In previous years we had disrupted the genes encoding the pertussis toxin sensitive G proteins Gi2, Gi1, Gi3 and Go. Conditional knockouts for Gi2 and Go were also generated. Double knockouts involving Gi2 and Go are lethal. We expect to learn from combining Gi2 with Gi3 and enhanced survival of Go KO mice by removing the floxed genes at various times after birth. Breeding programs have been set up to add cre recombinase under several specific promoters so as to remove the genes both generally in all issues or in specific cell types such as in dopaminergic neurons to remove Go or lymphocytes to remove Gi from Gi3 KO mice. Most phenotypic studies are done in collaboration with outside investigators, A second aim of this project is to establish the inter-relationship among the Gs, Gi and Gbeta-gamma components of the G protein signaling system, in as much as to learn how they regulate the different adenylyl cyclase (AC) isoforms. Previous studies by others have used recombinant adenylyl cyclases and studied their regulation by purified G proteins activated by non-hydolyzable GTP (GTP-gamma-S). We are using a different approach: we express the different ACs in HEK cells, isolate their membranes - with the properly folded and active enzyme - add to the membranes constitutively active Gs-alpha (GsalphaQ227L)which stimulates all nine AC isoforms (without need of adding a non-hydolyzable GTP) and then study changes in activity due to addition of constitutively active PTX-sentitive G protein alphas (Gi1, Gi2, Gi3, Go1 or Go2),all prepared in myristoylated form as described in our previous Annual Report and do so without and with co-addition of either Gbeta-gamma (prepared in and purified from SF9 cells, or Ca2+/Calmodulin (CaCaM) synthesized in E coli. WE hope inthsi way to establish once and for all whether Go's are inhibitory regulators of ACs and to what extent this may be dependent on an AC subtype. We continued under the guidance of Dr. Yanshun Liu (staff scientist) to work on the co-crystalization of rhodopsin with its cognate G protein transducin. Progress was poor and we terminated this aspect of your activities. We continue collaborating with extramural scientists in the analysis of the phenotypes that arise in G protein deficient mice. The most notable finding this year, in collaboration with Prof. Ahnert-Hilger in Berlin, has been the discovery that the Go2 G protein regulate1d axonal growth. In collaboration with Andrew Tinker in London, we examined and published the relative roles of Gs and Gi in the regulation of the cardiac sinoatrial pacemaker cells. With Prof Bernd Nuernberg in Duesseldorf, and Dr. Mireille Montcouquiol in Bordeaux, we described the role of Gi2 in the asymetric positioning of the primary cilium in hair cells of the inner ear. The fact that Gi2 is required establishes unequivocally that a G protein participates in developmental events that are triggered by non-canonical signaling of Wnt and Hedgehog.

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Liao, Yanhong; Abramowitz, Joel; Birnbaumer, Lutz (2014) The TRPC family of TRP channels: roles inferred (mostly) from knockout mice and relationship to ORAI proteins. Handb Exp Pharmacol 223:1055-75
Minetti, Giulia C; Feige, Jerome N; Bombard, Florian et al. (2014) G?i2 signaling is required for skeletal muscle growth, regeneration, and satellite cell proliferation and differentiation. Mol Cell Biol 34:619-30
Seo, Kinya; Rainer, Peter P; Shalkey Hahn, Virginia et al. (2014) Combined TRPC3 and TRPC6 blockade by selective small-molecule or genetic deletion inhibits pathological cardiac hypertrophy. Proc Natl Acad Sci U S A 111:1551-6
Tano, Jean-Yves; Solanki, Sumeet; Lee, Robert H et al. (2014) Bone marrow deficiency of TRPC3 channel reduces early lesion burden and necrotic core of advanced plaques in a mouse model of atherosclerosis. Cardiovasc Res 101:138-44
Oboti, Livio; Pérez-Gómez, Anabel; Keller, Matthieu et al. (2014) A wide range of pheromone-stimulated sexual and reproductive behaviors in female mice depend on G protein G?o. BMC Biol 12:31
Ball, Christopher B; Rodriguez, Karina F; Stumpo, Deborah J et al. (2014) The RNA-binding protein, ZFP36L2, influences ovulation and oocyte maturation. PLoS One 9:e97324
Kochukov, Mikhail Y; Balasubramanian, Adithya; Abramowitz, Joel et al. (2014) Activation of endothelial transient receptor potential C3 channel is required for small conductance calcium-activated potassium channel activation and sustained endothelial hyperpolarization and vasodilation of cerebral artery. J Am Heart Assoc 3:
Köhler, David; Devanathan, Vasudharani; Bernardo de Oliveira Franz, Claudia et al. (2014) G?i2- and G?i3-deficient mice display opposite severity of myocardial ischemia reperfusion injury. PLoS One 9:e98325
Choi, Chan-Il; Yoon, Sang-Phil; Choi, Jung-Mi et al. (2014) Simultaneous deletion of floxed genes mediated by CaMKII?-Cre in the brain and in male germ cells: application to conditional and conventional disruption of Go?. Exp Mol Med 46:e93
Ilatovskaya, Daria V; Palygin, Oleg; Chubinskiy-Nadezhdin, Vladislav et al. (2014) Angiotensin II has acute effects on TRPC6 channels in podocytes of freshly isolated glomeruli. Kidney Int 86:506-14

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