Recent studies suggest that there are at least two RGS proteins, EGL-10 and EAT-16, that can negatively regulate GOA-1 (Goa) and EGL-30 (Gqa), respectively, and that especially EAT-16 has an important role in mediating cross talk between GOA-1 and EGL-30. At this point, however, it is not known where and how the Gb subunits function in the GOA-1 and EGL-30 signaling network. Previously, we described the characterization of the second G protein b subunit in C. elegans, GPB-2. We found that, unlike
gpb-1,
gpb-2 is not an essential gene even though, like
gpb-1,
gpb-2 is expressed during development, in the nervous system, and in muscle cells. A loss-of-function mutation in
gpb-2 produces a variety of neuronal behaviors including delayed egg-laying and reduced pharyngeal pumping. Here, we focused on the functional role of GPB-2 in GOA-1 and EGL-30 signaling. We found that
gpb-2(
pk751);
dgk-1(
nu62) (
dgk-1 encodes a diacylglycerol kinase which is a potential downstream effector of GOA-1) and
gpb-2(
pk751)
goa-1(
pk62) double mutants result in a synthetic phenotypelethality at the larval stage (>95%). Moreover, the synthetic lethal phenotype of
gpb-2(
pk751)
goa-1(
pk62) is suppressed when we reduce EGL-30 activity by constructing an
egl-30(
n686)
gpb-2(
pk751)
goa-1(
pk62) triple mutant. No suppression is observed when we reduce EGL-8 activitya potential downstream effector of EGL-30. These results show that GPB-2 genetically interacts with the GOA-1 and EGL-30 pathway. GPB-2 is most similar to the divergent mammalian Gb 5 subunit, which has been shown to mediate a specific interaction with a Gg subunit-like (GGL) domain of RGS proteins. We examined the possibility that GPB-2 can physically interact with the GGL-domain of EGL-10 and EAT-16, and the Gg subunits, GPC-1 and GPC-2. Yeast two-hybrid analysis shows that GPB-2 binds to the GGL-domain of EGL-10 and EAT-16 and the G protein g subunits, GPC-1 and GPC-2. We found similar results with GPB-1. These findings suggest that there is no specificity for GPB-2 and GPB-1 in their ability to bind to EGL-10, EAT-16, GPC-1 or GPC-2 in vitro. Double-mutants of
gpb-2(
pk751)
eat-16(
sy438) and
gpb-2(
pk751);
egl-10(
md176) and
gpb-2(
pk751)
eat-16(
sy438);
egl-10(
md176) triple mutants are indistinguishable from
gpb-2 mutant animals. These results suggest that GPB-2 acts downstream of, or parallel to EGL-10 and EAT-16, also consistent with EGL-10 and EAT-16 being needed for GPB-2 function and vice versa. If this is the case, then loss of both
eat-16 and
egl-10 should result in animals resembling the phenotype of
gpb-2 mutant animals. Indeed, we observed that
eat-16(
sy438) in an
egl-10(
md176) genetic background resulted in animals that are similar to the
gpb-2 mutant phenotype. Thus,
eat-16 and
egl-10 act in parallel pathways, and in addition, our results provide evidence that GPB-2 genetically and physically interacts with both EGL-10 and EAT-16 to regulate the GOA-1 and EGL-30 signaling pathway.