Research objectives and content
G-protein coupled receptors (GPCRs) belong to a large family of membrane molecules that play a central role in signal transduction. The large scale production of GPCRs for their structural characterisation has been attempted in various systems like baculovirus infecting insect cells, yeast or bacteria... but scarcely with success. The major problem encountered is the low efficiency of expression of GPCRs at the cell surface. This could be due to an overloading of the protein insertion machinery leading to the accumulation of those proteins inside the cell without being translocated to the plasma membrane. Two main pathways of protein targeting to the membrane are well described in Escherichia coli: the Sec and the SRP pathway. Recent published data suggest that the SRP machinery of E.coli, which is largely studied in the host laboratory, is the main pathway that target membrane proteins to the cell membrane. We will first characterise the targeting and insertion pathway of GPCRs in E.coli. This study will be done with the vasopressin receptor V 1 a, a prototype of GPCR of great interest in the pharmacological industry and which the host laboratory is currently trying to overexpress in E.coli. The second step will be to co-overexpress the Vla receptor and its export machinery, using molecular biology and co-expression systems, to improve the targeting of the Vla receptors at the E.coli membrane. This new strategy of overexpression of GPCRs structural studies will be extended to other membrane receptors. Training content objective, benefit and expected impact)
Due to the importance of GPCRs in a large variety of biological processes and the need for pharmaceutical research of knowing their structural features to discover some new drugs, lot of efforts are developped for the determination of their 3D-stmctures. Nowadays, the limiting step for determining the structure of GPCRs is the availability of high amounts for 2D and 3D crystallisation. This however, is hampered by the low level of exoression of those receptors in all systems studied to date. The improvement of protein expression at the cell! surface by increasing the activity of the export machinery will represent a powerful strategy that can be extended to the majority of the GPCRs as well as other membrane proteins. On a personal side, my previous research experiences focussed on membrane protein-lipids interactions and this project represent a very good opportunity to strengthen my background in molecular biology and membrane protein biochemistry and to enter the field of structural studies.
Links untie industry / industrial relevance (22)
The group of I. Sinning has a collaboration with BASF in Ludwigshafen for the bcl complex (Franz Rue and an initial contact has been established with ASTRA, Sweden (T. Lundqvist) and with BAYER, Germany G. Kleymann) for GPCRs. Whether these contacts will be strengthened in the future depends on the progress ofthe projects.