Identification and enzymatic deglycosylation of the myometrial oxytocin receptor using a radioiodinated photoreactive antagonist.
To identify and characterize oxytocin receptors, a 125I-labeled photoreactive oxytocin antagonist was synthesized. The specific oxytocin antagonist [1-(beta-mercapto-beta,beta- cyclopentamethylenepropionic acid), 2-O-methyltyrosine,4-threonine,8- ornithine,9-tyrosylamide]oxytocin ([Mca,Tyr(O-Me)2,Thr4,Orn8,Tyr9-NH2]oxytocin) described by Elands et al. (Elands, J., Barberis, C., Jard, S., Tribollet, E., Dreifuss, J.-J., Bankowski, K., Manning, M., and Sawyer, W. H. (1987) Eur. J. Pharmacol. 147, 192-207) bound to the guinea pig uterine oxytocin receptor with high affinity (apparent Kd = 0.74 nM). The introduction of a 4-azidophenylamidino group at Orn8 resulted in the photoreactive ligand [Mca1,Tyr(O-Me)2,Thr4,Orn(4-azidophenylamidino)8,Tyr9- NH2]oxytocin, which retained the high binding affinity (Kd = 0.69 nM) of the parent compound. The photoreactive antagonist monoiodinated at Tyr9 had approximately double (Kd = 0.39 nM) the affinity of the photoreactive antagonist and several times that of oxytocin (Kd = 2.6 nM) for the guinea pig uterine oxytocin receptor. In photo-affinity labeling experiments using myometrial membranes obtained from guinea pigs during late pregnancy, the 125I-labeled photoreactive antagonist specifically labeled a protein with an apparent molecular mass of between 68 and 80 kDa: the labeling of this protein was completely suppressed by a 100-fold molar excess of oxytocin and oxytocin receptor-specific agonists, but not by vasopressin analogues specific for V1 or V2 receptors or by other peptide hormones. The ability of oxytocin to suppress labeling was decreased in the presence of guanosine 5'-O-(thiotriphosphate) or in the absence of Mn2+. Digestion of the photolabeled oxytocin receptor with endoglycosidase F gave rise to a protein with an apparent molecular mass of 38 +/- 2 kDa. The endoglycosidase F effect and the lack of endoglycosidase H action show that the myometrial oxytocin receptor is highly glycosylated with asparagine-linked complex oligosaccharide chains. Our results suggest that the radioiodinated photoreactive oxytocin antagonist could be a helpful tool in the isolation and further characterization of the oxytocin receptor.
Kojro
E
E
Max-Planck-Institut für Biophysik, Frankfurt am Main, Federal Republic of Germany.
Hackenberg
M
M
Zsigo
J
J
Fahrenholz
F
F
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D001667:Binding, Competitive; D002413:Cations, Divalent; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006026:Glycoside Hydrolases / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006168:Guinea Pigs; D007457:Iodine Radioisotopes; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009215:Myometrium / Q000378:metabolism*; D010121:Oxytocin / Q000378:metabolism*; D011247:Pregnancy; D011945:Receptors, Angiotensin / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D018045:Receptors, Oxytocin; D014316:Tritium
D000345:Affinity Labels; D002413:Cations, Divalent; D007457:Iodine Radioisotopes; D011945:Receptors, Angiotensin; D018045:Receptors, Oxytocin; D014316:Tritium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010121:Oxytocin; D006026:Glycoside Hydrolases