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.
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