Mutations That Rescue the Paralysis of Caenorhabditis elegans ric-8 (Synembryn) Mutants Activate the Gαs Pathway and Define a Third Major Branch of the Synaptic Signaling Network

To identify hypothesized missing components of the synaptic Gαo-Gαq 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 Gαq pathway activation. Through high-resolution mapping followed by sequence analysis, we show that these mutations affect four genes. Two activate the Gαq pathway through gain-of-function mutations in Gαq; however, all of the remaining mutations activate components of the Gαs pathway, including Gαs, adenylyl cyclase, and protein kinase A. Pharmacological assays suggest that the Gαs 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 Gαs pathway activating mutations. Using transgene induction studies, we show that activating the Gαs 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, Gαs pathway activation is sufficient to rescue ric-8(md303)'s paralysis. Our results appear to link RIC-8 (synembryn) and a third major Gα pathway, the Gαs pathway, with the previously discovered Gαo and Gαq pathways of the synaptic signaling network.