Worm Breeder's Gazette 15(5): 47 (February 1, 1999)

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 second G protein beta subunit in C. elegans

Femke Simmer, Hendrik C. Korswagen, Gert Jansen, Ronald H.A. Plasterk

The Netherlands Cancer Institute, Division of Molecular Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

Heterotrimeric G proteins couple cell surface receptors and effector proteins in G protein coupled signal transduction. In C. elegans several G protein subunits have been identified. There are 20 G alpha subunits, two G beta subunits and two G gamma subunits.

The first G beta protein subunit (GPB-1) is 86% identical to mammalian G protein beta subunits. Zwaal et al. (Cell 86, 619-629, 1996) analyzed the role of the gpb-1 gene in C. elegans. gpb-1 is broadly expressed, in muscle cells and neurons. Mutants homozygous for the gpb-1 deletion allele die soon after hatching. When the maternal contribution of GPB-1 is also excluded, the animals die during embryogenesis due to randomization of spindle orientations in early cell divisions. Overexpression of GPB-1 results in reduced locomotion and egg-laying.

Recently a second G protein beta subunit (gpb-2; F52A8.2) was identified in the genome sequence. This beta subunit only shares 48% identity with GPB-1 and the mammalian G beta subunits one to four. However it has 65% identity to the fifth mammalian G beta subunit, a somewhat diverged G beta subunit which was identified recently. To analyze the function of gpb-2 we determined the expression pattern and generated loss- and gain-of-function mutants.

Two gpb-2::GFP fusions, containing the first three exons with 2 kb upstream or the first five exons with 1.5 kb upstream fused in frame to GFP, were used to analyze the expression pattern. GFP staining was observed in practically all neurons and muscle cells. We generated a loss of function mutant using target selected gene inactivation. The deletion allele (pk571) misses the last five exons; exon four to eight, and 1.5 kb downstream of the gene. This probably is a null allele. Gain-of-function animals were generated that carry the wild type gpb-2 gene at elevated copy number as a transgene. The transgene was integrated by gamma-irradiation.

Both gain- and loss-of-function mutation did not affect viability. Based on the expression pattern and the phenotypic appearance the mutant animals were tested for egg-laying and locomotion. Locomotion was assayed as the number of body bends per 15 seconds. Loss-of-function animals showed significant decreased locomotion, gain-of-function animals behaved like wild type. Egg laying was assayed as the number of unlaid eggs in the uterus. Loss-of-function animals showed significantly increased numbers of eggs in their uterus, whereas gain-of-function animals looked like wild type. We also analyzed the developmental stage of newly laid eggs. The newly laid eggs produced by gpb-2 animals were in a late developmental stage, they contained post comma embryos, whereas gain-of-function animals laid eggs with embryos of 9-cell to comma stage like wild type.

We confirmed that the phenotypes observed are the result of the loss-of-function mutation by introducing the wild type gpb-2 gene in loss-of-function animals. This rescued the reduced muscle activity.

Our results show that gpb-2 regulates muscle activity: Loss-of-function reduces the activity of both body wall and egg-laying muscles. Although gpb-1 and gpb-2 are expressed in the same cells and affect similar activities, they do not seem to have redundant functions, and it remains to be determined how they differ in specificity.