Extracellular domain of prolactin receptor from bovine mammary gland: expression in Escherichia coli, purification and characterization of its interaction with lactogenic hormones.
The cDNA of the extracellular domain of bovine prolactin receptor (bPRLR-ECD) was cloned and expressed at high yield as an insoluble protein in Escherichia coli. This protein was solubilized, refolded and purified to > 98% homogeneity yielding 80 mg of monomeric fraction per 2 litres of induced culture. Its molecular mass was 25.7 kDa, as determined by SDS-PAGE in the absence of reducing agent and 24 kDa by gel filtration on a Superdex column. Binding experiments revealed that bPRLR-ECD binds to human (h) GH (hGH) with high affinity, whereas its affinity for ovine (o) or bovine (b) prolactins (PRLs) was lower and for bovine placental lactogen (bPL) very low. The affinity of bPRLR-ECD for the latter three hormones was, however, much higher than that of membrane-embedded or solubilized bPRLR. The stoichiometries of interaction of bPRLR-ECD with hGH, oPRL, bPRL and bPL were determined by gel-filtration chromatography. Even at a 3:1 ECD excess, only 1:1 complexes were detected at microM concentrations of ECD and ligand. At an up to 32-fold dilution, the complexes with oPRL, bPRL, and particularly bPL, underwent progressive dissociation, whereas the complex with hGH remained stable. Although all four hormones exhibited nearly identical activity in the Nb2 lymphoma cell bioassay, the ability of bPRLR-ECD to inhibit hormonal mitogenic activities differed, generally reflecting its affinity for the respective hormones. In view of these and previous results, we suggest that, unlike in the GH:GHR-ECD interaction, neither the stoichiometry of interaction of bovine or other PRLR-ECDs nor the affinity constants can predict the biological potency of the different lactogenic hormones.
Tchelet
A
A
Department of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot, Israel.
Staten
N R
NR
Creely
D P
DP
Krivi
G G
GG
Gertler
A
A
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002850:Chromatography, Gel; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005260:Female; D013006:Growth Hormone / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010928:Placental Lactogen / Q000378:metabolism; D011388:Prolactin / Q000378:metabolism*; D051381:Rats; D011981:Receptors, Prolactin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D013045:Species Specificity
D017931:DNA Primers; D011981:Receptors, Prolactin; D011994:Recombinant Proteins; D011388:Prolactin; D013006:Growth Hormone; D010928:Placental Lactogen