PHOTOAFFINITY-LABELING OF THE HUMAN BRAIN CHOLECYSTOKININ RECEPTOR OVEREXPRESSED IN INSECT CELLS - SOLUBILIZATION, DEGLYCOSYLATION AND PURIFICATION

The human cholecystokinin B (CCKB) receptor was expressed in Sf9 cells by infection with recombinant baculovirus. For immunodetection a c-my c epitope tag (EQKLISEEDL) was fused at the amino-terminus of the CCKB receptor. In a second construct an additional hexa-histidine tag was introduced at the C-terminus of the CCKB receptor to enable employment of metal affinity chromatography. The two receptor constructs were ex pressed at densities of 6.0 +/- 1.1 pmol/mg protein and 7.2 +/- 1.1 pm ol/mg protein, respectively which are 100-200-fold higher compared wit h the receptor amounts found in natural sources. Saturation of the bin ding sites with [H-3]propionyl-CCK8 revealed K-d values of 4.5 +/- 0.5 nM and 7.8 +/- 0.6 nM for the CCKB receptor without or with histidine tag. In SDS/PAGE and subsequent immunodetection the histidine-tagged CCKB receptor migrated as a 55-kDa band, whereas the CCKB receptor wit hout C-terminal modification revealed apparent molecular masses of 45 kDa and 49 kDa, The differences in the mass values observed for the tw o constructs suggest that the histidine tag could protect the CCKB rec eptor against proteolytical degradation from its C-terminus, Furthermo re two new photoreactive derivatives of cholecystokinin octapeptide re sidues 26-33 (CCK8) with high labeling efficiency and specificity for the cholecystokinin receptor subtype B were developed: [H-3]BzBz-des-M et28-[p-NH(2)Bz29]-CCK8 and [H-3]BzBz-biotinyl-des-Met28-[p-NH(2)Bz29] -CCK8. Both contain the p-benzoylbenzoyl (BzBz) residue at the N-termi nus for photoactivation and a p-aminobenzoyl (p-NH(2)BZ) residue inste ad of Met28-Gly29 in cholecystokinin. Enzymatic deglycosylation of the CCKB receptor with N-glycosidase F after photoaffinity labeling demon strated that the CCKB receptor with three potential glycosylation site s was slightly glycosylated, amounting to a molecular mass of about 4 kDa. Using the biotinylated cholecystokinin derivative the photoaffini ty-labeled CCKB receptor could be purified 1260-fold by a two-step pro cedure including affinity chromatography on a streptavidin/avidin agar ose matrix, For purification of the native receptor, an improved solub ilization protocol for the CCKB receptor using dodecyl beta-D-maltopyr anoside was developed. The solubilized CCKB receptors with C-terminal histidine tag retained their ligand binding characteristics after chro matography on a nickel affinity matrix.