Role of rhodopsin N-terminus in structure and function of rhodopsin-bitter taste receptor chimeras |
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| Authors: | Sai Prasad Pydi Raja Chakraborty Rajinder Pal Bhullar Prashen Chelikani |
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| Affiliation: | 3. School of Biological Sciences, University of Essex, Wivenhoe Park, Essex CO4 3SQ, United Kingdom;4. Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215;1. Department of Chemistry, Bohai University, Jinzhou 121000, PR China;2. Department of Food and Pharmaceutical Engineering, Suihua University, Suihua 152061, PR China;1. School of Energy and Environmental Engineering, Hebei University of Technology, Tianjin 300401, China;2. Key Laboratory of Efficient Utilization of Low and Medium Grade Energy (Tianjin University), Ministry of Education of China, Tianjin 300072, China;1. School of Mechanical Engineering, Hebei University of Technology, Tianjin, 300130, China;2. Hebei Key Laboratory of Smart Sensing and Human-Robot Interaction, Tianjin, 300401, China;3. School of Architecture and Design, Hebei University of Technology, Tianjin, 300401, China;4. Institute of Biophysics, Hebei University of Technology, Tianjin, 300401, China;1. Laboratory of Genetics and Molecular Hematology (LIM31), University of São Paulo School of Medicine, Av.Dr.Enéas de Carvalho Aguiar, 155, 1st floor, room 43, 05403-000, São Paulo/SP, Brazil;2. Federal University of Latin American Integration-UNILA, Life and Nature Science Institute, Av. Tarquinio Joslin dos Santos, 1000, Sala 105, CEP: 85870-901, Foz do Iguacu, Parana/PR, Brazil;3. Cell Culture and Wound Healing Research Laboratory, Division of Plastic Surgery, Hospital das Clínicas, University of São Paulo Medical School, Av. Dr. Arnaldo, 455, 1st floor, 05403-000, São Paulo/SP, Brazil;4. Department of Earth and Exact Sciences, Federal University of São Paulo, São Paulo/SP, Brazil |
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| Abstract: | The bitter taste receptors (T2Rs) belong to the G protein-coupled receptor (GPCR) superfamily. In humans, bitter taste sensation is mediated by 25 T2Rs. Structure–function studies on T2Rs are impeded by the low-level expression of these receptors. Different lengths of rhodopsin N-terminal sequence inserted at the N-terminal region of T2Rs are commonly used to express these receptors in heterologous systems. While the additional sequences were reported, to enhance the expression of the T2Rs, the local structural perturbations caused by these sequences and its effect on receptor function or allosteric ligand binding were not characterized. In this study, we elucidated how different lengths of rhodopsin N-terminal sequence effect the structure and function of the bitter taste receptor, T2R4. Guided by molecular models of T2R4 built using a rhodopsin crystal structure as template, we constructed chimeric T2R4 receptors containing the rhodopsin N-terminal 33 and 38 amino acids. The chimeras were functionally characterized using calcium imaging, and receptor expression was determined by flow cytometry. Our results show that rhodopsin N-terminal 33 amino acids enhance expression of T2R4 by 2.5-fold and do not cause perturbations in the receptor structure. |
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