PREPARATION OF THE EXTRACELLULAR DOMAIN OF THE RABBIT PROLACTIN RECEPTOR EXPRESSED IN ESCHERICHIA-COLI AND ITS INTERACTION WITH LACTOGENIC HORMONES

The cDNA of the extracellular domain of the rabbit prolactin receptor (rbPRLR-ECD) was cloned in the prokaryotic expression vector pTrc99A t o enable its expression in Escherichia coli after induction with isopr opyl-1-thio-beta-D-galactopyranoside. The bacterially expressed rbPRLR -ECD protein, contained within the refractile body pellet, was solubil ized in 4.5 m urea, refolded, and purified on a Q-Sepharose column by stepwise elution with NaCl. The bioactive monomeric fraction was elute d in 0.05 m NaCl, yielding 15-20 mg/8 liters of induced culture. The p urified protein was >98% homogeneous, as shown by SDS-polyacrylamide g el electrophoresis in the presence or absence of reducing agent and by chromatography on a Superdex column. Its molecular mass was 25 kDa as determined by SDS-polyacrylamide gel electrophoresis in the absence o f reducing agent and 22 kDa as determined by gel filtration. Binding e xperiments revealed remarkable differences between rabbit and porcine prolactins (PRLs) and the other tested lactogenic hormones. Gel filtra tion was used to determine the stoichiometry of the rbPRLR-ECD interac tion with ovine, rabbit, and porcine PRLs, with human growth hormone a nd its truncated des-7 analogue, and with bovine placental lactogen (b PL) and des-13-bPL. The formation of only 1:1 complexes was indicated, except with bPL, for which a 2:1 complex was detected. Identical stoi chiometry was also obtained using excess radiolabeled rbPRLR-ECD in ge l filtration experiments. Interaction of I-125-labeled ovine PRL with rbPRLR-ECD secreted into conditioned medium by rbPRLR-ECD cDNA-transfe cted COS 7 cells also indicated formation of 1:1 molar complexes. Desp ite the differences in binding potency and stoichiometries of the inte raction with rbPRLR-ECD, all seven tested hormones were biologically a ctive in inducing PRL receptor-mediated casein synthesis in explants o f rabbit mammary gland. We therefore propose that the formation of the 1:2 complexes with soluble rbPRLR-ECD is not predictive of biological activity of the different lactogenic hormones.