IDENTIFICATION OF RAT SEROTONIN 5-HT2C RECEPTORS AS GLYCOPROTEINS CONTAINING N-LINKED OLIGOSACCHARIDES

Antibodies against a portion of the rat 5-HT2C receptor third intracel lular loop were generated and used to identify receptors solubilized f rom cell lines and rat brain. Western blots of CHAPS-soluble proteins were probed with affinity-purified anti-2C antibodies. The specificity of anti-2C was demonstrated with extracts prepared from NIH/3T3 fibro blasts which stably express functional rat 5-HT2C or 5-HT2A receptors. Extracts from the 5-HT2C cell line, but not the 5-HT2A cell line, con tained immunoreactive proteins with masses of 51-52 kDa and 58-68 kDa. In the brain, immunoreactive proteins were identified from choroid pl exus extracts with masses of 51 kDa and 58-62 kDa. The major 58-62 kDa and minor 51 kDa proteins were not detected in extracts prepared from the hippocampus, striatum, or frontal cortex using the same amount of CHAPS-soluble protein. These results are consistent with previous stu dies demonstrating that 5-HT2C receptor binding sites and mRNA are mos t abundant in choroid plexus. The association of asparagine-linked (N- linked) oligosaccharides with the receptors was examined next. The 5-H T2C receptor cell line (3T3/2C) was grown in the presence of tunicamyc ia to metabolically inhibit N-linked glycosylation. Proteins from the cell extracts were detected with masses of 40 and 41 kDa. Extracts pre pared from 3T3/2C cells (grown in the absence of tunicamycin) and from choroid plexus were incubated with N-glycosidase F to enzymatically r emove available N-linked sugars. Immunoreactive proteins were detected with masses of 41 and 42 kDa from 3T3/2C cells and 41 kDa from choroi d plexus. Neuraminidase, which cleaves sialic acid (N-acetylneuraminic acid) residues from glycoproteins, reduced the mass of the 51 and 58- 62 kDa proteins from the choroid plexus to 50 and 54-58 kDa. In contra st, the 51-52 and 58-68 kDa proteins from 3T3/2C cells were not affect ed by treatment with neuraminidase. These results demonstrate that 5-H T2C receptors contain N-linked sugars and suggest that sialic acid res idues associate with 5-HT2C receptors in the choroid plexus. The oligo saccharide moieties, which contribute up to similar to 30% of the rela tive mass as judged by SDS-polyacrylamide gel electrophoresis, may imp art functional properties to 5-HT2C receptors.