Expression, Purification, and Reconstitution of Receptor for Pituitary Adenylate Cyclase-activating Polypeptide

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 for125I-PACAP27 (K d = 155.3 pm) and was insensitive to guanosine 5′-O-3-thiotriphosphate (GTPγS), whereas the receptor in CHO membranes had a high affinity (K d = 44.4 pm) and was GTPγS sensitive. The receptor in Sf9 membranes was converted to a high affinity state (K d = 20–40 pm) following solubilization with digitonin. A large quantity (2 mg from 8 liters of insect cells) of the purified PACAP receptors (B max = 23.9 nmol/mg of protein) were obtained in a digitonin-induced high affinity state (K d = 17.3 pm) using biotinylated ligand affinity chromatography. The apparent molecular weight of the purified receptor (M r = 48,000) was smaller than that of the receptor from CHO cells (M r = 58,000) due to differences in asparagine-linked sugar chains. The purified receptor reverted to a low affinity state (K d = 182.6 pm) upon reconstitution into lipid vesicles, however, the receptor reconstituted with Gs protein had a high affinity (K d = 40.2 pm) and was GTPγS sensitive. [35S]GTPγS 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. 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 for125I-PACAP27 (K d = 155.3 pm) and was insensitive to guanosine 5′-O-3-thiotriphosphate (GTPγS), whereas the receptor in CHO membranes had a high affinity (K d = 44.4 pm) and was GTPγS sensitive. The receptor in Sf9 membranes was converted to a high affinity state (K d = 20–40 pm) following solubilization with digitonin. A large quantity (2 mg from 8 liters of insect cells) of the purified PACAP receptors (B max = 23.9 nmol/mg of protein) were obtained in a digitonin-induced high affinity state (K d = 17.3 pm) using biotinylated ligand affinity chromatography. The apparent molecular weight of the purified receptor (M r = 48,000) was smaller than that of the receptor from CHO cells (M r = 58,000) due to differences in asparagine-linked sugar chains. The purified receptor reverted to a low affinity state (K d = 182.6 pm) upon reconstitution into lipid vesicles, however, the receptor reconstituted with Gs protein had a high affinity (K d = 40.2 pm) and was GTPγS sensitive. [35S]GTPγS 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. Pituitary adenylate cyclase-activating polypeptide (PACAP) 1The abbreviations used are: PACAP, pituitary adenylate cyclase-activating polypeptide; VIP, vasoactive intestinal polypeptide; GTPγS, 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. 1The abbreviations used are: PACAP, pituitary adenylate cyclase-activating polypeptide; VIP, vasoactive intestinal polypeptide; GTPγS, 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. was first discovered in 1989 as a novel hypothalamic hormone that increases adenylate cyclase activity in pituitary cells (1Miyata A. Arimura A. Dahl R.R. Minamino N. Uehara A. Jiang L. Culler M.D. Coy D.H. Biochem. Biophys. Res. Commun. 1989; 164: 567-574Crossref PubMed Scopus (1664) PACAP in PACAP38 with (1Miyata A. Arimura A. Dahl R.R. Minamino N. Uehara A. Jiang L. Culler M.D. Coy D.H. Biochem. Biophys. Res. Commun. 1989; 164: 567-574Crossref PubMed Scopus (1664) and PACAP27 with the A. Jiang L. Dahl R.R. Minamino N. Arimura A. Biochem. Biophys. Res. Commun. PubMed Scopus that the of PACAP and A. Jiang L. Dahl R.R. Arimura A. Biochem. Biophys. Res. Commun. PubMed Scopus Arimura A. Biochem. Biophys. Res. Commun. PubMed Scopus PACAP to in the to vasoactive intestinal polypeptide (1Miyata A. Arimura A. Dahl R.R. Minamino N. Uehara A. Jiang L. Culler M.D. Coy D.H. Biochem. Biophys. Res. Commun. 1989; 164: 567-574Crossref PubMed Scopus (1664) PACAP in the and A. A. A. Coy D.H. PubMed Scopus and a of as cell A. A. N. PubMed Scopus of from A. of pituitary (1Miyata A. Arimura A. Dahl R.R. Minamino N. Uehara A. Jiang L. Culler M.D. Coy D.H. Biochem. Biophys. Res. Commun. 1989; 164: 567-574Crossref PubMed Scopus (1664) M.D. PubMed Scopus of from cells A. Arimura A. PubMed Scopus A. Arimura A. PubMed Scopus of from cells N. Biochem. Biophys. Res. Commun. PubMed Scopus N. A. PubMed and Arimura A. PubMed The of PACAP by a receptor and a receptor The PACAP receptor the A. PubMed Scopus PubMed Scopus Arimura A. Biochem. Biophys. Res. Commun. PubMed Scopus PubMed Scopus Biochem. Biophys. Res. Commun. PubMed Scopus Biochem. Biophys. Res. Commun. PubMed Scopus L. PubMed Scopus PubMed Scopus and a novel PubMed Scopus and L. PubMed Scopus and PubMed Scopus in the receptor in the of in the L. PubMed Scopus PubMed Scopus of receptors to the receptor and into the receptor A. N. PubMed Scopus that from receptors that the of was obtained from the of PubMed Scopus molecular of the of receptors in the receptor to a in the PubMed Scopus the the of a receptor by in the and of the receptor receptors low in to that a large of the recombinant PubMed first that a was the of and receptors high receptors purified from the insect cells were functionally as were the receptors from PubMed The was used to however, a Biochem. PubMed Scopus in a of purified receptor due to in the solubilization and of of the receptor from bovine membranes in a high affinity state Arimura A. PubMed the of the recombinant receptor by the and the The recombinant purified in a high affinity PACAP and was functionally reconstituted with Gs protein in lipid The purified receptor to the of the and of the the PACAP receptor was in Sf9 insect cells the of a The was than of receptors from to PubMed A. PubMed Scopus A. A. A. PubMed Scopus PubMed Scopus PubMed PubMed The of a and in the PACAP the of the receptor to cell to the high of the and with the of the of the receptor in cells with the using cells and Biochem. Biophys. Res. Commun. PubMed Scopus PubMed Scopus that the of the to the high of the receptors in insect cells with the by and PACAP receptor to to Gs adenylate to protein by the to PACAP as increases in L. PubMed Scopus PubMed Scopus Biophys. PubMed Scopus and N. Biochem. Biophys. Res. Commun. PubMed Scopus A. PubMed Scopus The recombinant receptor in the Sf9 cell however, was not to as by low affinity and GTPγS in due to a in the The of and in insect cells were as and in Sf9 insect cells were the A. PubMed Scopus that the of protein in Sf9 insect cells PubMed Scopus that the of recombinant receptors to in insect cells to the receptor expressed a low as the receptor A. A. A. PubMed Scopus receptor PubMed Scopus and receptor PubMed were to in indicating to the the PubMed Scopus the receptor PubMed the receptor and the receptor PubMed not GTPγS in not that of the PACAP receptors in insect cells were not to recombinant PACAP receptor in the Sf9 cell membranes was purified in a The purified receptor had a protein to that purified from CHO cells but with sugar chains. The apparent molecular weight of (M r = was smaller than the molecular weight of the protein (M r = Biochem. Biophys. Res. Commun. PubMed Scopus A of the The however, in the that in a than with a molecular differences in sugar of the PACAP The PACAP receptor from CHO cells was to and high and sugar PubMed sugar but high sugar a PubMed with the from by that the receptor from CHO cells sugar chains. the PACAP receptor from Sf9 cells was by indicating that high sugar chains. 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Biochem. PubMed Scopus had [35S]GTPγS that the protein was [35S]GTPγS binding than was not to the of protein by PACAP to from the reconstituted the protein with the recombinant PACAP receptor from insect cells was to that with the recombinant receptor from CHO cells. the PACAP receptor purified from insect cells as functionally as that from CHO the recombinant PACAP receptor was purified from the Sf9 insect cells a large to The purified PACAP receptor was to the PACAP receptor purified from CHO cells in ligand binding and protein the sugar were The purified PACAP receptor a the and of Pituitary adenylate cyclase-activating polypeptide (PACAP) 1The abbreviations used are: PACAP, pituitary adenylate cyclase-activating polypeptide; VIP, vasoactive intestinal polypeptide; GTPγS, 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. 1The abbreviations used are: PACAP, pituitary adenylate cyclase-activating polypeptide; VIP, vasoactive intestinal polypeptide; GTPγS, 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. was first discovered in 1989 as a novel hypothalamic hormone that increases adenylate cyclase activity in pituitary cells (1Miyata A. Arimura A. Dahl R.R. Minamino N. Uehara A. Jiang L. Culler M.D. Coy D.H. Biochem. Biophys. Res. Commun. 1989; 164: 567-574Crossref PubMed Scopus (1664) PACAP in PACAP38 with (1Miyata A. Arimura A. Dahl R.R. Minamino N. Uehara A. Jiang L. Culler M.D. Coy D.H. Biochem. Biophys. Res. Commun. 1989; 164: 567-574Crossref PubMed Scopus (1664) and PACAP27 with the A. Jiang L. Dahl R.R. Minamino N. Arimura A. Biochem. Biophys. Res. Commun. PubMed Scopus that the of PACAP and A. Jiang L. Dahl R.R. Arimura A. Biochem. Biophys. Res. Commun. PubMed Scopus Arimura A. Biochem. Biophys. Res. Commun. PubMed Scopus PACAP to in the to vasoactive intestinal polypeptide (1Miyata A. Arimura A. Dahl R.R. Minamino N. Uehara A. Jiang L. Culler M.D. Coy D.H. Biochem. Biophys. Res. Commun. 1989; 164: 567-574Crossref PubMed Scopus (1664) PACAP in the and A. A. A. Coy D.H. PubMed Scopus and a of as cell A. A. N. PubMed Scopus of from A. of pituitary (1Miyata A. Arimura A. Dahl R.R. Minamino N. Uehara A. Jiang L. Culler M.D. Coy D.H. Biochem. Biophys. Res. Commun. 1989; 164: 567-574Crossref PubMed Scopus (1664) M.D. PubMed Scopus of from cells A. Arimura A. PubMed Scopus A. Arimura A. PubMed Scopus of from cells N. Biochem. Biophys. Res. Commun. PubMed Scopus N. A. PubMed and Arimura A. PubMed The of PACAP by a receptor and a receptor The PACAP receptor the A. PubMed Scopus PubMed Scopus Arimura A. Biochem. Biophys. Res. Commun. PubMed Scopus PubMed Scopus Biochem. Biophys. Res. Commun. PubMed Scopus Biochem. Biophys. Res. Commun. PubMed Scopus L. PubMed Scopus PubMed Scopus and a novel PubMed Scopus and L. PubMed Scopus and PubMed Scopus in the receptor in the of in the L. PubMed Scopus PubMed Scopus of receptors to the receptor and into the receptor A. N. PubMed Scopus that from receptors that the of was obtained from the of PubMed Scopus molecular of the of receptors in the receptor to a in the PubMed Scopus the the of a receptor by in the and of the receptor receptors low in to that a large of the recombinant PubMed first that a was the of and receptors high receptors purified from the insect cells were functionally as were the receptors from PubMed The was used to however, a Biochem. PubMed Scopus in a of purified receptor due to in the solubilization and of of the receptor from bovine membranes in a high affinity state Arimura A. PubMed the of the recombinant receptor by the and the The recombinant purified in a high affinity PACAP and was functionally reconstituted with Gs protein in lipid The purified receptor to the of the and of the the PACAP receptor was in Sf9 insect cells the of a The was than of receptors from to PubMed A. PubMed Scopus A. A. A. PubMed Scopus PubMed Scopus PubMed PubMed The of a and in the PACAP the of the receptor to cell to the high of the and with the of the of the receptor in cells with the using cells and Biochem. Biophys. Res. Commun. PubMed Scopus PubMed Scopus that the of the to the high of the receptors in insect cells with the by and PACAP receptor to to Gs adenylate to protein by the to PACAP as increases in L. PubMed Scopus PubMed Scopus Biophys. PubMed Scopus and N. Biochem. Biophys. Res. Commun. PubMed Scopus A. PubMed Scopus The recombinant receptor in the Sf9 cell however, was not to as by low affinity and GTPγS in due to a in the The of and in insect cells were as and in Sf9 insect cells were the A. PubMed Scopus that the of protein in Sf9 insect cells PubMed Scopus that the of recombinant receptors to in insect cells to the receptor expressed a low as the receptor A. A. A. PubMed Scopus receptor PubMed Scopus and receptor PubMed were to in indicating to the the PubMed Scopus the receptor PubMed the receptor and the receptor PubMed not GTPγS in not that of the PACAP receptors in insect cells were not to recombinant PACAP receptor in the Sf9 cell membranes was purified in a The purified receptor had a protein to that purified from CHO cells but with sugar chains. The apparent molecular weight of (M r = was smaller than the molecular weight of the protein (M r = Biochem. Biophys. Res. Commun. PubMed Scopus A of the The however, in the that in a than with a molecular differences in sugar of the PACAP The PACAP receptor from CHO cells was to and high and sugar PubMed sugar but high sugar a PubMed with the from by that the receptor from CHO cells sugar chains. the PACAP receptor from Sf9 cells was by indicating that high sugar chains. The of sugar however, that the PACAP receptor from Sf9 cells but not and The of high sugar with the from but with the to that the PACAP receptor from Sf9 cells sugar sugar Scopus than high sugar by of and of sugar as PubMed the of sugar not to the of sugar in the PACAP purified PACAP receptor upon were in receptors as PubMed and the receptor A. Biochem. PubMed Scopus that the receptor a A. the to a in A. PubMed Scopus The of receptor in high used as by Biochem. PubMed Scopus was that the receptor A. Biochem. PubMed Scopus in purified receptor had high affinity PACAP27 and PACAP38 but low affinity ligand binding were to of the receptor expressed in CHO cells. that the PACAP receptor in the high affinity state the that purified PACAP receptor a high affinity by Arimura A. PubMed was by the purified PACAP receptor into lipid The receptor reconstituted in lipid had low affinity in the of but high affinity in the of digitonin. The molecular of however, not not solubilization into a the receptor the of a of into not the receptor protein by the of the The d the purified receptor in a and the reconstituted receptor in the that solubilization the of digitonin. in the PACAP receptor by in the of ligand binding that the of from receptor to as A. PubMed and PubMed were using digitonin. The receptor with had high affinity ligand and GTPγS 1989; PubMed Scopus as in the the of in the solubilization of hormone PubMed and A. with in high affinity receptor PACAP receptor reconstituted with in lipid had high affinity in to that GTPγS, to protein by the protein the of that the purified PACAP receptor reconstituted in lipid high affinity The max the PACAP receptor was from The to the that in PACAP a low affinity state (K d = pm) and a low affinity state affinity max in the and of receptor in a low affinity that the CHO membranes PACAP receptor as as PACAP The of the PACAP receptors the high affinity state in the CHO membranes the of protein to the PACAP as as GTPγS the binding to the reconstituted The molecular of to the of from the protein that high affinity binding of PACAP27 the of whereas the of to by the PACAP receptor high affinity PACAP27 with PACAP binding to the receptor the binding the to the PACAP binding and A with in receptors as the receptor A. PubMed The the the of ligand binding of a high affinity was with a smaller of protein = with reconstitution and PubMed that a of the receptor of high the reconstitution of the receptor L. PubMed Scopus receptor A. PubMed a smaller protein converted a of the reconstituted receptors into the high affinity the PACAP receptor reconstituted with recombinant receptor = not a high affinity not the that the recombinant PACAP receptor from insect cells to and reconstitution with of to the of protein and the in the PACAP the reconstituted PACAP receptor and was by the in [35S]GTPγS [35S]GTPγS binding in the of was due to the of from the The of the in [35S]GTPγS binding to and PubMed Scopus that of the receptor with Gs a in [35S]GTPγS binding to membranes in the of but not in the of high of in [35S]GTPγS binding to Gs protein in the of to the from the high [35S]GTPγS binding in that the in [35S]GTPγS binding to the CHO cell membranes in the of not to Gs but to low (EC50 of pm) and low in the CHO membranes [35S]GTPγS binding a receptor of the [35S]GTPγS binding to the reconstituted was enhanced by PACAP27 and PACAP38 in the of The of PACAP27 was to d from binding in [35S]GTPγS binding a high was with the receptor antagonist A. Biochem. PubMed Scopus had [35S]GTPγS that the protein was [35S]GTPγS binding than was not to the of protein by PACAP to from the reconstituted the protein with the recombinant PACAP receptor from insect cells was to that with the recombinant receptor from CHO cells. the PACAP receptor purified from insect cells as functionally as that from CHO the recombinant PACAP receptor was purified from the Sf9 insect cells a large to The purified PACAP receptor was to the PACAP receptor purified from CHO cells in ligand binding and protein the sugar were The purified PACAP receptor a the and of the the PACAP receptor was in Sf9 insect cells the of a The was than of receptors from to PubMed A. PubMed Scopus A. A. A. PubMed Scopus PubMed Scopus PubMed PubMed The of a and in the PACAP the of the receptor to cell to the high of the and with the of the of the receptor in cells with the using cells and Biochem. Biophys. Res. Commun. PubMed Scopus PubMed Scopus that the of the to the high of the receptors in insect cells with the by and The PACAP receptor to to Gs adenylate to protein by the to PACAP as increases in L. PubMed Scopus PubMed Scopus Biophys. PubMed Scopus and N. Biochem. Biophys. Res. Commun. PubMed Scopus A. PubMed Scopus The recombinant receptor in the Sf9 cell however, was not to as by low affinity and GTPγS in due to a in the The of and in insect cells were as and in Sf9 insect cells were the A. PubMed Scopus that the of protein in Sf9 insect cells PubMed Scopus that the of recombinant receptors to in insect cells to the receptor expressed a low as the receptor A. A. A. PubMed Scopus receptor PubMed Scopus and receptor PubMed were to in indicating to the the PubMed Scopus the receptor PubMed the receptor and the receptor PubMed not GTPγS in not that of the PACAP receptors in insect cells were not to The recombinant PACAP receptor in the Sf9 cell membranes was purified in a The purified receptor had a protein to that purified from CHO cells but with sugar chains. The apparent molecular weight of (M r = was smaller than the molecular weight of the protein (M r = Biochem. Biophys. Res. Commun. PubMed Scopus A of the The however, in the that in a than with a molecular differences in sugar of the PACAP The PACAP receptor from CHO cells was to and high and sugar PubMed sugar but high sugar a PubMed with the from by that the receptor from CHO cells sugar chains. the PACAP receptor from Sf9 cells was by indicating that high sugar chains. The of sugar however, that the PACAP receptor from Sf9 cells but not and The of high sugar with the from but with the to that the PACAP receptor from Sf9 cells sugar sugar Scopus than high sugar by of and of sugar as PubMed the of sugar not to the of sugar in the PACAP The purified PACAP receptor upon were in receptors as PubMed and the receptor A. Biochem. PubMed Scopus that the receptor a A. the to a in A. PubMed Scopus The of receptor in high used as by Biochem. PubMed Scopus was that the receptor A. Biochem. PubMed Scopus in The purified receptor had high affinity PACAP27 and PACAP38 but low affinity ligand binding were to of the receptor expressed in CHO cells. that the PACAP receptor in the high affinity state the that purified PACAP receptor a high affinity by Arimura A. PubMed was by the purified PACAP receptor into lipid The receptor reconstituted in lipid had low affinity in the of but high affinity in the of digitonin. The molecular of however, not not solubilization into a the receptor the of a of into not the receptor protein by the of the The d the purified receptor in a and the reconstituted receptor in the that solubilization the of digitonin. in the PACAP receptor by in the of ligand binding that the of from receptor to as A. PubMed and PubMed were using digitonin. The receptor with had high affinity ligand and GTPγS 1989; PubMed Scopus as in the the of in the solubilization of hormone PubMed and A. with in high affinity receptor The PACAP receptor reconstituted with in lipid had high affinity in to that GTPγS, to protein by the protein the of that the purified PACAP receptor reconstituted in lipid high affinity The max the PACAP receptor was from The to the that in PACAP a low affinity state (K d = pm) and a low affinity state affinity max in the and of receptor in a low affinity that the CHO membranes PACAP receptor as as PACAP The of the PACAP receptors the high affinity state in the CHO membranes the of protein to the PACAP as as GTPγS the binding to the reconstituted The molecular of to the of from the protein that high affinity binding of PACAP27 the of whereas the of to by the PACAP receptor high affinity PACAP27 with PACAP binding to the receptor the binding the to the PACAP binding and A with in receptors as the receptor A. PubMed The the the of ligand binding The of a high affinity was with a smaller of protein = with reconstitution and PubMed that a of the receptor of high the reconstitution of the receptor L. PubMed Scopus receptor A. PubMed a smaller protein converted a of the reconstituted receptors into the high affinity the PACAP receptor reconstituted with recombinant receptor = not a high affinity not the that the recombinant PACAP receptor from insect cells to and reconstitution with of to the of protein and the in the PACAP the reconstituted PACAP receptor and was by the in [35S]GTPγS [35S]GTPγS binding in the of was due to the of from the The of the in [35S]GTPγS binding to and PubMed Scopus that of the receptor with Gs a in [35S]GTPγS binding to membranes in the of but not in the of high of in [35S]GTPγS binding to Gs protein in the of to the from the high [35S]GTPγS binding in that the in [35S]GTPγS binding to the CHO cell membranes in the of not to Gs but to low (EC50 of pm) and low in the CHO membranes [35S]GTPγS binding a receptor of the [35S]GTPγS binding to the reconstituted was enhanced by PACAP27 and PACAP38 in the of The of PACAP27 was to d from binding in [35S]GTPγS binding a high was with the receptor antagonist A. Biochem. PubMed Scopus had [35S]GTPγS that the protein was [35S]GTPγS binding than was not to the of protein by PACAP to from the reconstituted the protein with the recombinant PACAP receptor from insect cells was to that with the recombinant receptor from CHO cells. the PACAP receptor purified from insect cells as functionally as that from CHO cells. the recombinant PACAP receptor was purified from the Sf9 insect cells a large to The purified PACAP receptor was to the PACAP receptor purified from CHO cells in ligand binding and protein the sugar were The purified PACAP receptor a the and of to and and purified and of the protein