Around the onset of labor, uterine sensitivity to oxytocin (OT) increases tremendously. Although this is considered to reflect OT receptor (OTR) augmentation in myometrium, neither spatial expression of OTR nor the level of the receptor message during the course of pregnancy have been investigated at the molecular level. We examined the localization and expression of the OTR in human myometrium by means of in situ hybridization, immunohistochemistry, and Northern and Western blotting. In the term pregnant myometrium, OTR expressing smooth muscle cells are observed diffusely and heterogeneously. Some of the smooth muscle cells were expressed high levels of the receptor at the messenger RNA and protein level, and they were surrounded with cells weakly positive for the OTR or negative. The level of OTR transcripts increased according to the course of pregnancy. The receptor messenger RNA level reached over 300-fold at parturition compared with the nonpregnant myometrium. In the myometrium at 32 weeks of gestation and not in labor, a relatively large amount (about 100-fold) of the receptor message was expressed. In the nonpregnant myometrium, significant amount of the receptor protein was revealed by Western blotting. We also found that the receptor protein was augmented at term and after the onset of labor. These findings indicated that the expression of OTR changes dynamically at the transcription and protein level during pregnancy and that its expression is heterogeneous in the term myometrium.

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.

Abstract. The cDNA of the common a-subunit of human glycoprotein hormone was mutated by site directed mutagenesis in the CMGCC region composed of cysteine-methionine-glycine-cysteine-cysteine (position 28-32). The cDNA of wild-type human thyrotropin (hTSH) f3-subunit and that of wild-type or mutant common a-subunits were co-transfected into COS-I cells. The concentration of hTSH determined by two immunoradiometric assay systems was detectable in culture media of COS-I cells transfected with wild-type (CMGCC) and a mutant (CMGCC) a-subunits but not four other mutants (YMGCC) (CMRCC) (CMACC) (CMGCC). The present data with the other studies on wild-type or mutant glycoprotein hormones support our hypothesis that an amino acid motif of "C-X-G-X-C" in the common a-(CMGCC in human) and fJ-(CAGYC in human) subunits play an important role in biosynthesis of glycoprotein hormones in all species.