Oxytocin Receptor Dimerization

Objective: Investigating the existence form of oxytocin receptor(OTR) in vitro and in vivo, determining the effect of mutant OTR that 112,187 cystein residues mutate to ser on the OTR multimer, ligand binding affinity and endocytosis.Methods: The OTR existence form was investigated with the immunoprecipitation,Western Blotting ; The effect of mutant OTR on OTR multimer,ligand binding affinity and endocytosis was determined with immunoprecipitation,Western Blotting,immunofluorescence and radioligand binding assay.Results: ⑴ The OTR monomer , which apparent molecular weight is 47kDa, is commonly glycosylated, and the glycosylated OTR monomer molecular weight is 55kDa ～ 80kDa as reported. In our experiment, the OTR , which was expressed in CHO cells or extracted from Wistar Rat brain and uterus, existed mainly as multimer which consists of tetramer(MW: 244kDa), trimer(MW: 183kDa) and dimer(MW: 122kDa) in the higher concentration SDS and non-reducing circumstance. All multimers disappeared in the presence of reducing reagent of DTT or β-ME, and OTR migrated as monomer. ⑵ the mutant OTR, which the 112,187 cystein residues mutate to ser respectively or simultaneously, existed mainly as multimer whichconsists of tetramer(MW: 244kDa), trimer(MW: 183kDa) and dimer(MW: 122kDa) in the higher concentration SDS and non-reducing circumstance. All multimers disappeared in the presence of reducing reagent of DTT or β-ME, and OTR migrated as monomer. ⑶ In the radioligand binding assay, the wild type OTR bind antagonist 125I-OVT in high affinity(Kd=147.9pM), but all three mutant OTR can not bind 125I-OVT(m112187,m112,m187 Kd value as followed:1.15×1012 pM,1.12×1012 pM, 1.16×1012 pM), the same as the wild type OTR in the existence of DTT(Kd=1.35×1012 pM). ⑷ The wild type OTR internalized while continuous exposure to agonist oxytocin for 30min. However, the mutant OTR can not internalize in the same conditions. The wild type OTR in the existence of DTT can not intaernalize, too. Conclusions: ⑴ OTR existed mainly as multimer in vitro and in vivo. ⑵ the monomer of OTR multimer connected covalently(disulfide bond ). ⑶ OTR form homologous multimer. ⑷ The 112 and 187 cystein residues are not crucial for OTR multimer formation. ⑸ The 112 and 187 cystein residues of OTR are crucial for receptor-ligand binding. ⑹ The 112 and 187 cystein residues of OTR are important for receptor endocytosis...