Direct identification of the agonist binding site in the human brain cholecystokinin-B receptor

In investigating the agonist binding site of the human brain cholecystokinin(B) receptor (CCK(B)R), we employed the direct protein chemical approach using a photoreactive tritiated analogue of sulfated cholecystokinin octapeptide, which contains the p-benzoylbenzoyl moiety at the N-terminus, followed by purification of the affinity-labeled receptor to homogeneity. This probe bound specifically, saturably, and with high affinity (K(D) = 1.2 nM) to the CCK(B)R and has full agonistic activity. As the starting material for receptor purification, we used stably transfected HEK 293 cells overexpressing functional CCK(B)R. Covalent labeling of the WGA- lectin-enriched receptor revealed a 70-80 kDa glycoprotein with a protein core of about 50 kDa. Identification of the agonist binding site was achieved by the application of subsequent chemical and enzymatical cleavage to the purified receptor. A radiolabeled peptide was identified by Edman degradation amino acid sequence analysis combined with MALDI-TOF mass spectrometry. The position of the radioactive probe within the identified peptide was determined using combined tandem electrospray mass spectrometry and peptide mapping. The probe was covalently attached within the sequence L