1) Name – Title of the research project in CCHD
Michael Freissmuth - Trafficking of G protein-coupled receptors and of neurotransmitter transporters through the secretory pathway
2) Coordinates of the Faculty Member
Centre of Biomolecular Medicine and Pharmacology, Institute of Pharmacology, Medical Univ. Vienna, Währinger Straße 13a, A-1090 Vienna; Tel +43 1 42 77 64171; Fax +43 1 42 77 9641; email: michael.freissmuth@meduniwien.ac.at.
3) Keywords
G Protein-coupled receptors, neurotransmitter transporters, GABA-transporter-1 (GAT1); A2A-receptor; synapse-associated protein-102
4) Research interest of the Faculty Member
The work of Dr. Freissmuth was originally focussed on understanding how G protein-coupled receptors (GPCR's) activate G proteins, how GPCR's discriminate between related G proteins and how this knowledge can be exploited to search for inhibitors of G proteins. More recently, he has focused on alternative signalling routes, i.e. in a manner independent of heterotrimeric G proteins. This has led to the identification of several molecules that are involved in trafficking of the receptor to and from the membrane and to candidate mecha¬nisms that allow for local confinement of the receptor on the cell surface. The second broad area of interest is to understand neurotransmitter transporters. These transporters are crucial for termination and maintenance of synaptic communication because they clear the synaptic cleft from and replenish the synaptic vesicles with neurotransmitter. These transporters are of general interest because they are the site of action of drugs (e.g. antidepressants), of abused compounds (e.g. amphetamines) and because mutations and genetic poly¬mor¬phisms are associated with diseases (e.g. attention-deficit hyperactivity disease, depression). The transporters are specifically targeted to the rim of the synapse; understanding how this specific targeting is achieved is a challenging neurobiological problem.
5) Collaborations within CCHD
There is a long standing collaboration with S. Boehm (10 joint publications) on signalling via G protein-coupled receptors; this collaboration will continue in the CCHD. A collaborative effort has also been established with G. Superti-Furga (joint publication). Here, we will collaborate with Giulio Superti-Furga's group to identify proteins that are associated with GPCR's and with transporters in specific compartments. Within CCHD - with the PhD-student Amulya Shrivastave as liaison - we are currently collaborating with W. Sieghart on the cellular compartment where complexes between A1-receptors and ionotropic receptors assemble and how selected GPCR's can trigger degradation of GABAA-receptor subunits. The resulting ER stress is being investiga¬ted with reagents and with the conceptual help of members of B. Binder's group, who will also provide support for the generation of knock-in mice. Methods for single dye tracking are developed in collaboration with H. Stockinger. The compartment in which trafficking-deficient GAT1-mutants are trapped will be investigated in collaboration with H. Lassmann, who will provide access to electron microscopy. We will provide access to knowhow and reagents (in the GPCR field) for the projects of H. Esterbauer and T. Stulnig.
6) Collaborating research groups where PhD Students can perform their research stay
Daniel Choquet; UMR 5091 CNRS-Université de Bordeaux 2, Physiologie Cellulaire de la Synapse; Institut François Magendie, rue Camille Saint Saëns; 33077 Bordeaux Cédex
Tel +33 5 57 57 40 90; Fax +33 5 57 57 40 82; email: dchoquet@u-bordeaux2.fr
Joël Bockaert; Institute of Functional Genomics; CNRS UMR5203 - INSERM U661 UM1 - UM2; 141 rue de la Cardonille; 34094 Montpellier Cedex 5; France; Tel: +33 467142930
Fax: +33 467542432; email: Joel.Bockaert@igf.cnrs.fr
7) Industrial partners (if applicable)
Insights that are of potential value for applied pharmacology are pursued in collaboration with AOP Pharma, a pharmaceutical company specializing in orphan indications. The collaborati¬ve efforts have led to a patent that was licensed to AOP (second medical use of bortezomib for the treatment of cystic fibrosis - further development not pursued because of an FDA war¬ning letter). Another joint patent application (on ER export of CFTR) is currently being drafted that emanates from insights related to the work done within CCHD.
8) Know-how and infrastructure of the research group
This research group identified erk1/2 activation as an alternative signalling pathway of the A2A-receptor (Sexl et al., 1997), showed that it was independent of the cognate G protein Gs (Seidel et al., 1999) but required ARNO/ARF6 (Gsandtner et al., 2005) and occurred in a spatially segregated compartment (Charalambous et al., 2008). More recently, USP4 was identified as an interactor, which regulates ER export of the A2A-receptor by catalyzing its deubiquination (Milojevic et al., 2006) – the first example of this type of regulation in the GPCR field. The second concept to be pursued emanates form the observation that oligomer formation is a prerequisite for ER export of neurotransmitter transporters (Scholze et al., 2002). By creating a collection of mutated versions of GAT1, motifs were identified that recruit components of the ER export machinery and that specify the transit through the early secretory pathway (Farhan et al., 2007; Farhan et al. 2008; Reiterer et al., 2008).
Members of this group have access to a core facility equipped with microscopes for FRET and confocal microscopy, for single dye tracking and for LRET (lanthanide resonance energy transfer) that combines the resources of vascular biology, physology and pharmacology in the recently formed Center of Physiology and Pharmacology (FRET microscope, single dye tracking and LRET are our contribution).
The lab has all the standard equipment cellular and molecular biology and biochemcal pharmacology (including two cell culture hoods, two incubators, bacterial shakers, high speed and ultracentrifuges, FPLC and other chromatography systems for protein purification; animal facility for keeping mice under pathogen free conditions etc.).
