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J Biol Chem, Vol. 273, Issue 25, 15464-15473, June 19, 1998

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 Ohtaki, 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

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.

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