Structure-function studies of the eighth hydrophobic domain of a serotoninreceptor

The most prominent structural feature of the G protein-coupled receptor sup erfamily is their seven hydrophobic domains, which are postulated to form m embrane-spanning alpha helices, Some members of the G protein-coupled recep tor family, specifically several serotonin (5-HT) receptors, possess eight hydrophobic domains. The importance of this extra hydrophobic domain, locat ed at the N terminus of the receptor, is unknown. This question was address ed by deleting the extra hydrophobic region from the 5-HT2C receptor and co mparing its function and topology with those of the wild-type receptor. Imm unofluorescence microscopy was used to determine the location of the N term inus of the epitope-tagged wild-type and mutant receptors, The N terminus o f both receptors was extracellular, suggesting that the extra hydrophobic d omain does not change the topology of this receptor and is unlikely to be a membrane-spanning alpha helix. Radioligand-binding studies in transfected cells and expression studies in Xenopus oocytes demonstrated that seven hyd rophobic domains were sufficient for normal function in these assays. Inter estingly, the mutant receptor, now containing seven hydrophobic domains, is expressed at higher levels in transfected cells than the wild-type recepto r containing eight hydrophobic domains, suggesting that the extra hydrophob ic domain does impact the activity of this receptor by regulating its expre ssion.