Recombinant extracellular domain of rabbit growth hormone receptor and biological activity of somatogenic hormones.
The cDNA of the extracellular domain of rabbit growth hormone receptor (rbGHR-ECD) was cloned in the prokaryotic expression vector pMON, to enable its expression in Escherichia coli after induction with nalidixic acid. The bacterially expressed rbPRLR-ECD protein, contained within the refractile-body pellet, was solubilized in 4.5 M urea, refolded, and purified on a Q-Sepharose column, pH 8, by stepwise elution with NaCl. The bioactive monomeric 28-kDa fraction was eluted in 0.15 M NaCl, yielding 50 mg/2.5 l of induced culture. The purified protein was over 98% homogeneous, as shown by SDS-PAGE in the presence or absence of reducing agent, and by chromatography on a Superdex column. Gel filtration was used to determine the stoichiometry of rbGHR-ECD's interaction with human (h), ovine (o), chicken (ch) and common carp (cc) GHs and with bovine (b) and caprine (c) placental lactogens (PLs). The formation of 2:1 complexes was indicated in all cases. Binding experiments using radiolabelled oGH as a ligand revealed it to be the most effective competitor, followed by bPL, cPL, hGH chGH and ccGH, with respective IC50 values of 0.27, 0.94, 1.55, 2.13, 41.9 and 51.2 nM. Rabbit GHR-ECD inhibited the bPL-inducible proliferation of FDC-P1 cells stably transfected with rbGHR and Nb2 cells possessing rat PRLR. The biological activity of oGH, hGH, cPL, bPL, chGH and ccGH was tested in the FDC-P1 cells stably transfected with rbGHR and yielded the respective EC50 values (in nM) of 0.024, 0.023, 0.021, 0.24, 4.71 and 0.49. These results indicate remarkable discrepancies between the binding capacities and biological activities: the possible reasons for these findings are discussed.
Sakal
E
E
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food, and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel.
Chapnik-Cohen
N
N
Belair
L
L
Djiane
J
J
Gertler
A
A
D000818:Animals; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D013006:Growth Hormone / Q000378:metabolism*; D019382:Human Growth Hormone / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008970:Molecular Weight; D010928:Placental Lactogen / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011869:Radioligand Assay; D011986:Receptors, Somatotropin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D018076:DNA, Complementary; D008024:Ligands; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D019382:Human Growth Hormone; D013006:Growth Hormone; D010928:Placental Lactogen