Site-Directed Mutagenesis of Recombinant Bovine Placental Lactogen at Lysine-73 Leads to Selective Attenuation of Its Somatogenic Activity1

• Published in: Endocrinology (Philadelphia) Vol. 138; no. 10; pp. 4069 - 4080
• Main Authors: Helman, Daniel, Staten, Nicholas R, Byatt, John, Grosclaude, Jeanne, McKinnie, Russell E, Djiane, Jean, Gertler, Arieh
• Format: Journal Article
• Language: English
• Published: Endocrine Society 01-10-1997
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/endo.138.10.5419

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 β-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 koff rather than decreased
kon 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.