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Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum
Published on: September 20, 2011
Chemoattractant-induced Ras activation during Dictyostelium aggregation
Helmut Kae1, Chinten James Lim, George B Spiegelman
1Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z3.
Abstract:
Ras proteins are highly conserved molecular switches that regulate cellular response to external stimuli. Dictyostelium discoideum contains an extensive family of Ras proteins that function in regulation of mitosis, cytoskeletal function and motility, and the onset of development. Little is known about the events that lead to the activation of Ras proteins in Dictyostelium, primarily owing to a lack of a biochemical assay to measure the levels of activated Ras. We have adapted an assay, used successfully to measure activated Ras in mammalian cells, to monitor activation of two Dictyostelium Ras proteins, RasC and RasG. We have found that the Ras-binding domain (RBD) of mammalian Raf1 was capable of binding to the activated form of RasG, but not to the activated form of RasC; however, the RBD of Schizosaccharomyces pombe Byr2 was capable of binding preferentially to the activated forms of both RasC and RasG. Using this assay, we discovered that RasC and RasG showed a rapid and transient activation when aggregation-competent cells were stimulated with the chemoattractant cAMP, and this activation did not occur in a number of cAMP signalling mutants. These data provide further evidence of a role for both RasC and RasG in the early development of Dictyostelium.
Insights
Researchers developed a new assay to measure activated Ras proteins in Dictyostelium discoideum. This study reveals rapid, transient activation of RasC and RasG proteins in response to cAMP signaling during early development.
Area of Science:
- Cellular signaling and molecular biology
- Developmental biology in Dictyostelium discoideum
Background:
- Ras proteins act as molecular switches regulating cellular responses.
- Dictyostelium discoideum possesses a diverse Ras protein family involved in key cellular processes.
- Limited understanding of Ras protein activation mechanisms in Dictyostelium due to assay limitations.
Purpose of the Study:
- To adapt a biochemical assay for measuring activated Ras proteins in Dictyostelium.
- To investigate the activation patterns of RasC and RasG proteins in response to cAMP.
- To elucidate the role of RasC and RasG in early Dictyostelium development.
Main Methods:
- Adaptation of a mammalian cell-based assay to measure activated Ras proteins.
- Utilized Ras-binding domains (RBDs) from mammalian Raf1 and S. pombe Byr2 for detection.
- Stimulated aggregation-competent Dictyostelium cells with cAMP and monitored Ras activation.
Main Results:
- Mammalian Raf1 RBD bound activated RasG but not RasC; S. pombe Byr2 RBD bound both RasC and RasG.
- RasC and RasG exhibited rapid, transient activation upon cAMP stimulation in wild-type cells.
- This activation was absent in several cAMP signaling mutants, indicating pathway dependency.
Conclusions:
- The adapted assay effectively monitors Ras protein activation in Dictyostelium.
- RasC and RasG are rapidly activated by cAMP signaling during early Dictyostelium development.
- These findings support a crucial role for RasC and RasG in Dictyostelium morphogenesis and development.
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