Comprehensive analysis of hetrotrimeric G protein subunits for the purpose of elucidation and treatment of human diseases

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This project has yielded remarkable progresses in four different fields. [1] Comprehensive analysis of the G protein-coupling ability in various physiologically important receptors. Our original G protein hybrid method proved that somatostatin receptor type 3 (SSTR3) couples to Gαi, Gαq/11, Gα16, but not to Gαo, Gαz, Gα12, Gα13, or Gα14. Likewise, the study clarified that angiotensin II receptor type 1 (AT1) couples to all G proteins except for Gα13, and that AT2 does so to Gαi3. [2] Development of a new method to specify the G protein-coupling domains in receptors. Using our original method, we have identified two Gs coupling domains in the calcitonin receptor, in which one in the third intracellular loop plays a major role and another in the C-terminal tail an assisting role. By combining both methods [1] and [2], it is theoretically possible to analyze all kinds of G protein linkage in all receptors. [3] Analysis of novel biological functions mediated by G protein subunits. We have clarified that Gao subunit and associated Gβ2 subunit mediate the neurotoxic signal of the well-established familial Alzheimer's disease (FAD) gene V642I-APP through NADPH oxidase, and that this pathway is shared by other FAD genes K595N/M596L-APP and N141I-PS2. Unexpectedly, another FAD gene M146L-PS1 generates a neurotoxic signal in a similar framework, but differing only in details : M146L-PS1 activates an unknown target of pertussis toxin and then NO synthase. We have also found that LRP bound by ApoE4 (the most established AD risk factor) triggers a neurotoxic mechanism through any of Gαi, but not Gαo. [4] targeted disruption of Gβ2 subunit. We have clarified its genomic structure, constructed an appropriate targeting vector, and microinjected it to ES cells, using which chimeric mice have been born. We will continue their analysis.