Expression, Purification, and Reconstitution of Receptor for Pituitary Adenylate Cyclase-activating Polypeptide
LARGE-SCALE PURIFICATION OF A FUNCTIONALLY ACTIVE G PROTEIN-COUPLED RECEPTOR PRODUCED IN SF9 INSECT CELLS*

Tetsuya OhtakiDagger , Kazuhiro Ogi, Yasushi Masuda, Kaoru Mitsuoka§, Yoshinori Fujiyoshi§, Chieko Kitada, Hidekazu Sawada, Haruo Onda, and Masahiko Fujino

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

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 (GTPgamma S), whereas the receptor in CHO membranes had a high affinity (Kd = 44.4 pM) and was GTPgamma 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 GTPgamma S sensitive. [35S]GTPgamma 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.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha -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 beta -adrenergic and muscarinic receptors at high levels (5-30 pmol/mg). Recombinant beta -adrenergic receptors purified from the baculovirus-infected insect cells were functionally active as were the beta -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.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 (GTPgamma S) 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-beta -glucosaminyl) asparagine amidase (N-glycanase, recombinant), F. menigosepticum endo-beta -N-acetylglucosaminidase F2 (endoglycosidase F2), Streptomyces plicatus endo-beta -N-acetylglucosaminidase (endoglycosidase H, recombinant), and Streptococcus sp. sialidase (neuraminidase) were from Genzyme (Cambridge, MA). Xanthomonas manihotis beta -N-acetylglucosaminidase and X. manihotis alpha 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]GTPgamma S was obtained from NEN Life Science Products (Boston, MA).

Purified recombinant Gsalpha and purified brain Gbeta gamma were generous gifts from Dr. Kaori Wakamatsu (Gunma University). Gsalpha with a His10 tag sequence at its amino-terminal was expressed in Escherichia coli, purified using a Ni-NTA agarose (Qiagen, Germany) column, and treated with Factor Xa to remove the His10 tag sequence (30). Brain Gbeta gamma was purified as described previously (31). In the Gbeta gamma preparation, cholate was replaced with CHAPS using hydroxyapatite chromatography.

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 SRalpha 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, 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 M alpha -methylmannoside. The receptor was further applied to a hydroxyapatite column (1 ml) for concentration and detergent exchange. After washing the column with 10 ml of 20 mM Hepes, 1 M NaCl (pH 6.8) buffer, the receptor was eluted with 0.1% BIGCHAP (or CHAPS), 0.6 M potassium phosphate buffer (pH 7.6). The receptor was concentrated up to 1 mg/ml using a Centriplus 10 concentrator (Amicon Inc., Beverly, MA) and stored on ice. Although the receptor activity was stable for several months, protein-free insoluble materials developed over time. The receptor solution was centrifuged before use to remove the insolubilities.

Reconstitution 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 Gsalpha (80 pmol/8 µl), and purified brain Gbeta gamma (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.

GTPgamma S 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]GTPgamma 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 alpha 1-2,3-mannosidase (1 units) in 50 mM sodium citrate (pH 6.0), or X. manihotis beta -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).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 SRalpha 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).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1.   Scatchard plot analysis for saturation binding experiments with the membranes. Equilibrium binding of increasing concentration of 125I-PACAP27 to: A, the P1+2+3 membranes from the infected Sf9 cells (0.37 µg/ml); or B, that from the transformed CHO cells (PACR19 clone) (0.94 µg/ml) was determined in the BSA/TED or DG-BSA/TED buffer. Nonspecific binding was determined in the presence of 0.1 µM PACAP27. Binding data was plotted versus total ligand (inset) and analyzed by Scatchard plot.

The specific binding of 125I-PACAP27 to the CHO P1+2+3 membranes was sensitive to GTPgamma S in the BSA/TED buffer (Table I). The effect of GTPgamma S was more obvious in BSA/TED buffer supplemented with 5 mM MgCl2 (BSA/TED-Mg buffer). The specific binding to the Sf9 P1+2+3 membranes, however, was not sensitive to GTPgamma S in either condition (Table I).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Effect of GTPgamma S on 125I-PACAP binding to the membranes
Equilibrium binding of 100 pM 125I-PACAP27 to the Sf9 and CHO P1+2+3 membranes was determined in various buffers containing 20 µM GTPgamma S. Specific binding was derived by subtracting nonspecific binding from total binding. Results were shown in percent of control specific binding determined in the absence of GTPgamma S.

Thus, the recombinant PACAP receptor in the Sf9 cell membranes was of low affinity and GTPgamma S insensitive, suggesting that the receptor was not coupled to G protein. On the other hand, the high affinity ligand binding with GTPgamma S sensitivity indicates that the PACAP receptor in the CHO cell membranes is coupled to G protein.

Lack of Functional Coupling to G Protein in Sf9 Cell Membranes-- Agonist-dependent stimulation of [35S]GTPgamma S 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]GTPgamma S binding occurring in the absence of the agonist. The binding of [35S]GTPgamma S to the CHO P1+2+3 membranes increased markedly in the presence of 1 µM PACAP27 (Table II). The increase in [35S]GTPgamma 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]GTPgamma 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]GTPgamma 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.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Agonist stimulation of [35S]GTPgamma S binding to the membranes
Binding of [35S]GTPgamma S to the Sf9 and CHO P1+2+3 membranes in the presence and absence of 1 µM PACAP27 was determined in the BSA/TED-Mg buffer containing 1 µM GDP and 150 mM NaCl and in the BSA/TED-Mg buffer containing 1 µM GDP.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   Agonist concentration dependence of [35S]GTPgamma S binding to the membranes. Binding of [35S]GTPgamma S to the P1+2+3 membranes from the transformed CHO cells (13.6 µg/ml) was determined in the BSA/TED-Mg buffer containing 1 µM GDP, 150 mM NaCl, and increasing concentration of indicated peptide. Increase in GTPgamma S binding was obtained by subtracting basal binding (16 pM) from total binding.

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 GTPgamma S in the DG-BSA/TED buffer (Table I).

These findings suggest that digitonin stabilizes the PACAP receptor in a high affinity state independent of G protein-coupling states. Higher estimation for Bmax values in the presence of digitonin suggests the existence of the PACAP receptor that does not have detectable affinity for 125I-PACAP27 in the absence of digitonin.

The Sf9 P0 membranes, the residue remaining after preparing the P1+2+3 membranes, contained a significant amount of the PACAP receptor (Bmax = 50-90 pmol/mg of protein, determined in the DG-BSA/TED buffer) with comparable Kd values, and thus were combined with the P1+2+3 membranes in the purification study. On average, 150 nmol of the receptor was produced in the combined membranes from an 8-liter culture of Sf9 cells (Table III). The CHO cells at 50,000 cm2 (8 units of Nunc Cell Factory) produced approximately 15 nmol of the receptor in the P1+2+3 membranes (300 mg of membrane protein).

                              
View this table:
[in this window]
[in a new window]
 
Table III
Purification of the recombinant PACAP receptor from an 8-liter culture of the insect cells
Results are mean ± S.E. of the result from three batches of purification.

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 epsilon -amino residues in PACAP38. The apparent affinity of the biotinylated ligand mixture, however, was three times higher than the affinity of the previous biotinylated ligand, [biotin-Cys28]PACAP27, developed for the purification of the bovine brain PACAP receptor (28). The present ligands improved the yield of the affinity chromatography step (Fig. 3A). More than 60% of the receptor was recovered in the acidic eluate (Table III) using a small amount of biotinylated PACAP38 (ligand:receptor = 4:1) for affinity chromatography (Fig. 3A), although only 20-40% of the receptor was recovered in the previous affinity chromatography using a larger amount of [biotin-Cys28]PACAP27 (ligand:receptor = 30:1) (28, 36).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Purification of the PACAP receptor by biotinylated PACAP38/avidin-Affi-Gel 10 and lentil lectin-Sepharose 4B chromatography. A, recombinant PACAP receptor (144 nmol) solubilized from the combined Sf9 P1+2+3 and P0 membranes (8-liter culture) was adsorbed onto avidin-Affi-Gel 10 (80 ml) via biotinylated PACAP38 (600-700 nmol), eluted with 0.2% digitonin, 1 M NaCl, 10% glycerol, magnesium acetate buffer (pH 4.0) and neutralized with 1 M Tris/HCl (pH 7.5). Ligand (100 pM 125I-PACAP27) binding activity in each fraction (10 ml) was assayed at 33,000-fold dilution. B, the pooled fractions (3.5 mg of protein) from biotinylated PACAP38/avidin-Affi-Gel 10 chromatography were loaded onto a lentil lectin-Sepharose 4B column (9 ml). After washing the column with 0.2% digitonin, 0.5 M NaCl, 20 mM Tris buffer (pH 7.5), the receptor was eluted with the same buffer including 0.5 M alpha -methylmannoside. Ligand binding activity in each fraction (2.5 ml) was assayed at 33,000- and 3,300-fold dilution.

The affinity-purified receptor was further separated into two fractions by lentil lectin-Sepharose 4B chromatography (Fig. 3B). More than 60 to 70% of the affinity-purified receptor was recovered in eluate with alpha -methylmannoside, whereas the remainder was found in the flow-through fraction. The alpha -methylmannoside-eluted fraction, yielding approximately 2 mg from an 8-liter culture (Table III), was used for the subsequent functional studies after exchanging excess digitonin with another detergent with a higher critical micelle concentration, such as BIGCHAP or CHAPS, using hydroxyapatite chromatography. BIGCHAP had the second least denaturing effect on the purified receptor among the detergents tested.

The PACAP receptor expressed in the CHO cells was also solubilized with digitonin and purified using biotinylated PACAP38/avidin Affi-Gel 10 affinity chromatography. The lectin affinity chromatography step was not included in this purification. Approximately 0.1 to 0.2 mg of the purified receptor was obtained from 8 units of Nunc Cell Factory.

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).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   Binding experiment with the purified PACAP receptor. A, equilibrium binding of increasing concentration of 125I-PACAP27 to the purified PACAP receptor from the infected Sf9 cells (1.8 ng/ml) or from the transformed CHO cells (1.4 ng/ml) was determined in the DG-BSA/TED buffer. Nonspecific binding was determined in the presence of 0.1 µM PACAP27. Binding data was plotted versus total ligand (inset) and was analyzed using Scatchard plot. B, equilibrium binding of 125I-PACAP27 (100 pM) to the purified PACAP receptor (2.6 ng/ml) from the infected Sf9 cells was determined in the DG-BSA/TED buffer including increasing concentration of indicated peptide.

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.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 5.   SDS-PAGE of the purified receptor and deglycosylated receptor. The purified PACAP receptor (0.5 µg) from the infected Sf9 insect cells and from the transformed CHO cells were digested with: A, N-glycanase, endoglycosidase F2, endoglycosidase H; B, X. manihotis alpha 1-2,3-mannosidase, X. manihotis beta -N-acetylglucosaminidase, and Streptococcus sp. sialidase. The digested and undigested PACAP receptors were analyzed using SDS-PAGE in the absence (A) and presence (A and B) of dithiothreitol.

Both receptors mobilized faster under non-reducing conditions than under reducing conditions (Fig. 5A), suggesting that these receptor proteins have intramolecular disulfide linkages in their structure. This result was compatible with the amino acid sequence deduced by cDNA cloning (19). The human PACAP receptor has 15 Cys residues, 7 of which are located in the amino-terminal extracellular domain, 3 in the extracellular loops, 4 in the transmembrane domains, and 1 in the intracellular loop (19). In addition to a conserved disulfide bond linking the first and second extracellular loops, several disulfide bonds are presumably formed in the amino-terminal extracellular domain.

Higher molecular weight bands, probably dimers, tetramers, and octamers were observed regardless of the presence of the reducing reagent (Fig. 5A). Trimeric bands were not observed, however, suggesting that the PACAP receptor might form dimer units but not randomly sized oligomers. The oligomerization was not related to the expression system, because oligomer bands were found with the PACAP receptor from CHO cells.

The PACAP receptor deglycosylated by digestion with N-glycanase had a sharper band at Mr = 43,000 (Fig. 5A). The PACAP receptor from CHO cells presented a similar deglycosylated band (Fig. 5A). Treatment with O-glycanase following neuraminidase pretreatment did not influence the mobility of either receptor preparation (data not shown). This indicates that the difference in the apparent molecular weight of the PACAP receptor from different expression systems is due mainly to different asparagine-linked (N-linked) sugar chains.

Endoglycosidase H digestion did not influence the electrophoretic mobility of the PACAP receptor from Sf9 cells or CHO cells (Fig. 5A). Endoglycosidase F2 digested the PACAP receptor from Sf9 cells, but did not digest the PACAP receptor from CHO cells (Fig. 5A). The mobility of the PACAP receptor from Sf9 cells changed after digestion with X. manihotis alpha 1-2,3-mannosidase (Fig. 5B), but not after digestion with Streptococcus sp. sialidase or X. manihotis beta -N-acetylglucosaminidase (Fig. 5B). On the other hand, the mobility of the PACAP receptor from CHO cells increased only after digestion with Streptococcus sp. sialidase (Fig. 5B).

Ligand Binding Properties of the Reconstituted PACAP Receptor-- The purified receptor from insect cells was reconstituted with and without recombinant Gsalpha /bovine brain Gbeta gamma at the molar ratio of 1:4:8 (receptor:Gsalpha :Gbeta gamma ) in crude brain lipid vesicles. Saturation binding experiments performed in the BSA/TED buffer demonstrated that the purified PACAP receptor required Gsalpha /Gbeta gamma for expressing high affinity for PACAP27 when it was reconstituted into lipid vesicles (Fig. 6A). The dissociation constant of the reconstituted receptor without Gsalpha /Gbeta gamma (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 Gsalpha /Gbeta gamma 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).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 6.   Scatchard plot analysis for saturation binding experiments with reconstituted receptor. The purified PACAP receptor (20 pmol) from the Sf9 cells was reconstituted with or without recombinant Gsalpha (80 pmol)/bovine brain Gbeta gamma (160 pmol) in lipid vesicles. Equilibrium binding of increasing concentration of 125I-PACAP27 to the reconstitute (2400-fold dilution) with or without Gsalpha /Gbeta gamma was determined in the BSA/TED buffer (A: bullet , open circle ), in the BSA/TED buffer containing 100 µM GTPgamma S (A: black-triangle), or in the DG-BSA/TED buffer (B). Binding data was plotted versus total ligand (inset) and analyzed using Scatchard plot.

The specific binding of 125I-PACAP27 to the reconstitute with Gsalpha /Gbeta gamma was very sensitive to guanine nucleotides, GTPgamma S, GTP, and GDP but not to GMP and ATP (Fig. 7). In the presence of GTPgamma S, the reconstitute with Gsalpha /Gbeta gamma had decreased affinity for 125I-PACAP27 (Kd = 112.9 ± 18 pM) (Fig. 6A). The Bmax value also decreased to the same range as observed in the reconstitute without Gsalpha /Gbeta gamma (Fig. 6A). This result indicates that G protein-uncoupling causes a decrease in both affinity and apparent receptor concentration.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of nucleotide on the 125I-PACAP27 binding to the reconstituted PACAP receptor. Equilibrium binding of 100 pM 125I-PACAP27 to the purified PACAP receptor reconstituted with Gsalpha /Gbeta gamma (prepared as in Fig. 6, 2400-fold dilution) was determined in the BSA/TED buffer (open symbols) or in the DG-BSA/TED buffer (closed) including increasing concentrations of the indicated nucleotide. Nonspecific binding was determined in the presence of 0.1 µM PACAP27. Percent of control specific binding was plotted versus nucleotide concentration.

In the presence of digitonin, the reconstitutes with and without Gsalpha /Gbeta gamma had comparable high affinity for 125I-PACAP27. The Kd value for the reconstitute with Gsalpha /Gbeta gamma was 40.6 ± 2.5 pM and that without Gsalpha /Gbeta gamma was 43.6 ± 1.3 pM (Fig. 6B). These values were comparable to the value (Kd = 44.6 ± 2.1 pM, Fig. 1A) of the PACAP receptor in the Sf9 P1+2+3 membranes determined in the DG-BSA/TED buffer. The specific binding of 125I-PACAP27 to the reconstitute with Gsalpha /Gbeta gamma was not affected by GTPgamma S in the DG-BSA/TED buffer (Fig. 7). The result confirmed that the PACAP receptor had high affinity for PACAP27 regardless of G protein-coupling states when a low concentration of digitonin was included in the binding buffer.

The receptor concentration from the Bmax value (Fig. 6B) indicated that 50 to 80% of the initial receptor was recovered in the reconstitute. The Bmax value of the reconstitute with Gsalpha /Gbeta gamma determined in the BSA/TED buffer (Fig. 6A) was similar to that obtained in the DG-BSA/TED buffer (Fig. 6B), indicating that most of the reconstituted receptors were coupled to G protein and existed in the high affinity state.

G Protein Activation by the Reconstituted PACAP Receptor-- Agonist-dependent stimulation of [35S]GTPgamma S binding to the reconstitute was determined to investigate functional coupling of the recombinant PACAP receptor to G protein. Spontaneous [35S]GTPgamma 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]GTPgamma 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]GTPgamma S binding (Fig. 9).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 8.   Time course of [35S]GTPgamma S binding to the reconstitute in the presence and absence of PACAP27. Binding of [35S]GTPgamma S to the reconstitute with Gsalpha /Gbeta gamma (prepared as in Fig. 6, 2200-fold dilution) for the indicated incubation period was determined in the BSA/TED-Mg buffer with or without 1 µM PACAP27.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 9.   Effect of GDP on [35S]GTPgamma S binding to the reconstitute. Binding of [35S]GTPgamma S to the reconstitute with Gsalpha /Gbeta gamma (prepared as in Fig. 6, 2200-fold dilution) in the presence of the indicated concentration of GDP was determined in the BSA/TED-Mg buffer with or without 1 µM PACAP27.

The increase in [35S]GTPgamma S binding was dependent on agonist concentration (Fig. 10). Taking the maximal [35S]GTPgamma S binding level as [35S]GTPgamma S binding at 1 µM PACAP27, the EC50 values obtained were 9.4 ± 0.5 pM for PACAP38 and 42.5 ± 10 pM for PACAP27. These peptides did not increase [35S]GTPgamma S binding to the reconstitute lacking the PACAP receptor (Fig. 10, inset). Thus, the increase in [35S]GTPgamma S binding by PACAP is mediated by the reconstituted PACAP receptor. There was no significant difference in basal [35S]GTPgamma S binding between the reconstitutes with and without the PACAP receptor, suggesting that the PACAP receptor by itself did not activate G proteins. Antagonist peptide, PACAP(6-38) (37), did not increase the binding of [35S]GTPgamma S (Fig. 10). The reconstitute with the PACAP receptor purified from the CHO cells provided a similar increase in [35S]GTPgamma S binding and EC50 value (63.5 ± 13 pM for PACAP27 in Fig. 10). This suggests that the coupling efficiency of the PACAP receptor produced in Sf9 cells is comparable to that of the PACAP receptor produced in CHO cells.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 10.   Agonist concentration dependence of [35S]GTPgamma S binding to the reconstitute. The purified PACAP receptor from the Sf9 cells (20 pmol, open symbols) or the purified receptor from the CHO cells (20 pmol, closed) was reconstituted with Gsalpha (80 pmol)/Gbeta gamma (160 pmol) in lipid vesicles. Binding of [35S]GTPgamma S to the reconstitute (2200-fold dilution) was determined in the BSA/TED-Mg buffer with increasing concentration of the indicated peptide. A similar experiment was performed using the reconstituted Gsalpha (80 pmol)/Gbeta gamma (160 pmol) without the PACAP receptor (inset). Increase in GTPgamma S binding was obtained by subtracting basal binding (20 pM) from total binding.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 SRalpha 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 GTPgamma S 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 beta -adrenergic receptor and the muscarinic receptor (30 pmol/mg) (26), did not exhibit GTPgamma 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 alpha -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 Manalpha 1right-arrow3(Manalpha 1right-arrow6)Manbeta 1right-arrow4GlcNAc2 (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 beta -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 GTPgamma 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 Gsalpha /Gbeta gamma in lipid vesicles had high affinity for PACAP27, in contrast to that without Gsalpha /Gbeta gamma . GTPgamma S, known to inhibit receptor/G protein coupling by destabilizing the G protein trimer, almost completely neutralized the effect of Gsalpha /Gbeta gamma , demonstrating that the purified PACAP receptor reconstituted in lipid vesicles requires Gsalpha /Gbeta gamma 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 GTPgamma S 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 alpha -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:Gsalpha :Gbeta gamma  = 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 beta 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 Gialpha /bovine brain Gbeta gamma (PACAP receptor Gialpha :Gbeta gamma  = 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 Gsalpha /Gbeta gamma 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 Gsalpha /Gbeta gamma was further studied by determining the agonist-dependent increase in [35S]GTPgamma S binding. Spontaneous [35S]GTPgamma S binding occurring in the absence of agonist stimulation was very slow, probably due to the slow dissociation rate of bound GDP from the Gsalpha subunit. The addition of GDP strongly diminished the PACAP-dependent increase in [35S]GTPgamma S binding to Gsalpha /Gbeta gamma . Wieland and Jakobs (62) described that agonist activation of the beta -adrenergic receptor interacting with Gs proteins induces a slight increase in [35S]GTPgamma 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]GTPgamma 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]GTPgamma S binding usually encountered in crude membrane fractions. It is thus presumed that the PACAP-dependent increase in [35S]GTPgamma 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]GTPgamma S binding for 60 min being only about 50 pM at a receptor concentration of 700 pM).

On the other hand, [35S]GTPgamma S binding to the reconstituted Gsalpha /Gbeta gamma 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]GTPgamma 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]GTPgamma S binding. These results indicated that the G protein activating machinery was properly reconstituted. Inverse agonist activity, decreasing [35S]GTPgamma 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 Gsalpha /Gbeta gamma . 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.

    ACKNOWLEDGEMENTS

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.

    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.

Dagger 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; GTPgamma S, 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.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Miyata, A., Arimura, A., Dahl, R. R., Minamino, N., Uehara, A., Jiang, L., Culler, M. D., and Coy, D. H. (1989) Biochem. Biophys. Res. Commun. 164, 567-574[Medline] [Order article via Infotrieve]
  2. Miyata, A., Jiang, L., Dahl, R. R., Kitada, C., Kubo, K., Fujino, M., Minamino, N., and Arimura, A. (1990) Biochem. Biophys. Res. Commun. 170, 643-648[Medline] [Order article via Infotrieve]
  3. Kimura, C., Ohkubo, S., Ogi, K., Hosoya, M., Itoh, Y., Onda, H., Miyata, A., Jiang, L., Dahl, R. R., Stibbs, H. H., Arimura, A., and Fujino, M. (1990) Biochem. Biophys. Res. Commun. 166, 81-89[Medline] [Order article via Infotrieve]
  4. Ogi, K., Kimura, C., Onda, H., Arimura, A., and Fujino, M. (1990) Biochem. Biophys. Res. Commun. 173, 1271-1279[Medline] [Order article via Infotrieve]
  5. Arimura, A., Somogyvári-Vigh, A., Miyata, A., Mizuno, K., Coy, D. H., and Kitada, C. (1991) Endocrinology 129, 2787-2789[Abstract]
  6. Arimura, A., Somogyvari-Vigh, A., Weill, C., Fiore, R. C., Tatsuno, I., Bay, V., and Brenneman, D. E. (1994) Ann. N. Y. Acad. Sci. 739, 228-243[Medline] [Order article via Infotrieve]
  7. Cavallaro, S., Copani, A., D'Agata, V., Musco, S., Petralia, S., Ventra, C., Stivala, F., Travali, S., and Canonico, P. L. (1996) Mol. Pharmacol. 50, 60-66[Abstract]
  8. Culler, M. D., and Paschall, C. S. (1991) Endocrinology 129, 2260-2262[Abstract]
  9. Tatsuno, I., Somogyvari-Vigh, A., Mizuno, K., Gottschall, P. E., Hidaka, H., and Arimura, A. (1991) Endocrinology 129, 1797-1804[Abstract]
  10. Matsumoto, H., Koyama, C., Sawada, T., Koike, K., Hirota, K., Miyake, A., Arimura, A., and Inoue, K. (1993) Endocrinology 133, 2150-2155[Abstract]
  11. Watanabe, T., Masuo, Y., Matsumoto, H., Suzuki, N., Ohtaki, T., Masuda, Y., Kitada, C., Tsuda, M., and Fujino, M. (1992) Biochem. Biophys. Res. Commun. 182, 403-411[Medline] [Order article via Infotrieve]
  12. Watanabe, T., Shimamoto, N., Takahashi, A., and Fujino, M. (1995) Am. J. Physiol. 269, E903-E909[Abstract/Free Full Text]
  13. Yada, T., Sakurada, M., Ihida, K., Nakata, M., Murata, F., Arimura, A., and Kikuchi, M. (1994) J. Biol. Chem. 269, 1290-1293[Abstract/Free Full Text]
  14. Pisegna, J. R., and Wank, S. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6345-6349[Abstract]
  15. Hashimoto, H., Ishihara, T., Shigemoto, R., Mori, K., and Nagata, S. (1993) Neuron 11, 333-342[Medline] [Order article via Infotrieve]
  16. Hosoya, M., Onda, H., Ogi, K., Masuda, Y., Miyamoto, Y., Ohtaki, T., Okazaki, H., Arimura, A., and Fujino, M. (1993) Biochem. Biophys. Res. Commun. 194, 133-143[CrossRef][Medline] [Order article via Infotrieve]
  17. Morrow, J. A., Lutz, E. M., West, K. M., Fink, G., and Harmar, A. J. (1993) FEBS Lett. 329, 99-105[CrossRef][Medline] [Order article via Infotrieve]
  18. Svoboda, M., Tastenoy, M., Ciccarelli, E., Stiévenart, M., and Christophe, J. (1993) Biochem. Biophys. Res. Commun. 195, 881-888[CrossRef][Medline] [Order article via Infotrieve]
  19. Ogi, K., Miyamoto, Y., Masuda, Y., Habata, Y., Hosoya, M., Ohtaki, T., Masuo, Y., Onda, H., and Fujino, M. (1993) Biochem. Biophys. Res. Commun. 196, 1511-1521[CrossRef][Medline] [Order article via Infotrieve]
  20. 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]
  21. Pisegna, J. R., and Wank, S. A. (1996) J. Biol. Chem. 271, 17267-17274[Abstract/Free Full Text]
  22. Chatterjee, T. K., Sharma, R. V., and Fisher, R. A. (1996) J. Biol. Chem. 271, 32226-32232[Abstract/Free Full Text]
  23. Laburthe, M., Couvineau, A., Gaudin, P., Maoret, J.-J., Rouyer-Fessard, C., and Nicole, P. (1996) Ann. N. Y. Acad. Sci. 805, 94-109[Medline] [Order article via Infotrieve]
  24. Unger, V. M., Hargrave, P. A., Baldwin, J. M., and Schertler, G. F. X. (1997) Nature 389, 203-206[CrossRef][Medline] [Order article via Infotrieve]
  25. Donnelly, D. (1997) FEBS Lett. 409, 431-436[CrossRef][Medline] [Order article via Infotrieve]
  26. Parker, E. M., Kameyama, K., Higashijima, T., and Ross, E. M. (1991) J. Biol. Chem. 266, 519-527[Abstract/Free Full Text]
  27. Doi, T., Hiroaki, Y., Arimoto, I., Fujiyoshi, Y., Okamoto, T., Satoh, M., and Furuichi, Y. (1997) Eur. J. Biochem. 248, 139-148[Abstract]
  28. Ohtaki, T., Masuda, Y., Ishibashi, Y., Kitada, C., Arimura, A., and Fujino, M. (1993) J. Biol. Chem. 268, 26650-26657[Abstract/Free Full Text]
  29. Ohtaki, T., Watanabe, T., Ishibashi, Y., Kitada, C., Tsuda, M., Gottschall, P. E., Arimura, A., and Fujino, M. (1990) Biochem. Biophys. Res. Commun. 171, 838-844[Medline] [Order article via Infotrieve]
  30. Tanaka, T., Kohno, T., Kinoshita, S., Mukai, H., Itoh, H., Ohya, M., Miyazawa, T., Higashijima, T., and Wakamatsu, K. (1998) J. Biol. Chem. 273, 3247-3252[Abstract/Free Full Text]
  31. Roof, D. J., Applebury, M. L., and Sternweis, P. C. (1985) J. Biol. Chem. 260, 16242-16249[Abstract/Free Full Text]
  32. Ohtaki, T., Ogi, K., Kitada, C., Hinuma, S., and Onda, H. (1996) Ann. N. Y. Acad. Sci. 805, 590-594[Medline] [Order article via Infotrieve] (abstr.)
  33. Masuda, Y., Sugo, T., Kikuchi, T., Kawata, A., Satoh, M., Fujisawa, Y., Itoh, Y., Wakimasu, M., and Ohtaki, T. (1996) J. Pharmacol. Exp. Ther. 279, 675-685[Abstract]
  34. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  35. Schaffner, W., and Weissmann, C. (1973) Anal. Biochem. 56, 502-514[Medline] [Order article via Infotrieve]
  36. Ohtaki, T., Kitada, C., and Onda, H. (1996) in Biomethods 7: A Laboratory Guide to Biotin-Labeling in Biomolecule Analysis (Meier, T., and Fahlenholz, F., eds), pp. 45-63, Birkhäuser Verlag AG, Basel, Switzerland
  37. 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]
  38. Butkerait, P., Zheng, Y., Hallak, H., Graham, T. E., Miller, H. A., Burris, K. D., Molinoff, P. B., and Manning, D. R. (1995) J. Biol. Chem. 270, 18691-18699[Abstract/Free Full Text]
  39. Mills, A., Allet, B., Bernard, A., Chabert, C., Brandt, E., Cavegn, C., Chollet, A., and Kawashima, E. (1993) FEBS Lett. 320, 130-134[CrossRef][Medline] [Order article via Infotrieve]
  40. Ng, G. Y. K., George, S. R., Zastawny, R. L., Caron, M., Bouvier, M., Dennis, M., and O'Dowd, B. F. (1993) Biochemistry 32, 11727-11733[Medline] [Order article via Infotrieve]
  41. Mulheron, J. G., Casañas, S. J., Arthur, J. M., Garnovskaya, M. N., Gettys, T. W., and Raymond, J. R. (1994) J. Biol. Chem. 269, 12954-12962[Abstract/Free Full Text]
  42. Quehenberger, O., Prossnitz, E. R., Cochrane, C. G., and Ye, R. D. (1992) J. Biol. Chem. 267, 19757-19760[Abstract/Free Full Text]
  43. Miyamoto, Y., Habata, Y., Ohtaki, T., Masuda, Y., Ogi, K., Onda, H., and Fujino, M. (1994) Biochim. Biophys. Acta 1218, 297-307[Medline] [Order article via Infotrieve]
  44. Delporte, C., Poloczek, P., de Neef, P., Vertongen, P., Ciccarelli, E., Svoboda, M., Herchuelz, A., Winand, J., and Robberecht, P. (1995) Mol. Cell. Endocrinol. 107, 71-76[CrossRef][Medline] [Order article via Infotrieve]
  45. Wehmeyer, A., and Schulz, R. (1997) J. Neurochem. 68, 1361-1371[Medline] [Order article via Infotrieve]
  46. Trimble, R. B., and Tarentino, A. L. (1991) J. Biol. Chem. 266, 1646-1651[Abstract/Free Full Text]
  47. Altman, F. (1996) Trends Glycosci. Glycotechnol. 8, 101-114
  48. Borjigin, J., and Nathans, J. (1994) J. Biol. Chem. 269, 14715-14722[Abstract/Free Full Text]
  49. Nekrasova, E., Sosinskaya, A., Natochin, M., Lancet, D., and Gat, U. (1996) Eur. J. Biochem. 238, 28-37[Abstract]
  50. Potter, L. T., Ballesteros, L. A., Bichajian, L. H., Ferrendelli, C. A., Fisher, A., Hanchett, H. E., and Zhang, R. (1991) Mol. Pharmacol. 39, 211-221[Abstract]
  51. Michel, H., Oesterhelt, D., and Henderson, R. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 338-342[Abstract]
  52. Sagné, C., Isambert, M.-F., Henry, J.-P., and Gasnier, B. (1996) Biochem. J. 316, 825-831[Medline] [Order article via Infotrieve]
  53. Cubero, A., and Malbon, C. C. (1984) J. Biol. Chem. 259, 1344-1350[Abstract/Free Full Text]
  54. Sheikh, S. P., Hansen, A. P., and Williams, J. A. (1991) J. Biol. Chem. 266, 23959-23966[Abstract/Free Full Text]
  55. Grigoriadis, D. E., Zaczek, R., Pearsall, D. M., and De Souza, E. B. (1989) Endocrinology 125, 3068-3077[Abstract]
  56. Hazum, E., Schvartz, I., Waksman, Y., and Keinan, D. (1986) J. Biol. Chem. 261, 13043-13048[Abstract/Free Full Text]
  57. Faubeta ner, A., Heinz-Erian, P., Klier, C., and Roscher, A. A. (1991) J. Biol. Chem. 266, 9442-9446[Abstract/Free Full Text]
  58. Haga, K., Haga, T., and Ichiyama, A. (1986) J. Biol. Chem. 261, 10133-10140[Abstract/Free Full Text]
  59. Onaran, H. O., Costa, T., and Rodbard, D. (1993) Mol. Pharmacol. 43, 245-256[Abstract]
  60. Florio, V. A., and Sternweis, P. C. (1985) J. Biol. Chem. 260, 3477-3483[Abstract]
  61. Cerione, R. A., Codina, J., Benovic, J. L., Lefkowitz, R. J., Birnbaumer, L., and Caron, M. G. (1984) Biochemistry 23, 4519-4525[Medline] [Order article via Infotrieve]
  62. Wieland, T., and Jakobs, K. H. (1994) Methods Enzymol. 237, 3-13[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.