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Muscarinic acetylcholine receptor M3 modulates odorant receptor activity via inhibition of β-arrestin-2 recruitment.

Jiang Y, Li YR, Tian H, Ma M, Matsunami H - Nat Commun (2015)

Bottom Line: We previously reported that type 3 muscarinic acetylcholine receptors (M3-Rs) physically interact with odorant receptors (ORs) to promote odour-induced responses in a heterologous expression system.However, it is not known how M3-Rs affect the ability of olfactory sensory neurons (OSNs) to respond to odours.These results suggest a role for acetylcholine in modulating olfactory processing at the initial stages of signal transduction in the olfactory system.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA [2] University Program of Genetics and Genomics, Duke University, Duke, North Carolina 27710, USA.

ABSTRACT
The olfactory system in rodents serves a critical function in social, reproductive and survival behaviours. Processing of chemosensory signals in the brain is dynamically regulated in part by an animal's physiological state. We previously reported that type 3 muscarinic acetylcholine receptors (M3-Rs) physically interact with odorant receptors (ORs) to promote odour-induced responses in a heterologous expression system. However, it is not known how M3-Rs affect the ability of olfactory sensory neurons (OSNs) to respond to odours. Here, we show that an M3-R antagonist attenuates odour-induced responses in OSNs from wild-type, but not M3-R-, mice. Using a novel molecular assay, we demonstrate that the activation of M3-Rs inhibits the recruitment of β-arrestin-2 to ORs, resulting in a potentiation of odour-induced responses in OSNs. These results suggest a role for acetylcholine in modulating olfactory processing at the initial stages of signal transduction in the olfactory system.

No MeSH data available.


Related in: MedlinePlus

The third intracellular loop of M3-R is important for OR potentiation(A) Chimera construction. The third intracellular loop of M2-R and M3-R were exchanged to generate two chimeras: M2-M3L, the chimeric M2-R with the i3 loop replaced by that of M3-R, and M3-M2L, the M3-R with its i3 loop replaced by that of M2-R. (B) β-arrestin-2 recruitment and cAMP response of two ORs with the coexpression of M2, M3 or chimeras. For both ORs, M3-M2L showed less blocking of β-arrestin-2 recruitment to OR as compared to M3-R. In contrast, M2-M3L showed marked blocking of β-arrestin-2 recruitment. For both ORs, M2-M3L showed substantial potentiation on OR-mediated cAMP responses. ***: p<0.01. Two-way ANOVA for β-arrestin-2 recruitment comparisons: OR-S6, M3 vs M2 (p<0.0001), M3 vs M3-M2L (p<0.0001), M2 vs M2-M3L (p<0.0001), M2-M3L vs M3-M2L (p<0.0001); Olfr62, M3 vs M2 (p<0.0001), M3 vs M3-M2L (p<0.0001), M2 vs M2-M3L (p<0.0001), M2-M3L vs M3-M2L (p<0.0001). Two-ANOVA for cAMP response comparisons: OR-S6, M3 vs M2 (p<0.0001), M3 vs M3-M2L (p<0.0001), M2 vs M2-M3L (p<0.0001), M2-M3L vs M3-M2L (p<0.0001); Olfr62, M3 vs M2 (p=0.002), M3 vs M3-M2L (p=0.001), M2 vs M2-M3L (p<0.0001), M2-M3L vs M3-M2L (p<0.0001). n=3, error bars indicate standard errors. (C) Model for how M3-Rs potentiates OR activation. M3-Rs physically interacts with the OR. The third intracellular loop (i3) of M3-R is important for M3-Rs to inhibit β-arrestin-2 recruitment to activated ORs, and this inhibition is enhanced in the presence of M3-R agonists such as e.g., acetylcholine (ACh). Through the inhibition of β-arrestin-2 recruitment at ORs, M3-R promotes G-protein coupling of ORs and augments OR activation.
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Figure 10: The third intracellular loop of M3-R is important for OR potentiation(A) Chimera construction. The third intracellular loop of M2-R and M3-R were exchanged to generate two chimeras: M2-M3L, the chimeric M2-R with the i3 loop replaced by that of M3-R, and M3-M2L, the M3-R with its i3 loop replaced by that of M2-R. (B) β-arrestin-2 recruitment and cAMP response of two ORs with the coexpression of M2, M3 or chimeras. For both ORs, M3-M2L showed less blocking of β-arrestin-2 recruitment to OR as compared to M3-R. In contrast, M2-M3L showed marked blocking of β-arrestin-2 recruitment. For both ORs, M2-M3L showed substantial potentiation on OR-mediated cAMP responses. ***: p<0.01. Two-way ANOVA for β-arrestin-2 recruitment comparisons: OR-S6, M3 vs M2 (p<0.0001), M3 vs M3-M2L (p<0.0001), M2 vs M2-M3L (p<0.0001), M2-M3L vs M3-M2L (p<0.0001); Olfr62, M3 vs M2 (p<0.0001), M3 vs M3-M2L (p<0.0001), M2 vs M2-M3L (p<0.0001), M2-M3L vs M3-M2L (p<0.0001). Two-ANOVA for cAMP response comparisons: OR-S6, M3 vs M2 (p<0.0001), M3 vs M3-M2L (p<0.0001), M2 vs M2-M3L (p<0.0001), M2-M3L vs M3-M2L (p<0.0001); Olfr62, M3 vs M2 (p=0.002), M3 vs M3-M2L (p=0.001), M2 vs M2-M3L (p<0.0001), M2-M3L vs M3-M2L (p<0.0001). n=3, error bars indicate standard errors. (C) Model for how M3-Rs potentiates OR activation. M3-Rs physically interacts with the OR. The third intracellular loop (i3) of M3-R is important for M3-Rs to inhibit β-arrestin-2 recruitment to activated ORs, and this inhibition is enhanced in the presence of M3-R agonists such as e.g., acetylcholine (ACh). Through the inhibition of β-arrestin-2 recruitment at ORs, M3-R promotes G-protein coupling of ORs and augments OR activation.

Mentions: If M3-Rs directly potentiates OR activity, we expect that specific functional domains of M3-R would be critical for its interaction with ORs and/or β-arrestin-2. The long third intracellular loop (i3) is the most divergent domain among the muscarinic receptor family members, and is important for M3-R function 33, 34, 35, 36. We hypothesized that the i3 loop may be necessary for the ability of M3-R to inhibit β-arrestin-2 recruitment of ORs. To test this hypothesis, we interchanged the entire i3 loop of M3-R with that of M2-R and examined how the two resulting chimeric muscarinic receptors modulate OR function. M3-M2L refers to the chimeric M3-R with its i3 loop replaced by that of M2-R, whereas M2-M3L refers to the chimeric M2-R with its i3 loop replaced by that of M3-R (Fig. 10A). Interestingly, we found that M3-M2L behaved like M2-R and was unable to inhibit β-arrestin-2 recruitment to ORs. In contrast, M2-M3L decreased β-arrestin-2 recruitment to ORs, mimicking the effect of M3-R (p<0.0001 for M2 vs M3, M2 vs M2-M3L, M3 vs M3-M2L, M2-M3L vs M3-M2L, two-way ANOVA, Fig. 10B, left). Consistently, M2-M3L, like the native M3-R, potentiates OR-mediated cAMP responses, while M3-M2L does not (p<0.001 for M2 vs M3, M2 vs M2-M3L, M3 vs M3-M2L, M2-M3L vs M3-M2L, two-way ANOVA, Fig. 10B, right). Taken together, these results indicate that the i3 loop of M3-R is necessary and sufficient for M3-Rs to inhibit β-arrestin-2 recruitment and potentiate cAMP responses of ORs upon activation, lending further support to our model (Fig. 10C).


Muscarinic acetylcholine receptor M3 modulates odorant receptor activity via inhibition of β-arrestin-2 recruitment.

Jiang Y, Li YR, Tian H, Ma M, Matsunami H - Nat Commun (2015)

The third intracellular loop of M3-R is important for OR potentiation(A) Chimera construction. The third intracellular loop of M2-R and M3-R were exchanged to generate two chimeras: M2-M3L, the chimeric M2-R with the i3 loop replaced by that of M3-R, and M3-M2L, the M3-R with its i3 loop replaced by that of M2-R. (B) β-arrestin-2 recruitment and cAMP response of two ORs with the coexpression of M2, M3 or chimeras. For both ORs, M3-M2L showed less blocking of β-arrestin-2 recruitment to OR as compared to M3-R. In contrast, M2-M3L showed marked blocking of β-arrestin-2 recruitment. For both ORs, M2-M3L showed substantial potentiation on OR-mediated cAMP responses. ***: p<0.01. Two-way ANOVA for β-arrestin-2 recruitment comparisons: OR-S6, M3 vs M2 (p<0.0001), M3 vs M3-M2L (p<0.0001), M2 vs M2-M3L (p<0.0001), M2-M3L vs M3-M2L (p<0.0001); Olfr62, M3 vs M2 (p<0.0001), M3 vs M3-M2L (p<0.0001), M2 vs M2-M3L (p<0.0001), M2-M3L vs M3-M2L (p<0.0001). Two-ANOVA for cAMP response comparisons: OR-S6, M3 vs M2 (p<0.0001), M3 vs M3-M2L (p<0.0001), M2 vs M2-M3L (p<0.0001), M2-M3L vs M3-M2L (p<0.0001); Olfr62, M3 vs M2 (p=0.002), M3 vs M3-M2L (p=0.001), M2 vs M2-M3L (p<0.0001), M2-M3L vs M3-M2L (p<0.0001). n=3, error bars indicate standard errors. (C) Model for how M3-Rs potentiates OR activation. M3-Rs physically interacts with the OR. The third intracellular loop (i3) of M3-R is important for M3-Rs to inhibit β-arrestin-2 recruitment to activated ORs, and this inhibition is enhanced in the presence of M3-R agonists such as e.g., acetylcholine (ACh). Through the inhibition of β-arrestin-2 recruitment at ORs, M3-R promotes G-protein coupling of ORs and augments OR activation.
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Figure 10: The third intracellular loop of M3-R is important for OR potentiation(A) Chimera construction. The third intracellular loop of M2-R and M3-R were exchanged to generate two chimeras: M2-M3L, the chimeric M2-R with the i3 loop replaced by that of M3-R, and M3-M2L, the M3-R with its i3 loop replaced by that of M2-R. (B) β-arrestin-2 recruitment and cAMP response of two ORs with the coexpression of M2, M3 or chimeras. For both ORs, M3-M2L showed less blocking of β-arrestin-2 recruitment to OR as compared to M3-R. In contrast, M2-M3L showed marked blocking of β-arrestin-2 recruitment. For both ORs, M2-M3L showed substantial potentiation on OR-mediated cAMP responses. ***: p<0.01. Two-way ANOVA for β-arrestin-2 recruitment comparisons: OR-S6, M3 vs M2 (p<0.0001), M3 vs M3-M2L (p<0.0001), M2 vs M2-M3L (p<0.0001), M2-M3L vs M3-M2L (p<0.0001); Olfr62, M3 vs M2 (p<0.0001), M3 vs M3-M2L (p<0.0001), M2 vs M2-M3L (p<0.0001), M2-M3L vs M3-M2L (p<0.0001). Two-ANOVA for cAMP response comparisons: OR-S6, M3 vs M2 (p<0.0001), M3 vs M3-M2L (p<0.0001), M2 vs M2-M3L (p<0.0001), M2-M3L vs M3-M2L (p<0.0001); Olfr62, M3 vs M2 (p=0.002), M3 vs M3-M2L (p=0.001), M2 vs M2-M3L (p<0.0001), M2-M3L vs M3-M2L (p<0.0001). n=3, error bars indicate standard errors. (C) Model for how M3-Rs potentiates OR activation. M3-Rs physically interacts with the OR. The third intracellular loop (i3) of M3-R is important for M3-Rs to inhibit β-arrestin-2 recruitment to activated ORs, and this inhibition is enhanced in the presence of M3-R agonists such as e.g., acetylcholine (ACh). Through the inhibition of β-arrestin-2 recruitment at ORs, M3-R promotes G-protein coupling of ORs and augments OR activation.
Mentions: If M3-Rs directly potentiates OR activity, we expect that specific functional domains of M3-R would be critical for its interaction with ORs and/or β-arrestin-2. The long third intracellular loop (i3) is the most divergent domain among the muscarinic receptor family members, and is important for M3-R function 33, 34, 35, 36. We hypothesized that the i3 loop may be necessary for the ability of M3-R to inhibit β-arrestin-2 recruitment of ORs. To test this hypothesis, we interchanged the entire i3 loop of M3-R with that of M2-R and examined how the two resulting chimeric muscarinic receptors modulate OR function. M3-M2L refers to the chimeric M3-R with its i3 loop replaced by that of M2-R, whereas M2-M3L refers to the chimeric M2-R with its i3 loop replaced by that of M3-R (Fig. 10A). Interestingly, we found that M3-M2L behaved like M2-R and was unable to inhibit β-arrestin-2 recruitment to ORs. In contrast, M2-M3L decreased β-arrestin-2 recruitment to ORs, mimicking the effect of M3-R (p<0.0001 for M2 vs M3, M2 vs M2-M3L, M3 vs M3-M2L, M2-M3L vs M3-M2L, two-way ANOVA, Fig. 10B, left). Consistently, M2-M3L, like the native M3-R, potentiates OR-mediated cAMP responses, while M3-M2L does not (p<0.001 for M2 vs M3, M2 vs M2-M3L, M3 vs M3-M2L, M2-M3L vs M3-M2L, two-way ANOVA, Fig. 10B, right). Taken together, these results indicate that the i3 loop of M3-R is necessary and sufficient for M3-Rs to inhibit β-arrestin-2 recruitment and potentiate cAMP responses of ORs upon activation, lending further support to our model (Fig. 10C).

Bottom Line: We previously reported that type 3 muscarinic acetylcholine receptors (M3-Rs) physically interact with odorant receptors (ORs) to promote odour-induced responses in a heterologous expression system.However, it is not known how M3-Rs affect the ability of olfactory sensory neurons (OSNs) to respond to odours.These results suggest a role for acetylcholine in modulating olfactory processing at the initial stages of signal transduction in the olfactory system.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA [2] University Program of Genetics and Genomics, Duke University, Duke, North Carolina 27710, USA.

ABSTRACT
The olfactory system in rodents serves a critical function in social, reproductive and survival behaviours. Processing of chemosensory signals in the brain is dynamically regulated in part by an animal's physiological state. We previously reported that type 3 muscarinic acetylcholine receptors (M3-Rs) physically interact with odorant receptors (ORs) to promote odour-induced responses in a heterologous expression system. However, it is not known how M3-Rs affect the ability of olfactory sensory neurons (OSNs) to respond to odours. Here, we show that an M3-R antagonist attenuates odour-induced responses in OSNs from wild-type, but not M3-R-, mice. Using a novel molecular assay, we demonstrate that the activation of M3-Rs inhibits the recruitment of β-arrestin-2 to ORs, resulting in a potentiation of odour-induced responses in OSNs. These results suggest a role for acetylcholine in modulating olfactory processing at the initial stages of signal transduction in the olfactory system.

No MeSH data available.


Related in: MedlinePlus