Mutations in the G guanine nucleotide exchange factor RIC-8 cause strong paralysis and decreased neurotransmitter release due to disruption of the Gq and Gs pathways of the Synaptic Signaling Network. Using large-scale
ric-8 suppressor and hyperactive behavior genetic screens we found that mutations that activate either the Gq or Gs pathways suppress the strong paralysis of a
ric-8 reduction-of-function mutant. In addition to known components of the Gq and Gs pathways, our mutant collection from these screens has yielded mutations in
phlp-1 (Phosducin-Like Protein) and 8 unidentified stim genes that have the potential to reveal new components of the network. Here we focus on
phlp-1, represented by the single allele
ce266.
ce266 strikingly rescues the paralysis of both
ric-8(
md303) and
ric-8(
md1909). As a single mutant,
ce266 confers strongly hyperactive locomotion, constitutive egg-laying, and hypersensitivity to aldicarb (suggesting strongly increased neurotransmitter release). Its phenotype is very similar to mutants containing an activated Gq pathway or a knocked-out Go pathway. We used SNPs to map the mutation to a 118 Kb interval and then candidate gene sequencing to determine that the mutation affects PHLP-1, the sole C. elegans ortholog of Phosducin-like protein. In transgenic experiments, a fragment containing a wild type version of this gene rescues the mutants behavior to wild type levels. To our knowledge, this is the first ever loss-of-function mutation in Phosducin-Like Protein isolated in an animal system. Paradoxically,
ce266 mutants also show signs of a severely compromised Gq pathway. For example,
ce266 strongly increases, rather than rescues, the paralysis of
egl-30 (Gq) rf mutants, and a fraction of
ce266 single mutant larvae exhibit paralysis similar to
egl-30 nulls. Many, but not all,
ce266 single mutants recover and develop into hyperactive adults. Ingestion of
phlp-1 dsRNA has no effect on wild type or
rrf-3 mutants, but even a very small dose of
phlp-1 dsRNA in a
ce266 mutant greatly increases the fraction of paralyzed larvae. Explaining these results seems to require a model in which PHLP-1 is required for Gq signaling, but also exerts a strong inhibitory effect.
ce266 mutants also have ~4% embryonic lethality, which is increased to ~100% by feeding the
ce266 mutant
phlp-1 dsRNA. Interestingly, the
ce266 mutation significantly suppresses the partial embryonic lethality of both
ric-8(
md303) and
ric-8(
md1909), which suggests a role for PHLP-1 in regulating centrosome movements during early embryogenesis as we previously showed for RIC-8.