Identification of rat serotonin 5-HT2C receptors as glycoproteins containing N-linked oligosaccharides.
Antibodies against a portion of the rat 5-HT2C receptor third intracellular loop were generated and used to identify receptors solubilized from 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 fibroblasts 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, contained immunoreactive proteins with masses of 51-52 kDa and 58-68 kDa. In the brain, immunoreactive proteins were identified from choroid plexus 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 studies demonstrating that 5-HT2C receptor binding sites and mRNA are most abundant in choroid plexus. The association of asparagine-linked (N-linked) oligosaccharides with the receptors was examined next. The 5-HT2C receptor cell line (3T3/2C) was grown in the presence of tunicamycin to metabolically inhibit N-linked glycosylation. Proteins from the cell extracts were detected with masses of 40 and 41 kDa. Extracts prepared from 3T3/2C cells (grown in the absence of tunicamycin) and from choroid plexus were incubated with N-glycosidase F to enzymatically remove available N-linked sugars. Immunoreactive proteins were detected with masses of 41 and 42 kDa from 3T3/2C cells and 41 kDa from choroid 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 contrast, the 51-52 and 58-68 kDa proteins from 3T3/2C cells were not affected by treatment with neuraminidase. These results demonstrate that 5-HT2C receptors contain N-linked sugars and suggest that sialic acid residues associate with 5-HT2C receptors in the choroid plexus. The oligosaccharide moieties, which contribute up to approximately 30% of the relative mass as judged by SDS-polyacrylamide gel electrophoresis, may impart functional properties to 5-HT2C receptors.
Backstrom
J R
JR
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-6600, USA. Backst JR@ctrvax.Vanderbilt.edu
Westphal
R S
RS
Canton
H
H
Sanders-Bush
E
E
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D002831:Choroid Plexus / Q000378:metabolism; D006026:Glycoside Hydrolases; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D009928:Organ Specificity; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D044405:Receptor, Serotonin, 5-HT2C; D011985:Receptors, Serotonin / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry*; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry; D014162:Transfection
D000906:Antibodies; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D012333:RNA, Messenger; D044405:Receptor, Serotonin, 5-HT2C; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases