Structural Domains Required for Caenorhabditis elegans G Protein-coupled Receptor Kinase 2 (GRK-2) Function in Vivo

Wood, John J.; Wang, Jianjun; Benovic, Jeffrey L.; Ferkey, Denise M.

doi:10.1074/jbc.m111.336818

“…Thus, we tested whether the kinase activity of GRK-2 is required for proper locomotion and G q signaling by assaying whether a kinase-dead GRK-2[K220R] mutant [48,68] is capable of rescuing the grk-2(gk268) and egl-30(tg26); grk-2(gk268) mutants. Wild-type GRK-2 rescued the locomotion defect of grk-2(gk268) mutants (Fig 1C, Left), but the kinase-dead GRK-2[K220R], although it was properly expressed (Fig 2G), did not rescue either the locomotion defect or the suppression of activated G q (Fig 1C and 1D).…”

Section: Resultsmentioning

confidence: 99%

“…C . elegans has two GRKs: GRK-1 and GRK-2, orthologs of the GRK4/5/6 and GRK2/3 families respectively [48]. Mammalian GRK2 is ubiquitously expressed [49,50] and GRK2 knock-out mice die as embryos [51].…”

Section: Introductionmentioning

confidence: 99%

Dopamine negatively modulates the NCA ion channels in C. elegans

Topalidou

¹

,

Cooper

²

,

Pereira

³

et al. 2017

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The NALCN/NCA ion channel is a cation channel related to voltage-gated sodium and calcium channels. NALCN has been reported to be a sodium leak channel with a conserved role in establishing neuronal resting membrane potential, but its precise cellular role and regulation are unclear. The Caenorhabditis elegans orthologs of NALCN, NCA-1 and NCA-2, act in premotor interneurons to regulate motor circuit activity that sustains locomotion. Recently we found that NCA-1 and NCA-2 are activated by a signal transduction pathway acting downstream of the heterotrimeric G protein Gq and the small GTPase Rho. Through a forward genetic screen, here we identify the GPCR kinase GRK-2 as a new player affecting signaling through the Gq-Rho-NCA pathway. Using structure-function analysis, we find that the GPCR phosphorylation and membrane association domains of GRK-2 are required for its function. Genetic epistasis experiments suggest that GRK-2 acts on the D2-like dopamine receptor DOP-3 to inhibit Go signaling and positively modulate NCA-1 and NCA-2 activity. Through cell-specific rescuing experiments, we find that GRK-2 and DOP-3 act in premotor interneurons to modulate NCA channel function. Finally, we demonstrate that dopamine, through DOP-3, negatively regulates NCA activity. Thus, this study identifies a pathway by which dopamine modulates the activity of the NCA channels.

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“…In each case, expression of the mutant grk-2 cDNA under the control of the grk-2 promoter significantly rescued the slow locomotion of the grk-2(gk268) mutant (**, P<0.01; ***, P<0.001. Error disrupt specific activities of GRK-2, but do not disrupt GRK-2 protein expression or stability [48]. These mutations all affect conserved residues in well-characterized domains of GRK-2 [48].…”

Section: Gpcr-phosphorylation and Membrane Association Domains Of Grkmentioning

confidence: 99%

“…Error disrupt specific activities of GRK-2, but do not disrupt GRK-2 protein expression or stability [48]. These mutations all affect conserved residues in well-characterized domains of GRK-2 [48].…”

Section: Gpcr-phosphorylation and Membrane Association Domains Of Grkmentioning

confidence: 99%

“…Mammalian GRKs have been divided into three groups based on their sequences and function: 1) GRK1 and GRK7, 2) GRK2 and GRK3, and 3) GRK4, GRK5 and GRK6 [47]. C. elegans has two GRKs: GRK-1 and GRK-2, orthologs of the GRK4/5/6 and GRK2/3 families respectively [48]. Mammalian GRK2 is ubiquitously expressed [49,50] and GRK2 knock-out mice die as embryos [51].…”

Section: Introductionmentioning

confidence: 99%

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Dopamine Negatively Modulates the NCA Ion Channels inC. elegans

Topalidou

¹

,

Cooper

²

,

Pereira

³

et al. 2016

Preprint

View full text Add to dashboard Cite

The NALCN/NCA ion channel is a cation channel related to voltage-gated sodium and calcium channels. NALCN has been reported to be a sodium leak channel with a conserved role in establishing neuronal resting membrane potential, but its precise cellular role and regulation are unclear. The Caenorhabditis elegans orthologs of NALCN, NCA-1 and NCA-2, act in premotor interneurons to regulate motor circuit activity that sustains locomotion. Recently we found that NCA-1 and NCA-2 are activated by a signal transduction pathway acting downstream of the heterotrimeric G protein G q and the small GTPase Rho. Through a forward genetic screen, here we identify the GPCR kinase GRK-2 as a new player affecting signaling through the G q -Rho-NCA pathway. Using structure-function analysis, we find that the GPCR phosphorylation and membrane association domains of GRK-2 are required for its function. Genetic epistasis experiments suggest that GRK-2 acts on the D2-like dopamine receptor DOP-3 to inhibit G o signaling and positively modulate NCA-1 and NCA-2 activity. Through cell-specific rescuing experiments, we find that GRK-2 and DOP-3 act in premotor interneurons to modulate NCA channel function. Finally, we demonstrate that dopamine, through DOP-3, negatively regulates NCA activity. Thus, this study identifies a pathway by which dopamine modulates the activity of the NCA channels. Author summaryDopamine is a neurotransmitter that acts in the brain by binding seven transmembrane receptors that are coupled to heterotrimeric GTP-binding proteins (G proteins). Neuronal G proteins often function by modulating ion channels that control membrane excitability. Here we identify a molecular cascade downstream of dopamine in the nematode C. elegans that involves activation of the dopamine receptor DOP-3, activation of the G protein GOA-1, and inactivation of the NCA-1 and NCA-2 ion channels. We also identify a G protein-coupled receptor kinase (GRK-2) that inactivates the dopamine receptor DOP-3, thus leading to inactivation of GOA-1 and activation of the NCA channels. Thus, this PLOS Genetics | https://doi.org/10.1371/journal.pgen

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