Evidence for a role for the Dictyostelium Rap1 in cell viability and the response to osmotic stress

Abstract

The Dictyostelium genome contains a single rapA gene,which encodes a Rap1 monomeric G protein. As attempts at generating rapA-null Dictyostelium cells had been unsuccessful,expression of antisense RNA from the rapA gene under control of the folate repressible discoidin promoter was used to reduce cellular levels of the Rap1 protein. As Rap1 levels gradually decreased following antisense rapA RNA induction, growth rate and cell viability also decreased, a result consistent with the idea that rapA is an essential gene. The Rap1-depleted cells exhibited reduced viability in response to osmotic shock. The accumulation of cGMP in response to 0.4 M sorbitol was reduced after rapA antisense RNA induction and was enhanced in cells expressing the constitutively activated Rap1(G12V) protein, suggesting a role for Rap1 in the generation of cGMP. Dictyostelium Rap1 formed a complex with the Ras-binding domain of RalGDS only when it was in a GTP-bound state. This assay was used to demonstrate that activation of Rap1 in response to 0.4 M sorbitol occurred with initial kinetics similar to those observed for the accumulation of cGMP. Furthermore, the addition of 2 mM EDTA to osmotically shocked cells, a treatment that enhances cGMP accumulation, also enhanced Rap1 activation. These results suggest a direct role for Rap1 in the activation of guanylyl cyclase during the response to hyperosmotic conditions. Rap1 was also activated in response to low temperature but not in response to low osmolarity or high temperature.

Big roles for small GTPases in the control of directed cell movement

“…The small GTPase Rap1 was also found to promote cGMP production, although this was reported to occur in response to osmotic stress [160]. A role for Rap1 in the regulation of guanylate cyclase activity during chemotaxis therefore remains to be investigated.…”

Rap1b is required for normal platelet function and hemostasis in mice

“…Genetic analysis of Rap1 function in lower eukaryotes has revealed that Rap is critical for development, as loss-of-function mutations are lethal in Drosophila (3) and Dictyostelium (5). In mammals, the Rap family consists of 2 rap1 genes, rap1a and rap1b, encoding proteins that are 95% identical, and 2 rap2 genes encoding proteins that are 65% homologous with the Rap1 proteins.…”

Small molecule-enrichment analysis in response to osmotic stimuli in the intervertebral disc

“…RAP2A belongs to the RAS subfamily of small GTP-binding proteins, which are considered to control cell growth, differentiation and survival (Bokoch, 1993;Burgering and Bos, 1995). To date, there is no direct evidence of RAP2A ever regulating osmotic pressure; however, previous studies have demonstrated that Rap1 plays a direct role in the activation of guanylyl cyclase during the response to hyper-osmotic conditions (Kang et al, 2002). Because Rap2 and Rap1 proteins are 60% identical to each other (Reuther and Der, 2000;Takai et al, 2001), per- (Reuther and Der, 2000;Takai et al, 2001), per-, perhaps they share some common functions.…”