Heterologous functional expression system for odorant receptors

Hamana, Hiroshi; Shou-xin, Li; Breuils, L.; Hirono, Junzo; Sato, Takaaki

doi:10.1016/j.jneumeth.2009.09.024

Cited by 18 publications

(18 citation statements)

References 35 publications

(65 reference statements)

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“…In addition to activating the Gαs(olf)/AC pathway, heterologously expressed ORs are known to also couple to the promiscuous Gα15/16, an isoform of Gαq, to activate the PLC signaling pathway [20, 32]. With few exceptions [16, 33], these previous studies typically used fixed end point assays that rank ligands by their overall ability to activate a particular pathway [34, 35] and did not consider other response characteristics that may reflect ligand-GPCR and GPCR-G-protein interactions such as the degree of receptor activation, cellular amplification of the signal by other pathway components, and the transitory nature of the interactions that may affect the overall potency of the ligand. To circumvent some of these limitations, we adapted insight from previous studies of non-OR GPCRs where the kinetics of their Ca 2+ responses was used to explore various parameters of ligand-receptor interactions [36].…”

Section: Discussionmentioning

confidence: 99%

Ligand-selective activation of heterologously-expressed mammalian olfactory receptor

et al. 2014

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Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca2+ release and/or Ca2+ influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5 mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs.

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Section: Discussionmentioning

confidence: 99%

Ligand-selective activation of heterologously-expressed mammalian olfactory receptor

et al. 2014

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“…The chimeric Gα 15_olf has the Gα olf (a member of the G s class) C-terminal 376 KQYE motif instead of the corresponding 369 DEIN sequence present in Gα 15 (a member of the G q/11 class), and this change improves the rapidity of the response (2.2-fold shorter Ca 2+ response onset latency) as well as the response amplitude (1.7-fold), compared with Gα 15 [7,23]. As expected, EC 50 values for the most potent agonist of m OR-S6 showed no significant difference between Gα 15_olf and Gα 15 [23]. These results indicate that the observed improvements in kinetics are likely attributable to specific interactions at the C-terminal region of Gα 15_olf with ORs.…”

Section: Structural Features Of Helixmentioning

confidence: 99%

“…The replacement of Gα 15 369 DEIN with Gα olf 376 KQYE improved the response kinetics of m OR-S6 via the chimeric Gα 15_olf by shortening the onset latency 2.2-fold [7,23], but replacement of m OR-S6 Glu 302 with Arg 302 completely eliminated the effect of this mutation on m OR-S6-mediated Ca 2+ responses [7]. These findings clearly indicate that the second residue of helix 8 is a major determinant of the initial specific interaction with the target G protein that are essential for a rapid and robust response, rather than with Arg or Ala at the second position of helix 8 in ORs or non-target G proteins.…”

Section: The Second Residue Of Helix 8 Partially Governs the Hieramentioning

confidence: 99%

Functional Role of the C-Terminal Amphipathic Helix 8 of Olfactory Receptors and Other G Protein-Coupled Receptors

Sato

Kawasaki

Mine

et al. 2016

IJMS

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G protein-coupled receptors (GPCRs) transduce various extracellular signals, such as neurotransmitters, hormones, light, and odorous chemicals, into intracellular signals via G protein activation during neurological, cardiovascular, sensory and reproductive signaling. Common and unique features of interactions between GPCRs and specific G proteins are important for structure-based design of drugs in order to treat GPCR-related diseases. Atomic resolution structures of GPCR complexes with G proteins have revealed shared and extensive interactions between the conserved DRY motif and other residues in transmembrane domains 3 (TM3), 5 and 6, and the target G protein C-terminal region. However, the initial interactions formed between GPCRs and their specific G proteins remain unclear. Alanine scanning mutagenesis of the murine olfactory receptor S6 (mOR-S6) indicated that the N-terminal acidic residue of helix 8 of mOR-S6 is responsible for initial transient and specific interactions with chimeric Gα15_olf, resulting in a response that is 2.2-fold more rapid and 1.7-fold more robust than the interaction with Gα15. Our mutagenesis analysis indicates that the hydrophobic core buried between helix 8 and TM1–2 of mOR-S6 is important for the activation of both Gα15_olf and Gα15. This review focuses on the functional role of the C-terminal amphipathic helix 8 based on several recent GPCR studies.

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“…Hamana et al reported an improvement in the Ca 2+ response dynamics of ORs using Gα 15_olf , a chimeric Gα 15 in which the C‐terminal amino acids 369 DEIN were replaced with Gα olf 376 KQYE [18] (Fig. S3).…”

Section: Introductionmentioning

confidence: 99%

The N‐terminal acidic residue of the cytosolic helix 8 of an odorant receptor is responsible for different response dynamics via G‐protein

et al. 2015

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Interaction Homology model a b s t r a c tWe previously observed highly rapid and robust response of murine olfactory receptor S6 (mOR-S6) with chimeric Ga 15_olf , compared to Ga 15 . To identify residues responsible for this difference in response, mutations of the cytosolic helix 8 were analyzed in a heterologous functional expression system. The N-terminal hydrophobic core between helix 8 and TM1-2 of mOR-S6 is important for activation of both Ga 15_olf and Ga 15 . Point mutation of a helix 8 N-terminal acidic residue eliminated the differences in response dynamics via Ga. This result suggests that an N-terminal acidic residue of helix 8 is responsible for rapid response via Ga 15_olf .

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Heterologous functional expression system for odorant receptors

Cited by 18 publications

References 35 publications

Ligand-selective activation of heterologously-expressed mammalian olfactory receptor

Ligand-selective activation of heterologously-expressed mammalian olfactory receptor

Functional Role of the C-Terminal Amphipathic Helix 8 of Olfactory Receptors and Other G Protein-Coupled Receptors

The N‐terminal acidic residue of the cytosolic helix 8 of an odorant receptor is responsible for different response dynamics via G‐protein

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