Studies on the optimization of expression and purification & Functional characterization of Class C – GPCRs
G-protein coupled receptor (GPCRs) is a multigene family consisting of more than 1000 genes. They are the most abundant membrane proteins found on a cell surface and are involved in several signaling pathways. In a cell, the signal is transduced by diverse activating endogenous ligands binding on the extracellular surface. This results in the uncoupling of G-proteins from the cytoplasmic loops, leading to the activation of the second messengers. GPCRs have enormous therapeutic importance due to their involvement in basic physiological processes including sensory perception, neurotransmission, metabolism, hormonal balance, etc. Structural and biochemical data are the pre-requisites for designing the drugs involving GPCRs. The current study was focused on the optimization of expression, purification and functional characterization of class-C GPCRs involved in neurotransmission. The first part of the study was aimed at optimizing the expression and purification of the ligand binding extracellular domain (ECD) of rat metabotropic GABAB1b receptor (GBR1bNT) in the recombinant baculovirus (RBV) and E.coli expression systems. GBR1bNT was modeled based on the crystal structure coordinates of ECD of metabotropic glutamate receptor (mGluR). Depending on this GBR1bNT model, molecular cloning strategies were developed for the expression of GBR1bNT. In both systems, GBR1bNT was well expressed and purified. The second part of the study was aimed at biochemical characterization of a putative cholesterol binding motif (pCBM) in Drosophila metabotropic glutamate receptor (DmGluRA). This pCBM might be involved in regulating the binding of DmGluRA to glutamate with high affinity. During the study, it was inferred that a 12- amino-acid amphipathic peptide containing the pCBM might be crucial for the activity of the receptor. Upon conducting [3H]-glutamate binding and detergent-resistant-membrane (DRM) association studies on different truncation constructs of DmGluRA (1-910 amino acids), the N-terminal construct (1-624 amino acids) containing the ECD with one single pCBM was found to be capable of binding glutamate in high affinity state as observed with the full length DmGluRA. Together this study shows that 1) Using an interdisciplinary approach (computational and experimental strategies) GBR1bNT was efficiently expressed and purified in both E.coli and RBV expression systems and 2) the role of pCBM was studied in DmGluRA receptor regulation.
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