Mechanism of cAMP-induced Ca2+ influx in Dictyostelium: role of phospholipase A2

Abstract

cAMP-induced Ca2+ influx in Dictyostelium follows two pathways: a G-protein-dependent pathway where influx is reduced by 50-70% in Galpha2 and Gbeta-negative strains and a heterotrimeric G-protein-independent pathway. Using a pharmacological approach, we found that phospholipase A2 (PLA2) is the target of both pathways. The products of PLA2 activity, arachidonic acid (AA) and palmitic acid, induced Ca2+ influx to a similar extent as cAMP. Half-maximal activation occurred at 3 microM AA and saturation at 10 microM AA. The response to AA was quantitatively similar throughout early differentiation and thus independent of cAMP-receptor concentration. Synergy experiments revealed that cAMP and AA acted through identical pathways. The PLA2-activating peptide, a peptide with sequence similarity to the G-protein beta-subunit, activated Ca2+ influx. The G-protein-independent pathway was sensitive to genistein but not to blockers of protein kinase C and other kinases, suggesting that tyrosine kinase may directly or indirectly activate PLA2 in this case.

Release of Ca 2+ from the Endoplasmic Reticulum Contributes to Ca 2+ Signaling in Dictyostelium discoideum

“…This accords with the suggestion that chemoattractants such as cAMP may activate phospholipase A2, resulting in the production of long chain fatty acids and the release of Ca 2ϩ from intracellular stores, depleting them and thereby activating influx (45).…”

“…All work was done with Dictyostelium discoideum strain AX2 (2), with the mutant strains derived from it, HG1774 (calreticulin deficient), HG1770 (calnexin deficient), HG1772 (calreticulin and calnexin deficient) (35), and with aequorin-expressing transformants derived from them (HPF401 from AX2, HPF608 from HG1774, and HPF609 and HPF610 from HG1770). For axenic growth the amoebae were incubated as previously in shaken culture (150 rpm) at 23°C in axenic medium (45) or at 21°C in HL5 medium (38).…”

“…The cAR1 receptor was the first G-protein-coupled receptor shown to elicit some downstream events in a G-protein-independent manner including an influx of extracellular Ca 2ϩ (32,33,39,45) and activation of the mitogen-activated protein kinase ERK2 (25). During chemotaxis in a spatiotemporal attractant gradient, the cytosolic Ca 2ϩ elevation in response to an attractant occurs primarily in the rear cortex of the cell (59) where it may contribute to retraction of the rear and inhibition of inappropriate pseudopod extension in incorrect directions.…”

The regulation of cell motility and chemotaxis by phospholipid signaling

“…Previous studies in Dictyostelium have suggested that products of PLA2 action, such as arachidonic acid, affect chemoattractantinduced Ca 2+ influx and can trigger Ca 2+ influx directly (Schaloske and Malchow, 1997). In mammalian cells, arachidonic acid is involved in the release of Ca 2+ from internal stores by regulating calcium channels (Osterhout and Shuttleworth, 2000;Shuttleworth and Thompson, 1999).…”

Oscillatory signaling and network responses during the development of Dictyostelium discoideum

“…Ca 2+ is not required to activate PLA 2 (Chen et al, 2007), but PLA 2 inhibitors suppress cAMPdependent influx of Ca 2+ and addition of the putative PLA 2 product AA rescues Ca 2+ influx in the presence of PLA 2 inhibitors (Schaloske and Malchow, 1997). Nonetheless, cells genetically deficient for one of the several putative PLA 2 genes in Dictyostelium display normal Ca 2+ influx and addition of AA to pla2-null cells treated with LY2940004 partially rescues the ability of these cells to aggregate (Chen et al, 2007).…”