared by reacting PACAP38 with a 1.4 mol equivalent of biotinylating reagent with an active ester, biotin-(AC5)2-OSu, under dehydrated conditions. The biotinylated product was composed of heterogeneously biotinylated PACAP38 because of multiple
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Ligand Binding Properties of the Purified PACAP Receptor-- Saturation receptor binding experiments in the DG-BSA/TED buffer indicated that the purified PACAP receptor from the insect cells had a single class of high affinity binding sites with a Kd value of 17.3 ± 1.3 pM (Fig. 4A). The affinity of the purified receptor was slightly higher than the membranous PACAP receptor determined in the DG-BSA/TED buffer. The specific activity was 23.9 ± 1.5 nmol/mg of protein (Table III). Competitive binding experiments demonstrated that the purified receptor retained selectivity for PACAP27 (IC50 = 0.21 ± 0.02 nM) and PACAP38 (IC50 = 0.086 ± 0.005 nM) against VIP (IC50 = 0.37 ± 0.04 µM) (Fig. 4B). These binding properties were very similar to those of the purified PACAP receptor from the CHO cells (Kd = 14.7 ± 1.1 pM, Fig. 4A; IC50 = 0.20 ± 0.02 nM for PACAP27, 0.12 ± 0.008 nM for PACAP38, and 0.27 ± 0.01 µM for VIP, data not shown). Similar results have been obtained for the PACAP receptor purified from bovine brain (28).
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Biochemical Properties of the Purified PACAP Receptor-- The PACAP receptor purified from Sf9 cells showed a broad silver-stained band with Mr = 48,000 (Fig. 5A) in SDS-PAGE analysis, while the PACAP receptor purified from the CHO cells showed a major silver-stained band at Mr = 58,000 (Fig. 5A). Both receptor bands presented the same amino-terminal amino acid sequence of Met-His-Ser-Asp-(unidentified)-Ile-Phe-Lys-Lys-Glu-Gln-. This sequence corresponds to the previously reported amino-terminal amino acid sequence of purified bovine brain PACAP receptor (28). Therefore, insect cells as well as mammalian cells recognize and cleave the signal sequence of PACAP receptor correctly.
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Ligand Binding Properties of the Reconstituted PACAP
Receptor--
The purified receptor from insect cells was
reconstituted with and without recombinant Gs/bovine
brain G
at the molar ratio of 1:4:8
(receptor:Gs
:G
) in crude brain lipid vesicles.
Saturation binding experiments performed in the BSA/TED buffer
demonstrated that the purified PACAP receptor required
Gs
/G
for expressing high affinity for PACAP27 when
it was reconstituted into lipid vesicles (Fig.
6A). The dissociation constant
of the reconstituted receptor without Gs
/G
(Kd = 182.6 ± 26 pM, Fig.
6A) was similar to that of the membranous PACAP receptor in
the Sf9 P1+2+3 membranes (Kd = 155.3 ± 16.7 pM, Fig. 1A). In contrast,
the reconstituted receptor with Gs
/G
had a single
class of high affinity binding sites with a Kd value
of 40.2 ± 4.2 pM in the BSA/TED buffer (Fig.
6A). The dissociation constant was comparable to the value
of the membranous PACAP receptor in the CHO P1+2+3
membranes (Kd = 44.4 ± 2.2 pM,
Fig. 1B) but two times larger than the value of the purified receptor in a digitonin-solubilized form (Kd = 17.3 ± 1.3 pM, Fig. 4A).
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G Protein Activation by the Reconstituted PACAP
Receptor--
Agonist-dependent stimulation of
[35S]GTPS binding to the reconstitute was determined
to investigate functional coupling of the recombinant PACAP receptor to
G protein. Spontaneous [35S]GTP
S binding to the
reconstitute was very slow in the absence of PACAP27; however, it was
greatly enhanced in the presence of 1 µM PACAP27 (Fig.
8). The reagents GDP and NaCl, which
decrease the basal [35S]GTP
S binding level, were not
added to the reaction mixture because the addition of higher
concentrations of GDP diminished the agonist-dependent
stimulation of [35S]GTP
S binding (Fig.
9).
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DISCUSSION |
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In the present study, the human PACAP receptor was overexpressed
in Sf9 insect cells under the control of a strong polyhedrin promoter. The expression level (50-150 pmol/mg) was higher than those
of other G protein-coupled receptors so far reported (ranging from 0.5 to 100 pmol/mg) (26, 33, 38-42). The presence of a signal sequence and
many potential glycosylation sites in the PACAP receptor, favoring the
translocation of the receptor to cell membranes, presumably contributes
to the high expression level. Furthermore, trimming of the 5'- and
3'-noncoding regions along with the use of pAKKO vector having SR
promoter might increase the expression level of the receptor in
CHO/dhfr
cells compared with the previous
expression study using CHO-K1 cells and pRc/CMV expression vector (19).
Butkerait et al. (38) suggested that the shortening of the
5'-untranslated sequence to 11 authentic bases might be the reason for
their high expression level of the 5-HT1A receptors in
insect cells (5-34 pmol/mg) compared with the values reported by
others (0.15 and 3 pmol/mg).
The native PACAP receptor is believed to be coupled primarily to
Gs proteins for activating adenylate cyclase. Additional coupling to another G protein species is suggested by the pleiotropic cellular response to PACAP stimulation such as increases in
phosphoinositide turnover (20, 21, 43) and intracellular calcium ion
concentration (11, 44). The recombinant receptor in the Sf9 cell
membranes, however, was not coupled to endogenous G proteins as
evidenced by its low affinity and GTPS insensitivity in agonist
binding. This is most probably due to a deficiency in the appropriate G proteins. The concentrations of immunoreactive Gi2 and
Go proteins in High 5 insect cells were estimated as 10.7 and 0.5 pmol/mg, respectively, while those in Sf9 insect cells
were below the detection limit (45). This finding strongly suggests
that the expression level of Gs-like protein in Sf9
insect cells is also very low. Butkerait et al. (38)
suggested that the coupling of recombinant receptors to endogenous G
proteins in insect cells is related to the receptor expression level.
Receptors expressed at a low level, such as the D4 dopamine
receptor (5 pmol/mg) (39), serotonin 5-HT1B receptor (0.5 pmol/mg) (40), and 5-HT1A receptor (0.15 pmol/mg) (41),
were sensitive to guanine nucleotides in agonist binding, indicating
coupling to G protein. On the other hand, the 5HT1A
receptor (5-34 pmol/mg) (38), the formyl peptide receptor (27 pmol/mg)
(42), the
-adrenergic receptor and the muscarinic receptor (30 pmol/mg) (26), did not exhibit GTP
S sensitivity in agonist binding.
Therefore, it is not surprising that most of the PACAP receptors
overexpressed in insect cells were not coupled to endogenous G
proteins.
The recombinant PACAP receptor in the Sf9 cell membranes was
purified in a digitonin-solubilized form. The purified receptor had a
protein core similar to that purified from CHO cells but with different
N-linked sugar chains. The apparent molecular weight of the
N-glycanase digested band (Mr = 43,000) was smaller than the calculated molecular weight of the protein
core (Mr = 51,354) (19). A possible reason for
this discrepancy is proteolytic degradation of the carboxyl terminus
region. The carboxyl-terminal amino acid sequence could not, however,
be determined in the present study. Another possibility is that strong
hydrophobic interactions between membrane spanning -helices
restricted complete unfolding, resulting in a higher mobility than
soluble proteins with a similar molecular weight.
Glycosidase digestion studies suggested structural differences in the
N-linked sugar chains of the PACAP receptors. The PACAP receptor from CHO cells was resistant to endoglycosidase H and endoglycosidase F2. Endoglycosidase H digests high
mannose-type and hybrid-type sugar chains (46). Endoglycosidase
F2 cleaves biantennary complex-type sugar chains
preferentially, but also cleaves high mannose-type sugar chains at a
slower rate (46). Taken together with the result from digestion by
exoglycosidases, it is suggested that the receptor from CHO cells has
sialylated tri- or tetraantennary complex-type sugar chains. In
contrast, the PACAP receptor from Sf9 cells was digested by
endoglycosidase F2, indicating that it has biantennary
complex-type and/or high mannose-type sugar chains. The presence of
biantennary complex-type sugar chains, however, is somewhat
controversial, because exoglycosidase digestion studies indicate that
the PACAP receptor from Sf9 cells has only mannosyl residues at
non-reducing terminal but does not have NeuAc and GlcNAc residues. The
presence of high mannose-type sugar chains is compatible with the
result from mannosidase digestion but inconsistent with the resistance
to endoglycosidase H. Considering that the PACAP receptor from
Sf9 cells has truncated N-linked sugar chains
(paucimannosidic N-linked sugar chains) (47) rather than
high mannose-type sugar chains, this discrepancy could be explained by
possible difference between substrate specificity of
endoglycosidase H and endoglycosidase F2. Endoglycosidase H scarcely digests some kinds of paucimannosidic
N-linked sugar chains such as
Man1
3(Man
1
6)Man
1
4GlcNAc2 (46), while
the reactivity of endoglycosidase F2 on these sugar chains
is not revealed. Direct evidences are required to clarify the structure of N-linked sugar chains in the PACAP receptors.
The purified PACAP receptor presented several oligomer bands upon SDS-PAGE. Similar results were found in various G protein-coupled receptors such as rhodopsin (48) and the olfactory receptor (49). Pharmacological evidence also suggests that the m2-muscarinic receptor forms a dimeric structure (50). On the other hand, bacteriorhodpsin has been shown to form a trimeric structure in orthorhombic two-dimensional crystals (51). The physiological significance of receptor oligomerization observed in SDS-PAGE is still unclear, because high temperatures used during SDS-PAGE sample preparation might promote artificial oligomerization via hydrophobic interactions as described by Sagné et al. (52). In fact, it was reported that the olfactory receptor forms higher oligomers after prolonged boiling (49). Receptor oligomerization in physiological conditions should be further examined.
The purified receptor had high affinity for PACAP27 and PACAP38 but low affinity for VIP. These ligand binding properties were similar to those of the receptor expressed in CHO cells. It has been proposed that digitonin might stabilize the PACAP receptor in the high affinity state based on the observations that purified PACAP receptor has a high affinity by itself (28). This hypothesis was further substantiated by reconstituting the purified PACAP receptor into lipid vesicles. The receptor reconstituted in lipid vesicles alone had low affinity in the absence of digitonin but high affinity in the presence of digitonin. The molecular mechanism of digitonin action, however, is not yet clear. For example, it is not clear whether solubilization into a lipid/digitonin mixed micelle is required for stabilizing the receptor or if the insertion of a small amount of digitonin into membrane bilayers is sufficient. Also, it is not clear whether digitonin acts directly on the receptor protein or acts indirectly by changing the milieu of the membrane or micelle. The small difference found between Kd values for the purified receptor in a digitonin-solubilized form and the reconstituted receptor in the DG-BSA/TED buffer suggests that complete solubilization is required for the full effect of digitonin. Studies on possible conformational changes in the PACAP receptor induced by digitonin may aid in understanding the G protein-dependent regulation of ligand binding affinity.
It should be also noted that the effect of digitonin is different from
receptor to receptor. Some G protein-coupled receptors, such as
-adrenergic (53) and neuropeptide Y (54) receptors, were solubilized
successfully using digitonin. The corticotropin-releasing factor
receptor solubilized with digitonin had high affinity for its ligand
and no GTP
S sensitivity (55), as observed in the present study. In
contrast, the use of digitonin failed in the solubilization of
gonadotropin-releasing hormone (56) and B2 bradykinin (57)
receptors. Receptors solubilized with digitonin in high affinity states
are easier targets for receptor purification.
The PACAP receptor reconstituted with Gs/G
in
lipid vesicles had high affinity for PACAP27, in contrast to that
without Gs
/G
. GTP
S, known to inhibit receptor/G
protein coupling by destabilizing the G protein trimer, almost
completely neutralized the effect of Gs
/G
,
demonstrating that the purified PACAP receptor reconstituted in lipid
vesicles requires Gs
/G
for expressing high
affinity for PACAP27. The Bmax value also
decreased when the PACAP receptor was uncoupled from G protein. The
result leads to the hypothesis that there are at least two states in G
protein-uncoupled PACAP receptors, a low affinity state
(Kd = 100-200 pM) and a very low
affinity state without detectable affinity for 125I-PACAP27. Difference between the
Bmax values in the presence and absence of
digitonin represents G protein-uncoupled receptor in a very low
affinity state. It should thus be interpreted that the CHO membranes
contain G protein-uncoupled spare PACAP receptor as much as G
protein-coupled PACAP receptor. The number of the PACAP receptors at
the high affinity state (approximately 20-25 pmol/mg) in the CHO
membranes reflects the maximum amount of G protein available for
coupling to the PACAP receptor.
GDP as well as GTPS or GTP decreased the specific binding of
125I-PACAP27 to the reconstituted receptor. The molecular
mechanism of GDP action is thought to decrease the dissociation rate of GDP from the G protein
-subunit. Thus, this indicates that high affinity binding of PACAP27 requires the dissociation of GDP, whereas
the dissociation of GDP is believed to be promoted by agonist
stimulation. In other words, the PACAP receptor has high affinity for
PACAP27 when it is interacting with nucleotide-free G protein.
Therefore, PACAP binding to the receptor shifts the guanine nucleotide
binding equilibrium toward the nucleotide free state, leading to the
cooperative PACAP binding and GDP dissociation. A similar result with
GDP has been observed in other G protein-coupled receptors such as the
muscarinic receptor (58). The mathematical solution for the
multiequilibrium system explains the observed effect of GDP on ligand
binding (59).
The expression of a homogeneous high affinity was attained with a
smaller amount of G protein (PACAP receptor:Gs:G
= 1:4:8) compared with other reconstitution studies. Florio and Sternweis (60) described that approximately a 1000-fold excess of Go
protein versus the muscarinic receptor is required for
complete expression of high affinity. On the other hand, reconstitution
of the
2-adrenergic receptor (61) or muscarinic receptor
(58) at a smaller receptor:G protein ratio (1:1-1:20) converted only a
portion of the reconstituted receptors into the high affinity state. In
our preliminary experiments, the PACAP receptor reconstituted with
recombinant Gi
/bovine brain G
(PACAP receptor
Gi
:G
= 1:4:8) did not have a high affinity (data
not shown). Therefore, the present result implies that the recombinant
PACAP receptor from insect cells couples to Gs
/G
efficiently and preferentially. Further reconstitution studies with
various kinds of G proteins at different molar ratios will help us to
understand the specificity of G protein coupling and the signal
transduction mechanism in the PACAP receptor.
Functional coupling between the reconstituted PACAP receptor and
Gs/G
was further studied by determining the
agonist-dependent increase in [35S]GTP
S
binding. Spontaneous [35S]GTP
S binding occurring in
the absence of agonist stimulation was very slow, probably due to the
slow dissociation rate of bound GDP from the Gs
subunit.
The addition of GDP strongly diminished the PACAP-dependent
increase in [35S]GTP
S binding to
Gs
/G
. Wieland and Jakobs (62) described that
agonist activation of the
-adrenergic receptor interacting with
Gs proteins induces a slight increase in
[35S]GTP
S binding to erythrocyte membranes in the
absence of GDP, but not in the presence of high concentrations of GDP.
Taken together, an agonist-dependent increase in
[35S]GTP
S binding to Gs protein should be
determined in the absence of GDP, although this makes it difficult to
distinguish the agonist-dependent signal from the high
basal [35S]GTP
S binding usually encountered in crude
membrane fractions. It is thus presumed that the
PACAP-dependent increase in [35S]GTP
S
binding to the CHO cell membranes observed in the presence of GDP might
not reflect primary coupling to Gs proteins but secondary coupling to other G proteins. This interpretation may account for
PACAP's low potency (EC50 values of 580 pM)
and low efficacy in the CHO membranes (the accumulated
[35S]GTP
S binding for 60 min being only about 50 pM at a receptor concentration of 700 pM).
On the other hand, [35S]GTPS binding to the
reconstituted Gs
/G
was potently enhanced by
PACAP27 and PACAP38 in the absence of GDP. The EC50 value
of PACAP27 was comparable to its Kd value from
saturation binding experiments. An increase in
[35S]GTP
S binding reached a high level, which was
compatible with the receptor concentration. An antagonist peptide
PACAP(6-38) (37) had no effect on [35S]GTP
S binding.
These results indicated that the G protein activating machinery was
properly reconstituted. Inverse agonist activity, decreasing
[35S]GTP
S binding less than control levels, was not
observed for PACAP(6-38). This finding is related to the lack of G
protein activation by an agonist-vacant PACAP receptor. These are all attributed to very slow GDP dissociation from the reconstituted Gs
/G
. Furthermore, the G protein activation
observed with the recombinant PACAP receptor from insect cells was
similar to that observed with the recombinant receptor from CHO cells.
Thus, the PACAP receptor purified from insect cells is as functionally
active as that from CHO cells.
In conclusion, the human recombinant PACAP receptor was purified from the infected Sf9 insect cells on a large scale, yielding 1 to 2 mg. The purified PACAP receptor was comparable to the PACAP receptor purified from CHO cells in ligand binding and G protein activating properties, although the N-linked sugar chains were different. The purified PACAP receptor provides a good model for studying the structure, function, and regulatory mechanisms of G protein-coupled receptors.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Hisayoshi Okazaki, Yasuhiro Sumino, Kyozo Tsukamoto, and Tsutomu Kurokawa for their helpful discussions and encouragement. We thank Dr. Kaori Wakamatsu, Gunma University, for providing purified G proteins and critical reading of the manuscript. We thank Dr. Yoshihiro Ishibashi for protein sequencing.
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FOOTNOTES |
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* 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.
To whom correspondence and reprint requests should be addressed:
Discovery Research Laboratories I, Pharmaceutical Discovery Research
Div., Takeda Chemical Industries, Ltd., Wadai 10, Tsukuba, Ibaraki
300-4293, Japan. Tel.: 81-298-64-5003; Fax: 81-298-64-5000; E-mail:
Ohtaki_Tetsuya{at}Takeda.co.jp.
1
The abbreviations used are: PACAP, pituitary
adenylate cyclase-activating polypeptide; VIP, vasoactive intestinal
polypeptide; GTPS, guanosine 5'-O-3-thiotriphosphate;
BSA, bovine serum albumin; BIGCHAP,
N,N-bis(3-D-gluconamidopropyl)cholamide; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; biotin-(AC5)2-OSu,
5-[5-(N-succinimidyloxycarbonyl)penthylamido]hexyl-D-biotinamide; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel
electrophoresis.
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REFERENCES |
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