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Multiple signalling pathways connect chemoattractant receptors and calcium channels in Dictyostelium
Thomas Nebl1, Martha Kotsifas, Pauline Schaap
1Department of Microbiology, La Trobe University, Bundoora, Victoria 3083, Australia.
Abstract:
Dictyostelium mutants expressing aequorin were used to study and compare the roles of heterotrimeric G-proteins and the second messengers IP3 and cGMP in regulating folate- and cAMP receptor-activated [Ca2+]i signals. The calcium responses of vegetative cells to folate were dramatically impaired in Gbeta and Galpha4 null mutants but were restored with altered kinetics and temperature-sensitivity in Gbeta null mutants overexpressing wild type and temperature-sensitive Gbeta isoforms. Folic acid receptors thus mediate changes in [Ca2+]i via a Galpha4betagamma-dependent pathway. Neither folate nor cAMP-induced [Ca2+]i signals were significantly altered in PLC null transformants, but [Ca2+]i changes elicited by both attractants were significantly prolonged in two stmF mutants lacking cGMP-specific phosphodiesterase activity. This confirms an important role of cGMP in regulating receptor-activated Ca2+ uptake and/or extrusion systems. This cGMP-dependent part of the Ca2+ response to cAMP stimuli was developmentally down-regulated and all but disappeared by the time the cells reached full aggregation competence after 8 h of starvation. The results suggest that folate and cAMP receptor-activated [Ca2+]i signals are regulated in a complex manner via multiple signalling pathways, one that is G-protein- and cGMP-dependent (present at the vegetative and early poststarvation stage) and another that is G-protein-independent (dominant in fully aggregation-competent cells at approximately 8 h poststarvation).
Insights
Dictyostelium cells use G-proteins and cyclic GMP (cGMP) to control calcium (Ca2+) signals triggered by folate and cAMP. These pathways are crucial for early cell stages but become less important as cells aggregate.
Area of Science:
- Cellular signaling
- Biochemistry
- Molecular biology
Background:
- Dictyostelium discoideum serves as a model organism to investigate fundamental cellular processes.
- Calcium ions ([Ca2+]i) play a critical role in signal transduction pathways.
- Heterotrimeric G-proteins and second messengers like cyclic adenosine monophosphate (cAMP) and inositol trisphosphate (IP3) are key regulators of cellular responses.
Purpose of the Study:
- To elucidate the roles of G-proteins, IP3, and cGMP in regulating calcium signals activated by folate and cAMP receptors in Dictyostelium.
- To compare the distinct and overlapping functions of these signaling molecules in mediating cellular responses to different chemoattractants.
Main Methods:
- Utilized Dictyostelium mutants expressing aequorin to monitor intracellular calcium levels ([Ca2+]i).
- Generated and analyzed null mutants for G-protein subunits (Gbeta, Galpha4) and phospholipase C (PLC).
- Investigated mutants lacking cGMP-specific phosphodiesterase (stmF) and overexpressed G-protein isoforms.
Main Results:
- Folate receptor-mediated calcium signaling is dependent on a Galpha4betagamma pathway, as indicated by impaired responses in Gbeta and Galpha4 null mutants.
- cGMP plays a significant role in regulating calcium uptake/extrusion, with mutations affecting cGMP hydrolysis prolonging calcium signals.
- The contribution of cGMP to calcium responses diminishes during cellular development, becoming negligible in aggregation-competent cells.
Conclusions:
- Folate and cAMP receptor-activated calcium signals are regulated by complex, parallel pathways.
- A G-protein- and cGMP-dependent pathway is active in vegetative and early-starvation stages.
- A G-protein-independent pathway becomes dominant in fully aggregation-competent cells, suggesting developmental regulation of signaling.