We propose to establish a fundamental understanding of the critical structural and energetic driving forces at the molecular level in cytokine hormone-induced receptor activation and regulation processes. The endocrine class of cytokine hormones initiate signaling cascades by inducing receptor homodimerization in a programmed two step sequence. The current models are inadequate because they lack the functional data relating to the energetics of the binding of the second receptor - the critical regulatory step. We have developed new methods that can characterize and control the energetics of the regulatory step, setting the stage for new experiments to more broadly examine the structural and energetic criteria of cytokine-induced signaling processes. Based on extensive structural and mutagenesis research, we propose a new conceptual model for receptor homodimerization. We have determined that changes in Site 1 receptor binding affect the overall strengths and kinetics of Site2 receptor binding, as well as the specificity and binding characteristics of individual residues at the site. These cooperativity relationships were wholly unanticipated and the implications are significant with regard for engineering tunable properties into modified biomedically important proteins. Using growth hormone (OH) and prolactin (PR.L) systems, we organize our Specific Aims to address the crucial structural and biophysical issues relating to hormone-induced receptor dimerization at three levels. 1) Through structural and protein engineering studies, we propose to establish a detailed molecular basis for Site I -Site2 cooperativity, identify the structural elements through which the effects are transmitted, and determine the energetic and structural changes it induces. 2) Using newly developed phage display methods, we will select for hormone variants that display strong Site 1 -Site2 cooperativity and thus, regulate receptor homodimerization in controlled ways. This approach should allow development of biomedically important hormone analogs that can homodimerize defective receptors and circumvent immunogenicity problems. 3) As longer range goals, we will generalize our findings in the hGH system by undertaking a set of parallel protein engineering studies based on prolactin-like molecules.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Molecular and Cellular Biophysics Study Section (BBCA)
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Blondel, Olivier
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University of Chicago
Schools of Medicine
United States
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Walsh, Scott T R; Kossiakoff, Anthony A (2006) Crystal structure and site 1 binding energetics of human placental lactogen. J Mol Biol 358:773-84
Horn, James R; Kraybill, Brian; Petro, Elizabeth J et al. (2006) The role of protein dynamics in increasing binding affinity for an engineered protein-protein interaction established by H/D exchange mass spectrometry. Biochemistry 45:8488-98
Kouadio, Jean-Louis K; Horn, James R; Pal, Gabor et al. (2005) Shotgun alanine scanning shows that growth hormone can bind productively to its receptor through a drastically minimized interface. J Biol Chem 280:25524-32
Walsh, Scott T R; Sylvester, Juliesta E; Kossiakoff, Anthony A (2004) The high- and low-affinity receptor binding sites of growth hormone are allosterically coupled. Proc Natl Acad Sci U S A 101:17078-83
Bernat, Bryan; Sun, Miao; Dwyer, Mary et al. (2004) Dissecting the binding energy epitope of a high-affinity variant of human growth hormone: cooperative and additive effects from combining mutations from independently selected phage display mutagenesis libraries. Biochemistry 43:6076-84
Bernat, Bryan; Pal, Gabor; Sun, Miao et al. (2003) Determination of the energetics governing the regulatory step in growth hormone-induced receptor homodimerization. Proc Natl Acad Sci U S A 100:952-7
Walsh, Scott T R; Jevitts, Liz M; Sylvester, Juliesta E et al. (2003) Site2 binding energetics of the regulatory step of growth hormone-induced receptor homodimerization. Protein Sci 12:1960-70