IMMUNOLOGICAL RECOGNITION OF DIFFERENT FORMS OF THE NEUROTENSIN RECEPTOR IN TRANSFECTED CELLS AND RAT DRAIN

In this work, the molecular forms of the rat neurotensin receptor (NTR ) expressed in transfected Chinese hamster ovary (CHO) cells, in infec ted Sf9 insect cells and in rat cerebral cortex were immunologically d etected by means of an anti-peptide antibody raised against a fragment of the third intracellular loop of the receptor. Immunoblot experimen ts against a fusion protein indicated that the anti-peptide antibody r ecognized, under denaturing conditions, the corresponding amino acid s equence within the NTR. In immunoblot analysis of membranes from NTR-t ransfected CHO cells, high levels of immunoreactivity were observed be tween 60 and 72 kDa, while only a faint labelling was observed at 47 k Da, the molecular mass deduced for the rat NTR cDNA. The bands of high molecular mass were no longer observed after deglycosylation of membr ane proteins by peptide N-glycosidase F, indicating that they represen ted glycosylated forms of the receptor. Extracts of membranes derived from baculovirus-infected Sf9 insect-cells expressing the NTR provided a quite different immunoblot pattern, since the major band detected i n that case was at 47 kDa, the molecular size of the non-glycosylated receptor. Taken together, these data show that, while most of the NTR protein was glycosylated in CHO cells, it was unglycosylated in Sf9 in sect-cells. In addition, molecular sizes of the receptor proteins obse rved in these two cell lines differed from those obtained for the NTR endogenously expressed in the rat cerebral cortex of 7 day-old rats, w here bands at 56 and 54 kDa were detected. Binding experiments carried out on membrane preparations obtained from baculovirus-infected Sf9 c ells demonstrated that the immunogenic sequence was still accessible t o the antibody when the receptor was embedded in the cell membrane. Im munohistochemical studies carried out on both transfected CHO cells an d infected Sf9 cells confirmed this interpretation and further indicat ed that the antibody could be applied in the visualization of the rece ptor.