Worm Breeder's Gazette 16(4): 31 (October 1, 2000)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

The G protein beta subunit GPB-2 in Caenorhabditis elegans regulates the Go-alpha and Gq-alpha signaling network through interactions with the RGS proteins EGL-10 and EAT-16

Alexander M. van der Linden, Femke Simmer, Edwin Cuppen, Ronald H.A. Plasterk

Hubrecht Laboratory, Centre for Biomedical Genetics, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands

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 phenotypeŚlethality 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 activityŚa 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.