The role of N-terminal glycosylation in the human oxytocin receptor.

The human oxytocin receptor includes three N-glycosylation sites in its extracellular N-terminal domain. We have established permanent cell-lines in which the gene for the human oxytocin receptor (OTR) has been introduced into HeLa cells. These cells differ by the disruption of one or more of the N-terminal N-glycosylation sites by site-directed mutagenesis of the transfected OTR constructs. The binding capacity of each transfectant, calculated per mg membrane protein, was 5-17 times higher than that of human term myometrium. The pharmacological characteristics of the transfected wild-type OTR are very similar to those of native myometrial OTR. The mutation of N-glycosylation sites (Asn-X-Ser/Thr), namely OTR-D8N15N26 (Asn8-->Asp8), -N8D15N26(Asn15-->Asp15), -N8D15D26(Asn15-->Asp15, Asn26-->Asp26) and -D8N15D26 (Asn8-->Asp8, Asn26-->Asp26) appear to affect neither their dissociation constant (Kd), nor the affinities for various oxytocin related ligands. As a high level of cell surface binding was retained for each clone, receptor trafficking appears to be normal. This suggests that the full glycosylation of OTR observed in vivo is not essential for its activity. These results indicate also that these cell lines may prove very useful for pharmacological screening of oxytocin related products.