PREPARATION AND CHARACTERIZATION OF RECOMBINANT PROLACTIN RECEPTOR EXTRACELLULAR DOMAIN FROM RAT

Complementary (c)DNA of the extracellular domain of rat prolactin rece ptor (rPRLR-ECD) was cloned in the prokaryotic expression vector pTrc9 9A, and expressed in Escherichia coli following induction with isoprop yl-b-D-thiogalactopyranoside. The expressed rPRLR-ECD protein, contain ed within the refractile body pellet was solubilized in 4.5 M urea, re folded and purified on a Q-Sepharose column by stepwise elution with N aCl. Only similar to 10% of the expressed protein refolded as a monome ric fraction, yielding 5-6 mg/l of induced culture. The purified prote in was over 98% homogeneous, as shown by SDS-PAGE in the presence or a bsence of reducing agent, and by chromatography on a Superdex column. Its molecular mass, determined by SDS-PAGE in the absence of reducing agent, was 28 kDa, and by gel filtration, 25.6 kDa. Binding experiment s indicated high affinity for bovine placental lactogen (bPL) and huma n growth hormone (hGH) as compared to ovine to) or rat PRLs, Gel filtr ation was used to determine the stoichiometry of rPRLR-ECD's interacti on with these hormones. At a 5 mu M initial concentration of the hormo nes, formation of 2:1 (ECD:ligand) complexes was detected with bPL, hG H and oPRL whereas only 1:1 complex was formed with rPRL. Dilution (25 -fold) of these complexes did not affect the stoichiometry with bPL, w hereas with hGH a clear tendency towards dissociation of the initial 2 :1 complex to 1:1 complex was observed, This tendency was even stronge r in the case of oPRL. Although all four hormones exhibited nearly ide ntical activities in the Nb-2-11C lymphoma cell bioassay, the ability of the purified rat or rabbit PRLR-ECD to inhibit hormonal mitogenic a ctivity generally reflected their affinity for the respective hormones . In view of these and former results, we suggest that unlike in the G H:GHR-ECD interaction, the inability of lactogenic hormones to form a 1:2 complex with soluble recombinant PRLR-ECDs does not necessarily pr edicts lack of biological activity.