School of Science Division of Life Science 32 G Protein-coupled Receptors in Cell Signaling Supervisor: WONG Yung Hou / LIFS Student: KOT Yung Kei / BIBU Course: UROP 1100, Spring Stimulatory G protein alpha subunit (Gαs) has an important role in cell signaling of G protein-coupled receptors (GPCRs). Gs activates adenylyl cyclase (AC), producing cyclic AMP (cAMP) which is key player in downstream intracellular signaling pathway. In this study, a conditional controlled gene-inactivation system was introduced to HEK293 cell lines, the knock-in of Gαs gene-OFF system was verified by Western blotting analysis. GloSensor™ cAMP assay was employed to monitor the variation of forskolin and isoproterenolstimulated intracellular cAMP levels of various transfected live HEK293 cell lines of the conditional Gαs geneOFF system under the treatment of doxycycline and indole-3-acetic acid for 24 h. G Protein-coupled Receptors in Cell Signaling Supervisor: WONG Yung Hou / LIFS Student: LAI Chak Long / BCB-IRE Course: UROP 1100, Spring Since GPCRs can couple with more than one G protein families (Daaka et al., 1997), understanding the influence of the cellular levels of coupling partners on GPCR coupling selectivity could facilitate the development of more specific GPCR-targeted drugs. In this UROP project, six constructs (Gαi1-DD, Gαi1-DD with CI mutation, Gαi1-DD with QL mutation, Gαi2-DD, Gαi2-DD with CI mutation, Gαi2-DD with QL mutation) in pcDNA3.1 plasmid are cloned, and their functions are tested by western blot and cAMP assay. This system (Gαi-DD controlled by an inducible promoter) aims to modulate the abundance of Gαi precisely and quickly and study the downstream effects on GPCR coupling efficiency with Gαi, Gαs, and other coupling partners. G Protein-coupled Receptors in Cell Signaling Supervisor: WONG Yung Hou / LIFS Student: ZHANG Siqi / BCB Course: UROP 1100, Summer Some of the G protein-coupled receptors (GPCRs), such as beta-2 adrenergic receptor (β2-AR), are able to couple with both Gαs and Gαi, which have opposite intracellular functions in regulating cAMP levels. To investigate the binding preference of GPCRs in dual-coupling, the previous study constructed a plasmid with mini auxin-inducible degron-induced (mAID) protein degradation system on Gαs to induce conditional Gαs degradation in the presence of IAA. Through screening the cAMP level change after the addition of β2-AR agonist, this project finds that the mAID tag does not have a significant influence on Gαs normal function.
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