Limits...
Cell-Autonomous Gβ Signaling Defines Neuron-Specific Steady State Serotonin Synthesis in Caenorhabditis elegans.

Xu L, Choi S, Xie Y, Sze JY - PLoS Genet. (2015)

Bottom Line: We found that signaling through cell autonomous GPB-1 to the OCR-2 TRPV channel defines the baseline expression of 5-HT synthesis enzyme tryptophan hydroxylase tph-1 in ADF chemosensory neurons.These results revealed a Gβ-mediated signaling operating in differentiated cells to specify intrinsic functional properties, and indicate that baseline TPH expression is not a default generic serotonergic fate, but is programmed in a cell-specific manner in the mature nervous system.Cell-specific regulation of TPH expression could be a general principle for tailored steady state 5-HT synthesis in functionally distinct neurons and their regulation of innate behavior.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT
Heterotrimeric G proteins regulate a vast array of cellular functions via specific intracellular effectors. Accumulating pharmacological and biochemical studies implicate Gβ subunits as signaling molecules interacting directly with a wide range of effectors to modulate downstream cellular responses, in addition to their role in regulating Gα subunit activities. However, the native biological roles of Gβ-mediated signaling pathways in vivo have been characterized only in a few cases. Here, we identified a Gβ GPB-1 signaling pathway operating in specific serotonergic neurons to the define steady state serotonin (5-HT) synthesis, through a genetic screen for 5-HT synthesis mutants in Caenorhabditis elegans. We found that signaling through cell autonomous GPB-1 to the OCR-2 TRPV channel defines the baseline expression of 5-HT synthesis enzyme tryptophan hydroxylase tph-1 in ADF chemosensory neurons. This Gβ signaling pathway is not essential for establishing the serotonergic cell fates and is mechanistically separated from stress-induced tph-1 upregulation. We identified that ADF-produced 5-HT controls specific innate rhythmic behaviors. These results revealed a Gβ-mediated signaling operating in differentiated cells to specify intrinsic functional properties, and indicate that baseline TPH expression is not a default generic serotonergic fate, but is programmed in a cell-specific manner in the mature nervous system. Cell-specific regulation of TPH expression could be a general principle for tailored steady state 5-HT synthesis in functionally distinct neurons and their regulation of innate behavior.

No MeSH data available.


Related in: MedlinePlus

GPB-1 regulates baseline ADF tph-1::gfp expression in a GOA-1-dependent manner.(A) Both GOA-1 deletion (goa-1lf) and GTP-bound hyperactive GOA-1(Q205L) (goa-1gf) mutations elevated ADF tph-1::gfp compared to WT. gpb-1(yz71) and ocr-2 TRPV mutations suppressed tph-1::gfp in goa-1(lf) and goa-1(gf) backgrounds. (B) Dominant negative GDP-bound GOA-1(S47C) transgene (goa-1DN) diminished ADF tph-1 expression in three independent transgenic lines. goa-1(DN) transgenic animals remained capable of enhancing ADF tph-1::gfp when induced to form dauers or in tir-1(gf) background. For each assay, the value of ADF GFP fluorescence in dauers and that of mutants and transgenic animals is normalized to the value of WT animals under optimal conditions. Data represent the average of ≥ 2 trials ± SEM. The differences between WT and mutants are marked on the top of bar, and the differences between comparison groups are indicated, *p < 0.05, ** p < 0.01, *** p < 0.001, t-test for two group comparisons, and ANOVA for multi-group/condition comparisons. (C) ADF-specific marker Psrh-142::mCherry was expressed and showed characteristic ADF axon and dendritic morphology in goa-1(DN) transgenic animals. Animals at all developmental stages were analyzed, and images of L4 animals are shown. (D) A schematic of GPB-1 and GOA-1 interaction on tph-1 expression. Effector molecules compete with Goα for Gβ binding to drive tph-1 expression. In goa-1(lf) an goa-1(gf) mutants, the effector constitutively binds to Gβ. GDP-bound conformation of the GOA-1(DN) protein blocks the effector binding. GPB-1(G162E) obstructs the interactions with both GOA-1 and the effector, leading to constitutively diminished tph-1 regardless of the presence of GDP- or GTP-bound Goα.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4581872&req=5

pgen.1005540.g006: GPB-1 regulates baseline ADF tph-1::gfp expression in a GOA-1-dependent manner.(A) Both GOA-1 deletion (goa-1lf) and GTP-bound hyperactive GOA-1(Q205L) (goa-1gf) mutations elevated ADF tph-1::gfp compared to WT. gpb-1(yz71) and ocr-2 TRPV mutations suppressed tph-1::gfp in goa-1(lf) and goa-1(gf) backgrounds. (B) Dominant negative GDP-bound GOA-1(S47C) transgene (goa-1DN) diminished ADF tph-1 expression in three independent transgenic lines. goa-1(DN) transgenic animals remained capable of enhancing ADF tph-1::gfp when induced to form dauers or in tir-1(gf) background. For each assay, the value of ADF GFP fluorescence in dauers and that of mutants and transgenic animals is normalized to the value of WT animals under optimal conditions. Data represent the average of ≥ 2 trials ± SEM. The differences between WT and mutants are marked on the top of bar, and the differences between comparison groups are indicated, *p < 0.05, ** p < 0.01, *** p < 0.001, t-test for two group comparisons, and ANOVA for multi-group/condition comparisons. (C) ADF-specific marker Psrh-142::mCherry was expressed and showed characteristic ADF axon and dendritic morphology in goa-1(DN) transgenic animals. Animals at all developmental stages were analyzed, and images of L4 animals are shown. (D) A schematic of GPB-1 and GOA-1 interaction on tph-1 expression. Effector molecules compete with Goα for Gβ binding to drive tph-1 expression. In goa-1(lf) an goa-1(gf) mutants, the effector constitutively binds to Gβ. GDP-bound conformation of the GOA-1(DN) protein blocks the effector binding. GPB-1(G162E) obstructs the interactions with both GOA-1 and the effector, leading to constitutively diminished tph-1 regardless of the presence of GDP- or GTP-bound Goα.

Mentions: Gβ could serve as a regulator of Gα signaling that drives tph-1 expression. Alternatively, Gβ itself could function as the signaling molecule to define tph-1 expression in ADF neurons. We first considered the possibility that altered Gα-mediated signaling underscores diminished tph-1 expression in gpb-1 mutants. ADF expresses seven Gα genes. Loss-of-function mutants of egl-30, gpa-3, gpa-10, gpa-13 and odr-3 did not dramatically diminish ADF tph-1::gfp [10,25,27]. gsa-1(lf) is lethal, but RNAi of gsa-1 also did not diminish ADF tph-1::gfp (134±14%, N = 50) compared to empty vector control (N = 28). Further, two deletion alleles of Goα goa-1, n363 and sa734, both markedly elevated ADF tph-1::gfp [10,25] (Fig 6A). Repeated attempts failed to generate a goa-1(n363);gpb-1(yz71) double mutant. As both GOA-1 and GPB-1 function in early embryogenesis [19,20,30], eliminating GOA-1 in gpb-1(yz71) background could be lethal. We therefore generated a double mutant of ocr-2 and goa-1(n363), and found that ocr-2;goa-1(n363) double mutants displayed diminished ADF tph-1::gfp as in ocr-2 single mutants (Fig 6A), suggesting that the absence of GOA-1 protein elevated tph-1 expression by activating the OCR-2 TRPV pathway.


Cell-Autonomous Gβ Signaling Defines Neuron-Specific Steady State Serotonin Synthesis in Caenorhabditis elegans.

Xu L, Choi S, Xie Y, Sze JY - PLoS Genet. (2015)

GPB-1 regulates baseline ADF tph-1::gfp expression in a GOA-1-dependent manner.(A) Both GOA-1 deletion (goa-1lf) and GTP-bound hyperactive GOA-1(Q205L) (goa-1gf) mutations elevated ADF tph-1::gfp compared to WT. gpb-1(yz71) and ocr-2 TRPV mutations suppressed tph-1::gfp in goa-1(lf) and goa-1(gf) backgrounds. (B) Dominant negative GDP-bound GOA-1(S47C) transgene (goa-1DN) diminished ADF tph-1 expression in three independent transgenic lines. goa-1(DN) transgenic animals remained capable of enhancing ADF tph-1::gfp when induced to form dauers or in tir-1(gf) background. For each assay, the value of ADF GFP fluorescence in dauers and that of mutants and transgenic animals is normalized to the value of WT animals under optimal conditions. Data represent the average of ≥ 2 trials ± SEM. The differences between WT and mutants are marked on the top of bar, and the differences between comparison groups are indicated, *p < 0.05, ** p < 0.01, *** p < 0.001, t-test for two group comparisons, and ANOVA for multi-group/condition comparisons. (C) ADF-specific marker Psrh-142::mCherry was expressed and showed characteristic ADF axon and dendritic morphology in goa-1(DN) transgenic animals. Animals at all developmental stages were analyzed, and images of L4 animals are shown. (D) A schematic of GPB-1 and GOA-1 interaction on tph-1 expression. Effector molecules compete with Goα for Gβ binding to drive tph-1 expression. In goa-1(lf) an goa-1(gf) mutants, the effector constitutively binds to Gβ. GDP-bound conformation of the GOA-1(DN) protein blocks the effector binding. GPB-1(G162E) obstructs the interactions with both GOA-1 and the effector, leading to constitutively diminished tph-1 regardless of the presence of GDP- or GTP-bound Goα.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581872&req=5

pgen.1005540.g006: GPB-1 regulates baseline ADF tph-1::gfp expression in a GOA-1-dependent manner.(A) Both GOA-1 deletion (goa-1lf) and GTP-bound hyperactive GOA-1(Q205L) (goa-1gf) mutations elevated ADF tph-1::gfp compared to WT. gpb-1(yz71) and ocr-2 TRPV mutations suppressed tph-1::gfp in goa-1(lf) and goa-1(gf) backgrounds. (B) Dominant negative GDP-bound GOA-1(S47C) transgene (goa-1DN) diminished ADF tph-1 expression in three independent transgenic lines. goa-1(DN) transgenic animals remained capable of enhancing ADF tph-1::gfp when induced to form dauers or in tir-1(gf) background. For each assay, the value of ADF GFP fluorescence in dauers and that of mutants and transgenic animals is normalized to the value of WT animals under optimal conditions. Data represent the average of ≥ 2 trials ± SEM. The differences between WT and mutants are marked on the top of bar, and the differences between comparison groups are indicated, *p < 0.05, ** p < 0.01, *** p < 0.001, t-test for two group comparisons, and ANOVA for multi-group/condition comparisons. (C) ADF-specific marker Psrh-142::mCherry was expressed and showed characteristic ADF axon and dendritic morphology in goa-1(DN) transgenic animals. Animals at all developmental stages were analyzed, and images of L4 animals are shown. (D) A schematic of GPB-1 and GOA-1 interaction on tph-1 expression. Effector molecules compete with Goα for Gβ binding to drive tph-1 expression. In goa-1(lf) an goa-1(gf) mutants, the effector constitutively binds to Gβ. GDP-bound conformation of the GOA-1(DN) protein blocks the effector binding. GPB-1(G162E) obstructs the interactions with both GOA-1 and the effector, leading to constitutively diminished tph-1 regardless of the presence of GDP- or GTP-bound Goα.
Mentions: Gβ could serve as a regulator of Gα signaling that drives tph-1 expression. Alternatively, Gβ itself could function as the signaling molecule to define tph-1 expression in ADF neurons. We first considered the possibility that altered Gα-mediated signaling underscores diminished tph-1 expression in gpb-1 mutants. ADF expresses seven Gα genes. Loss-of-function mutants of egl-30, gpa-3, gpa-10, gpa-13 and odr-3 did not dramatically diminish ADF tph-1::gfp [10,25,27]. gsa-1(lf) is lethal, but RNAi of gsa-1 also did not diminish ADF tph-1::gfp (134±14%, N = 50) compared to empty vector control (N = 28). Further, two deletion alleles of Goα goa-1, n363 and sa734, both markedly elevated ADF tph-1::gfp [10,25] (Fig 6A). Repeated attempts failed to generate a goa-1(n363);gpb-1(yz71) double mutant. As both GOA-1 and GPB-1 function in early embryogenesis [19,20,30], eliminating GOA-1 in gpb-1(yz71) background could be lethal. We therefore generated a double mutant of ocr-2 and goa-1(n363), and found that ocr-2;goa-1(n363) double mutants displayed diminished ADF tph-1::gfp as in ocr-2 single mutants (Fig 6A), suggesting that the absence of GOA-1 protein elevated tph-1 expression by activating the OCR-2 TRPV pathway.

Bottom Line: We found that signaling through cell autonomous GPB-1 to the OCR-2 TRPV channel defines the baseline expression of 5-HT synthesis enzyme tryptophan hydroxylase tph-1 in ADF chemosensory neurons.These results revealed a Gβ-mediated signaling operating in differentiated cells to specify intrinsic functional properties, and indicate that baseline TPH expression is not a default generic serotonergic fate, but is programmed in a cell-specific manner in the mature nervous system.Cell-specific regulation of TPH expression could be a general principle for tailored steady state 5-HT synthesis in functionally distinct neurons and their regulation of innate behavior.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT
Heterotrimeric G proteins regulate a vast array of cellular functions via specific intracellular effectors. Accumulating pharmacological and biochemical studies implicate Gβ subunits as signaling molecules interacting directly with a wide range of effectors to modulate downstream cellular responses, in addition to their role in regulating Gα subunit activities. However, the native biological roles of Gβ-mediated signaling pathways in vivo have been characterized only in a few cases. Here, we identified a Gβ GPB-1 signaling pathway operating in specific serotonergic neurons to the define steady state serotonin (5-HT) synthesis, through a genetic screen for 5-HT synthesis mutants in Caenorhabditis elegans. We found that signaling through cell autonomous GPB-1 to the OCR-2 TRPV channel defines the baseline expression of 5-HT synthesis enzyme tryptophan hydroxylase tph-1 in ADF chemosensory neurons. This Gβ signaling pathway is not essential for establishing the serotonergic cell fates and is mechanistically separated from stress-induced tph-1 upregulation. We identified that ADF-produced 5-HT controls specific innate rhythmic behaviors. These results revealed a Gβ-mediated signaling operating in differentiated cells to specify intrinsic functional properties, and indicate that baseline TPH expression is not a default generic serotonergic fate, but is programmed in a cell-specific manner in the mature nervous system. Cell-specific regulation of TPH expression could be a general principle for tailored steady state 5-HT synthesis in functionally distinct neurons and their regulation of innate behavior.

No MeSH data available.


Related in: MedlinePlus