Neurotransmitters typically activate several types of receptors. Glutamate binds to ligand-gated ion channels (NMDA, AMPA, Kainate receptors) or G-protein coupled receptors (GPCRs): the metabotropic glutamate receptors (mGluRs). The inhibitory neurotransmitter -aminobutyric acid (GABA) activates ionotropic GABA_A and GABA_C and metabotropic GABAB receptors. While the ionotropic receptors are engaged in fast interneuronal transmission, the metabotropic receptors modulate neurotransmission. They are located on the pre- or postsynaptical membranes of neurons. Such a location allows these metabotropic receptors to influence the functional properties of the synapses. Thus, metabotropic receptors are targets for the design of new drugs and therapeutics for certain neurological disorders.
Current projects focus on the following topics:
- Structure-function relationships of the metabotropic glutamate (mGlu) and GABAB receptors
- Molecular dissection of mGlu and GABAB receptor interaction with G-proteins
- Function of proteins associated with GABAB receptor subunits

      The development of new compounds acting on the receptors depends on our knowledge of their structure and function. Metabotropic glutamate and GABAB receptors have a similar structure in which two proteins form a receptor complex. Metabotropic glutamate receptors are homomeric dimers while GABAB receptor is a heterodimer composed of two proteins called GB1 and GB2. These receptors belong to "Family 3 GPCR" with common structural and functional features. The interaction of agonists with the extracellular N-terminal portion of the receptors evokes changes in their conformation leading to the activation of the receptors. This conformational change is transmitted to the heptahelical transmembrane domains as a change of relative position of the intracellular portions of the receptors. The intracellular portions of the heptahelical domains mediate coupling to G-proteins. Our current research is aimed at investigating signalling mechanisms of the receptor complexes with an emphasis on mapping the molecular determinants that modulate the activation process. Newly designed drugs acting allosterically are one of the tools that we use in our research. Describing the mechanism of their positive or negative modulation of the receptors is done using site-directed mutagenesis, heterologous expression of the clones in mammalian cell lines with subsequent functional tests. Biochemistry and immunohistochemistry are tools used mostly as controls for the proper expression of the proteins.

Fig 1: 3D Model of the G - protein molecule is presented in white with the 5 extreme C-terminal residues important for coupling in dark blue. In red are residues we described as a novel site on G - protein that discriminates between mGlu receptors.

      The dimeric structure of the mGlu and GABAB receptors is also studied from the point of G-protein interaction with the heptahelical region of each protomer (that is each protein from the receptor complex). In a recent study we showed that a single heptahelical domain of one subunit (GB2) of the GABA_B receptor contains all determinants for G-protein activation. The dimeric structure of the GABA_B receptor is necessary for the activation process, but in this dimeric structure, once activated each subunit probably may act as an independent unit or even has another role in signalling. A recently proposed mechanism of activation of the metabotropic glutamate receptors would be in favor of such a notion. This model, based on the crystal structure of the open and closed states of the extracellular domains of the mGluRs, suggests that the relative position of the two subunits' heptahelical domains corresponds to the active or inactive state. Our data showing the GB2 subunit being capable of G-protein activation add new information to the spectrum of findings that are necessary for solving the puzzle connected with GPCR activation and transmission of this activation on G-proteins. Recently, we and other groups pointed at the GB2 subunit of the heteromeric complex as the subunit that activates G-proteins. The functional significance of the GB1 subunit transmembrane region is explored in our laboratory.
      Our ongoing collaborations with the laboratory of Jean Phillipe Pin and Laurent Fagni from the C.N.R.S. unit in Montpellier, headed by Joel Bockaert, and that of Bernhard Bettler at the University of Basel are critical for these projects.

Fig 2.: The Heterodimeric structure of GABAB receptor. Experiments with chimeric receptors proved that single heptahelical domain (HD of the GB2 subunit) activates G-proteins. Symbol G indicates subunit that is capable of activating G-proteins; symbol x is where mutation within intracellular portion of GB2 HD was done in order to disrupt coupling to G proteins. a) wild type receptor; b,c,d) chimerical and mutated receptors.

      Each receptor activates a specific subset of G-proteins. This interaction encodes which intracellular pathway will be modulated. Regions on the G proteins that are compatible with sequences at the receptors come into close proximity during the activation of the receptor and transmission of this activation on the G-proteins. This compatibility is decisive for G-protein selectivity of the receptors. We are mapping the contact sites between the receptors and G-proteins. Recently the extreme C-terminus of G -protein was shown to interact with the intracellular portions of mGluRs, specifically with the second intracellular loop of the heptahelical transmembrane domain. This is one of the most important contact points between these proteins. Other regions on the G-proteins are involved in the selectivity, and the partner sequences on the receptors will be described in detail.

      A yeast two-hybrid system is employed in order to identify proteins that interact with the GABAB receptor. Interactions of regulatory or transport proteins with the receptor GABAB complexes and relationships with the associated and signalling proteins are examined using recombinant proteins expressed in heterologous cells or in neuronal cultures. Biochemical, immunochemical and molecular biology tools are used to describe the interactions and the functional significance of these interactions. This project is carried out in collaboration with the laboratory of Dr. Pavel Osten at the Max-Planck Institute in Heidelberg.