Site-directed mutagenesis of recombinant bovine placental lactogen at lysine-73 leads to selective attenuation of its somatogenic activity.
Bovine placental lactogen (bPL) is capable of binding and transducing biological activity via somatogenic and lactogenic receptors. To modify this capability, three analogs, bPL(K73D), bPL(K73F) and bPL(K73A), mutated at position 73, and corresponding to R64 in human GH (hGH), were produced in Escherichia coli. Circular dichroic spectrum analyses indicated proper refolding in all cases. Biological activity of these analogs was tested in vitro. In a lactogenic-receptor-mediated Nb2 rat lymphoma cell bioassay, bPL and its analogs acted similarly. In another lactogenic bioassay that measures beta-casein synthesis by HC-11 mouse mammary-gland cells, the analogs were 30-40% as potent as bPL. In contrast, somatogenic receptor-mediated bioactivity in FDC-P1 cells transfected with either rabbit (rb) or hGH receptor (R) was almost completely abolished in these analogs. In receptor binding assays, the effect was more conspicuous and the mutations affected not only somatogenic but also lactogenic binding. Binding to rat (r) and rabbit PRL receptor extracellular domains (ECDs) or membrane-embedded receptors was only slightly changed, except for bPL (K73D), which displayed very low affinity. In somatogenic binding assays to intact IM-9 human lymphocytes, hGHR-ECD or bovine liver membranes, bPL (K73D) did not bind at all, and bPL(K73F) or bPL(K73A) binding was drastically reduced. Binding experiments performed in real time using a BIAcore apparatus revealed that the decreased binding could be mainly attributed to increased k(off) rather than decreased k(on) values. The complex with hGHR-ECD revealed a 2:1 stoichiometry with bPL, bPL(K73F) and bPL(K73A), although the complex with these analogs was less stable than with bPL, whereas bPL(K73D) scarcely assembled a 1:1 complex. In contrast, bPL and the three analogs formed stable 1:2 complexes with rPRL-ECD. These results suggest that position 73 in bPL is more important for somatogenic than lactogenic properties and concurs with results from other groups, which have shown that R64, the analogous amino acid in hGH holds the same differential importance with respect to somatogenic binding.
Helman
D
D
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel.
Staten
N R
NR
Byatt
J
J
Grosclaude
J
J
McKinnie
R E
RE
Djiane
J
J
Gertler
A
A
D000818:Animals; D002364:Caseins / Q000032:analysis / Q000096:biosynthesis; D002417:Cattle; D002460:Cell Line; D002850:Chromatography, Gel; D004926:Escherichia coli; D005260:Female; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D008223:Lymphoma / Q000737:chemistry / Q000378:metabolism / Q000473:pathology; D008239:Lysine / Q000737:chemistry*; D008321:Mammary Glands, Animal / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010928:Placental Lactogen / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D051381:Rats; D018000:Receptors, Peptide / Q000032:analysis / Q000378:metabolism / Q000502:physiology; D011981:Receptors, Prolactin / Q000032:analysis / Q000378:metabolism / Q000502:physiology; D011986:Receptors, Somatotropin / Q000032:analysis / Q000378:metabolism / Q000502:physiology; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D002364:Caseins; D018000:Receptors, Peptide; D011981:Receptors, Prolactin; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; C028202:placental lactogen receptor; D010928:Placental Lactogen; D008239:Lysine