title: Role of rhodopsin N-terminus in structure and function of rhodopsin-bitter taste receptor chimeras.
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.
authors.affiliation: Department of Oral Biology, University of Manitoba, and The Manitoba Institute of Child Health, Winnipeg MB, Canada R3E 0W4.
authors.forename: Sai Prasad
authors.initials: SP
authors.lastname: Pydi
authors.forename: Raja
authors.initials: R
authors.lastname: Chakraborty
authors.forename: Rajinder Pal
authors.initials: RP
authors.lastname: Bhullar
authors.forename: Prashen
authors.initials: P
authors.lastname: Chelikani
mesh_terms: D000595:Amino Acid Sequence; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000502:physiology; D012243:Rhodopsin / Q000737:chemistry*; D013649:Taste; D000098706:Taste Receptors, Type 2
chemical_list: D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D000098706:Taste Receptors, Type 2; D012243:Rhodopsin