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

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 I-125-PACAP27 (K-d = 155.3 pM) and was insensitive to guanosine 5'-O-3-thiotriphosphate (GTP gamma S), whereas the receptor in CHO membranes had a high affinity (K-d = 44.4 pM) and was GTP gamma S 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 G(s) protein had a high affinity (K-d = 40.2 pM) and was GTP gamma S sensitive. [S-35]GTP gamma S binding to the reconstituted G(s) protein was enhanced by PACAP27 and PACAP38 (EC50 = 42.8 and 9.4 pM, respectively) but not by antagonist PACAP(6-38), indicating that the purified receptor was functionally active.