The topic of our research is signalling mechanism of G-protein coupled receptors (GPCRs) dissected with molecular biology and biochemical tools.

1. Role of proteins associated with the cannabinoid and GABAb receptors in signalling GPCRs exist and function in concert with many scaffolding, trafficking, regulatory etc. proteins.

These proteins are interacting with the receptors’ C-termini or other parts. Our question was the role of heptahelical domain of GB1 subunit of GABAb receptor, since this subunit does not activate G-proteins. To answer this question we employed yeast two hybrid screen against intracellular portions of the receptor and detected several possible partners. In case of cannabinoid receptor we used the C-terminus as a bite.

For further evaluation of the protein-protein association this is followed by biochemistry(immunoprecipitation..) and fluorescence methodology studies (FRET, BRET, FRAP..).

Production of antibodies against the newly detected partners is part of the project. The actual role will be studied using letiviral system for delivery of mutant cDNAs into cultured cells (primary neuonal cultures) with the plan to deliver these into animals in single-cell genetic approach.

2. Structure-function relationship of dimeric metabotropic glutamate receptors and GABAb receptors.

The activation process of GPCRs from family C is asymetrical (see our recent EMBO J and JBC publication). This means that in dimeric receptors only one subunit reaches active state upon agonis stimulation. This finding opens many new questions that we plan to address. The most intriguing is the association of the dimeric receptor with the Gproteins.

Is the stoichiometry 2receptor subunits :

• 1 set of trimeric G-proteins or is the ratio different ?
• Are the G-proteins precoupled to tha receptor and redistributed during activation ?
• What is the mechanism of inverse agonists in respect to G-protein association with the receptor ?

Therefore these studies are aimed at explaining assymetrical functioning of these receptors.
This will be done using labeling of various intracellular receptors portions via tetracysteine-biarsenical tagging system of small fluorescent compounds like FlAsH (fluorescein arsenical hairpin binder) developed by R.Y. Tsien and traditional GFPbased chameleons. FRET changes upon activation of the receptor will be evaluated in respect of position of the sensors within each subunit and associated (activated) Gproteins.

M. Havlickova, J. Blahos, I. Brabet, J. Liu, B. Hruskova, L. Prézeau and J-P. Pin(2003):The second intracellular loop of metabotropic glutamate receptors recognizes C-termini of G protein -subunits..J. Biol. Chem:35063-35070