Heterotrimeric G proteins are signal transducing molecules that link extracellular signals received via transmembrane receptors to intracellular second messengers. In mammals, heterotrimeric G protein -subunits are classified into four families: Gi/Go; Gq; Gs and G12. In this study, we characterize a C. elegans G -subunit gene,
gpa-12, that is most similar to mammalian G12/ G13.
gpa-12 is expressed in pharyngeal muscle and hypodermal cells. Animals defective in
gpa-12 do not show any obvious phenotype. However, overexpression of a constitutively active form of
gpa-12 results in developmentally arrested larvae that eventually die. This developmental growth arrest is likely caused by a feeding defect, because the pharyngeal pumping rate of the arrested animals is almost reduced to zero. To elucidate the molecular nature of the signaling pathways in which G12 participate, we screened for suppressors of the activated GPA-12 phenotype. Therefore, we expressed the constitutively active form of
gpa-12 from a heat-shock promoter. Heat-shock treatment of L1 larvae results in a developmental growth arrest from which the animals eventually recover. A 5 min heat-shock is sufficient to induce a growth arrest, but the recovery time is less than for longer heat-shock treatments (up to 2 hr). Using EMS mutagenesis, we identified 44 extragenic suppressors that suppress the developmental growth arrest induced by 2 hr heat-shock. All 44 suppressors have a mutation in
tpa-1, a gene encoding a protein kinase C subunit. This strongly suggests that
tpa-1 is a mediator of G12 signaling.
tpa-1 encodes two isoforms, TPA-1A and TPA-1B. TPA-1A has four additional exons upstream compared to TPA-1B. Mutations in
tpa-1 were firstly identified to suppress the developmental growth arrest induced by the tumor-promoting phorbol ester PMA (Tabuse et al., 1995). All mutations found in our screen disrupt both isoforms, whereas none is located in one of the four additional exons of TPA-1A. This suggests that wildtype TPA-1B is required for the G12-induced growth arrest. This is consistent with the result that TPA-1B and GPA-12 are expressed in the same tissues. We also isolated several mutants that suppress the developmental growth arrest induced by 5 min heat-shock, but that do not or only moderately suppress the growth arrest induced by 2 hr heat- shock. Surprisingly, one of these suppressors has a mutation in the part of TPA-1 that is only present in TPA-1A, thereby disrupting TPA-1A but not TPA-1B. Altogether, these results indicate that both isoforms of TPA-1 are involved in G12-mediated signaling.