This Article Full Text Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Helman, D. Articles by Gertler, A. Search for Related Content PubMed PubMed Citation Articles by Helman, D. Articles by Gertler, A.

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

Institute of Biochemistry, Food Science and Nutrition (D.H., A.G.), Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel; Unite d’Endocrinologie Moleculaire (J.D.), Unite de Virologie et Immunologie Moleculaire (J.G.), Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex, France; and Animal Science Division and Searle (N.R.S., J.B., R.E.M.), c/o Monsanto Co., Department of Molecular Biology, St. Louis, Missouri 63198

Address all correspondence and requests for reprints to: Arieh Gertler, Institute of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel. E-mail: gertler{at}agri.huji.ac.il

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 ß-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.

This article has been cited by other articles:

				E. Biener, C. Martin, N. Daniel, S. J. Frank, V. E. Centonze, B. Herman, J. Djiane, and A. Gertler Ovine Placental Lactogen-Induced Heterodimerization of Ovine Growth Hormone and Prolactin Receptors in Living Cells Is Demonstrated by Fluorescence Resonance Energy Transfer Microscopy and Leads to Prolonged Phosphorylation of Signal Transducer and Activator of Transcription (STAT)1 and STAT3 Endocrinology, August 1, 2003; 144(8): 3532 - 3540. [Abstract] [Full Text] [PDF]

				A. Herman, C. Bignon, N. Daniel, J. Grosclaude, A. Gertler, and J. Djiane Functional Heterodimerization of Prolactin and Growth Hormone Receptors by Ovine Placental Lactogen J. Biol. Chem., February 25, 2000; 275(9): 6295 - 6301. [Abstract] [Full Text] [PDF]

				A. Herman, D. Helman, O. Livnah, and A. Gertler Ruminant Placental Lactogens Act as Antagonists to Homologous Growth Hormone Receptors and as Agonists to Human or Rabbit Growth Hormone Receptors J. Biol. Chem., March 19, 1999; 274(12): 7631 - 7639. [Abstract] [Full Text] [PDF]

				E Juarez-Aguilar, F Castro-Munozledo, N. Guerra-Rodriguez, D Resendez-Perez, H. Martinez-Rodriguez, H. Barrera-Saldana, and W Kuri-Harcuch Functional domains of human growth hormone necessary for the adipogenic activity of hGH/hPL chimeric molecules J. Cell Sci., January 9, 1999; 112(18): 3127 - 3135. [Abstract] [PDF]

				D. Helman, N. R. Staten, J. Grosclaude, N. Daniel, C. Nespoulous, J. Djiane, and A. Gertler Novel Recombinant Analogues of Bovine Placental Lactogen. G133K AND G133R PROVIDE A TOOL TO UNDERSTAND THE DIFFERENCE BETWEEN THE ACTION OF PROLACTIN AND GROWTH HORMONE RECEPTORS J. Biol. Chem., June 26, 1998; 273(26): 16067 - 16074. [Abstract] [Full Text] [PDF]