Molecular analysis of the interaction between the intracellular loops of the human serotonin receptor type 6 (5-HT6) and the α subunit of GS protein
Section snippets
Materials and methods
Materials. The human cDNA library was purchased from Novagen (Madison, WI, USA) and the sensor chip CM5 for the surface plasmon resonance analysis was obtained from BIAcore AB (Uppsala, Sweden). [3H]5-HT (specific activity, 30 mCi/mmol) was obtained from NEN Life Science (Boston, MA, USA), and 5-HT and Ro20-1724 were purchased from Sigma (St. Louis, MO, USA). Reagents for cell culture were purchased from Gibco (Paisley, UK). All of the other reagents were of reagent grade.
Construction of
Preparation of H-GαS, GST-iL2, GST-iL3, GST-CT, and GST-iL3 mutants
Two isoforms of GαS, GNAS1, and GNAS2 (NCBI Accession Nos. NP_536351 and NP_000507), were amplified from a human brain cDNA library. Both GαS cDNAs have the same sequence except that GNAS1 lacks 14 amino acids beginning at the 72nd residue, compared to GNAS2. The cDNA encoding GαS-1 (NP_536351) was used for the expression of H-GαS protein. About 20–30% of the bacterially expressed H-GαS was recovered as soluble protein (data not shown), and it was further purified successively on a Ni2+
Interaction between the iL3 region of the 5-HT6 receptor and GαS
Ligand binding followed by interactions with the α-subunit of heterotrimeric G-proteins initiates the signaling pathway of GPCRs. In this study, we have demonstrated that the third intracellular loop region (iL3) of the 5-HT6 receptor is critical for the interaction with GαS. There are four potential regions of the GPCR that could interact with the G-protein; iL1, iL2, iL3, and the cytosolic C-terminal tail. Among them, the most critical regions in determining the receptor/G-protein
Acknowledgments
We thank Dr. Bryan Roth of Case Western Reserve University for providing the human 5-HT6 cDNA. We also thank Dr. H.S. Jeon at the Korea Institute of Science and Technology for helpful advice on cell culture and transfection experiments, and Neuronex at Postech for helpful techniques on cAMP assay. This work was supported by grants from the 21C Frontier Research Program, Functional Proteomics Research, Ministry of Science and Technology, Korea.
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