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J Biol Chem, Vol. 273, Issue 25, 15464-15473, June 19, 1998
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 (GTP
S), whereas the receptor in
CHO membranes had a high affinity (Kd = 44.4 pM) and was GTPS 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 GTPS
sensitive. [35S]GTPS binding to the reconstituted
Gs protein was enhanced by PACAP27 and PACAP38
(EC50 = 42.5 and 9.4 pM, respectively) but not
by antagonist PACAP(6-38), indicating that the purified receptor was
functionally active.
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