Expression of the extracellular domain of the rat liver prolactin receptor and its interaction with ovine prolactin.
A clone of the extracellular domain of the rat liver prolactin receptor was generated by the RNA-based polymerase chain reaction, and the NH2-terminal 210 amino acids were expressed in HeLa cells using a vaccinia virus/T7 hybrid expression system. The protein was isolated from serum-free culture medium directly by chromatography on an ovine prolactin affinity column and yielded approximately 1.5 mg of protein/liter of suspension culture. The extracellular domain of the rat prolactin receptor inhibited the ovine prolactin-dependent mitogenesis of rat lymphoma Nb2 cells with an IC50 of 7.1 pM and bound 125I-labeled ovine prolactin with a Kd of 1.21 +/- 0.19 nM. In contrast, the binding of the 125I-labeled extracellular domain to ovine prolactin exhibited positive cooperativity with a Hill coefficient of 1.73. High pressure gel filtration chromatography was used to demonstrate the formation of a complex consisting of one molecule of ovine prolactin and two molecules of the extracellular domain of the rat prolactin receptor. Complex formation occurred with human growth hormone, but not with ovine growth hormone, a non-lactogen.
Hooper
K P
KP
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66160-7421.
Padmanabhan
R
R
Ebner
K E
KE
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D005260:Female; D007668:Kidney; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008223:Lymphoma; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011388:Prolactin / Q000302:isolation & purification / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D011981:Receptors, Prolactin / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D012756:Sheep; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D011981:Receptors, Prolactin; D011388:Prolactin