SITE-DIRECTED MUTAGENESIS OF RECOMBINANT BOVINE PLACENTAL-LACTOGEN ATLYSINE-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 modif y this capability, three analogs, bPL(K73D), bPL(K73F) and bPL(K73A), mutated at position 73, and corresponding to R64 in human GH (hGH), we re produced in Escherichia coli. Circular dichroic spectrum analyses i ndicated proper refolding in all cases. Biological activity of these a nalogs was tested in vitro. In a lactogenic-receptor-mediated Nb2 rat lymphoma cell bioassay, bPL and its analogs acted similarly. In anothe r lactogenic bioassay that measures beta-casein synthesis by HC-11 mou se mammary-gland cells, the analogs were 30-40% as potent as bPL. In c ontrast, somatogenic receptor-mediated bioactivity in FDC-P1 cells tra nsfected with either rabbit (rb) or hGH receptor (R) was almost comple tely abolished in these analogs. In receptor binding assays, the effec t was more conspicuous and the mutations affected not only somatogenic but also lactogenic binding. Binding to rat (r) and rabbit PRL recept or extracellular domains (ECDs) or membrane-embedded receptors was onl y slightly changed, except for bPL (K73D), which displayed very law af finity. 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 ex periments performed in real time using a BIAcore apparatus revealed th at the decreased binding could be mainly attributed to increased k(off ) rather than decreased k(on) values. The complex with hGHR-ECD reveal ed 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 thre e analogs formed stable 1:2 complexes with rPRL-ECD. These results sug gest that position 73 in bPL is more important for somatogenic than la ctogenic properties and concurs with results from other groups, which have shown that R64, the analogous amino acid in hGH holds the same di fferential importance with respect to somatogenic binding.