Mutations that rescue the paralysis of Caenorhabditis elegans ric-8 (synembryn) mutants activate the G alpha(s) pathway and define a third major branch of the synaptic signaling network.
To identify hypothesized missing components of the synaptic G alpha(o)-G alpha(q) signaling network, which tightly regulates neurotransmitter release, we undertook two large forward genetic screens in the model organism C. elegans and focused first on mutations that strongly rescue the paralysis of ric-8(md303) reduction-of-function mutants, previously shown to be defective in G alpha(q) pathway activation. Through high-resolution mapping followed by sequence analysis, we show that these mutations affect four genes. Two activate the G alpha(q) pathway through gain-of-function mutations in G alpha(q); however, all of the remaining mutations activate components of the G alpha(s) pathway, including G alpha(s), adenylyl cyclase, and protein kinase A. Pharmacological assays suggest that the G alpha(s) pathway-activating mutations increase steady-state neurotransmitter release, and the strongly impaired neurotransmitter release of ric-8(md303) mutants is rescued to greater than wild-type levels by the strongest G alpha(s) pathway activating mutations. Using transgene induction studies, we show that activating the G alpha(s) pathway in adult animals rapidly induces hyperactive locomotion and rapidly rescues the paralysis of the ric-8 mutant. Using cell-specific promoters we show that neuronal, but not muscle, G alpha(s) pathway activation is sufficient to rescue ric-8(md303)'s paralysis. Our results appear to link RIC-8 (synembryn) and a third major G alpha pathway, the G alpha(s) pathway, with the previously discovered G alpha(o) and G alpha(q) pathways of the synaptic signaling network.
Program in Molecular, Cell and Developmental Biology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA.
Michael A
MA
Schade
Nicole K
NK
Reynolds
Claudia M
CM
Dollins
Kenneth G
KG
Miller
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000502:physiology*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D017238:Genes, Helminth; D005816:Genetic Complementation Test; D020662:Guanine Nucleotide Exchange Factors; D015801:Helminth Proteins / Q000235:genetics / Q000502:physiology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D009435:Synaptic Transmission / Q000235:genetics; D019076:Transgenes
D029742:Caenorhabditis elegans Proteins; D020662:Guanine Nucleotide Exchange Factors; D015801:Helminth Proteins; D009687:Nuclear Proteins; C413418:RIC-8 protein, C elegans; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000262:Adenylyl Cyclases