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-myc 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 expressed at densities of 6.0 +/- 1.1 pmol/mg protein and 7.2 +/- 1.1 pmol/mg protein, respectively which are 100-200-fold higher compared with the receptor amounts found in natural sources. Saturation of the binding sites with [3H]propionyl-CCK8 revealed Kd 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 without C-terminal modification revealed apparent molecular masses of 45 kDa and 49 kDa. The differences in the mass values observed for the two constructs suggest that the histidine tag could protect the CCKB receptor against proteolytical degradation from its C-terminus. Furthermore two new photoreactive derivatives of cholecystokinin octapeptide residues 26-33 (CCK8) with high labeling efficiency and specificity for the cholecystokinin receptor subtype B were developed: [3H]BzBz-des-Met28-[p-NH2Bz29]-CCK8 and [3H]BzBz-biotinyl-des-Met28-[p-NH2Bz29]-CCK8. Both contain the p-benzoyl-benzoyl (BzBz) residue at the N-terminus for photoactivation and a p-aminobenzoyl (p-NH2BZ) residue instead of Met28-Gly29 in cholecystokinin. Enzymatic deglycosylation of the CCKB receptor with N-glycosidase F after photoaffinity labeling demonstrated that the CCKB receptor with three potential glycosylation sites was slightly glycosylated, amounting to a molecular mass of about 4 kDa. Using the biotinylated cholecystokinin derivative the photoaffinity-labeled CCKB receptor could be purified 1260-fold by a two-step procedure including affinity chromatography on a streptavidin/avidin agarose matrix. For purification of the native receptor, an improved solubilization protocol for the CCKB receptor using dodecyl beta-D-maltopyranoside was developed. The solubilized CCKB receptors with C-terminal histidine tag retained their ligand binding characteristics after chromatography on a nickel affinity matrix.
Gimpl
G
G
Max-Planck-Institut für Biophysik, Frankfurt, Germany.
Anders
J
J
Thiele
C
C
Fahrenholz
F
F
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D002460:Cell Line; D004274:DNA, Recombinant / Q000235:genetics; D000939:Epitopes / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000276:immunology; D011949:Receptors, Cholecystokinin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D018411:Spodoptera
D000345:Affinity Labels; D004274:DNA, Recombinant; D000939:Epitopes; D016271:Proto-Oncogene Proteins c-myc; D011949:Receptors, Cholecystokinin; D011993:Recombinant Fusion Proteins