Identification of Amino Acid Residues for Oxytocin Receptor Oligomerization
|School||Second Military Medical University|
|Keywords||oxytocin receptor multimer cysteine residues ligand binding affinity endocytosis|
The neurohypophyseal hormone, oxytocin(OT) , is synthesized in hypothalamic neurons and transported down axons of the posterior pituitary for secretion into blood. Besides the classical physiological function, milk-ejection and uterine smooth muscle contraction, OT has many other physiological functions including regulate central neural system and cardiovascular system. The pharmacological and physiological function of OT depend on oxytocin receptor(OTR),which distribute widely,coupling to Gq/11 and phospholipase C .OTR belongs to G-protein coupled receptors (GPCRs)family. The human OTR, which was first cloned, has 389 amino acid residues, including seven transmembrane α -helices, three intra-cellular loops and three extra-cellular loops. The cysteine residues in the first and second extra-cellular loops connect by a disulfide bond. It is important for maintaining OTR structure and ligand binding. Two other cysteine residues within the COOH-terminal domain are palmitoylation sites and form the fourth intra-cellular loop. It is probably the Gq/11 binding domain. The recent study has shown that OTR can form homo- and hetero- oligomerization, and ligand binding can not influence OTR oligomerization. These indicate that OTR oligomerization is constitutive . In the years past ,we found that OTR can form homo-oligomerization under physiological condition. The detergent can not influence OTR homo-oligomerization and multimers bands disappeared in the presence of reducing reagent. These results indicate that cysteine is crucial for OTR homo- oligomerization.From the OTR gene structure, we know that OTR contained fourteen cysteines residues, including two within intra-cellular loops, two within extra-cellular loops, seven located in transmembrane domain and three in COOH-terminal domain. We have proved that the mutants, which two cysteine residues in extra-cellular loops (112、 187)exchanged by serines, can form homo-oligomerization ,and ligand bindingand agonist-induced endocytosis of OTR mutants become obviously impaired, suggesting that two cysteine residues in extra-cellular loops involved in maintaining the conformation of OTR, mediating ligand binding but not contributing to oligomerization of OTR. So our goal is to investigate which is the crucial cysteine residues for OTR homo-oligomerization and the effect of these cysteine residues on OTR function.We constitute ten mutants by using point-mutation and then transfect them to CHO cells. The effect of mutant OTR on OTR multimer、 ligand binding affinity and endocytosis was observed by immunoprecipitation 、 Western Blotting 、 immunofluorescence and radioligand binding assay.The main results are as follows:(l)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 experiments, the OTR, which was extracted from SD rat brain and uterus, existed as monomer and multimer. The multimer is mainly consists of tetramer(MW: 244kDa), trimer(MW: 183kDa) and dimer(MW: 122kDa) in the higher concentration of SDS and non-reducing circumstance.(2)A11 multimers disappeared in the presence of reducing reagent of DTT or β -ME, and OTR transfered to monomer.(3)The OTR ,which is expressed in CHO cells, also have this characteristic. The molecular weight of monomer is 55kDa.The mutant OTR, which the 47、 59、 138、 142、 164、 219、 286、 322、 323、 345、 382 cysteine residues were mutate to serine respectively or simultaneously, existed as monomer and multimer, which mainly consists of tetramer(MW: 220kDa), in the higher concentration of SDS and non-reducing circumstance. All multimers disappeared in the presence of reducing reagent of DTT or β -ME, and OTR transfered to monomer.(4)The mutant OTR, which the 346 cysteine residue was mutated to serine, existed as monomer, either in the non-reducing circumstance or in the reducing circumstance.