Effects of prostaglandin E2 on the subcellular localization of Epac-1 and Rap1 proteins during Fcγ-receptor-mediated phagocytosis in alveolar macrophages

Abstract

Recent studies have demonstrated a central role for the exchange protein activated by cAMP (Epac) in the inhibition of Fcγ-receptor mediated phagocytosis and bacterial killing by prostaglandin E 2 (PGE 2 ) in macrophages. However, the subcellular localization of Epac, and its primary target Rap1, has yet to be determined in primary macrophages. Therefore, we used immunofluorescent techniques and phagosome isolation to localize Epac-1 and Rap1 in alveolar macrophages. Epac-1 was predominantly expressed on punctate and tubular membranes throughout the cell body; on the plasma membrane; and co-localized with microtubule organizing centers (MTOCs). Rap1 was abundant on punctate membranes, less abundant on plasma membrane, and also found on MTOCs. Following PGE 2 treatment, Epac-1, but not Rap1, accumulated on the nuclear envelope and disappeared from MTOCs. By immunofluorescent microscopy, both Epac-1 and Rap1 were seen to associate with phagosomes containing IgG-opsonized beads, but this association appeared weak, as we failed to observe such interactions in phagosomes isolated from cells at various timepoints after bead ingestion. Strikingly, however, Epac-1, but not Rap1, appeared to accumulate on maturing phagosomes, but only after PGE 2 treatment (or treatment with a selective Epac-1 agonist). This association was confirmed in isolated phagosome preparations. The changes in Epac-1 localization were too slow to account for the inhibitory effects of PGE 2 on phagocytosis. However, the appearance of Epac-1 on late phagosomes following PGE 2 treatment might be important for suppressing H 2 O 2 production and inhibiting the killing of intraphagosomal pathogens. The absence of Rap1 on late phagosomes suggests that the effect of Epac-1 might not require Rap1.

Intracellular cAMP Sensor EPAC: Physiology, Pathophysiology, and Therapeutics Development

“…Comparable results are witnessed in malaria-infested Kupffer cells where sporozoites defend themselves against these macrophages through a PKA-independent cAMP/ EPAC signal to abrogate respiratory burst (1053). In addition, PGE 2 and 007 treatment do not alter Rap1 localization in rat alveolar macrophages compared with EPAC phagosome localization which could alter EPAC function in these cells (108). The localization of constitutively active Rap1 compared with dominant-negative Rap1 N17 would also be of interest to determine if small populations of active Rap1-GTP are transiently associating with the phagosome and were undetected in the mentioned study, potentially explaining the discrepancy between microscopic and immunoprecipitation observations.…”

“…The dynamic cellular distribution of EPAC1 indicates that its cellular localization is highly regulated and has important functional implications. Not surprisingly, cAMP, in addition to directly activating EPAC1, is crucial in controlling EPAC1's subcellular targeting (108,834,845). For example, in primary rat alveolar macrophage (AM), EPAC1 is detected on punctate and tubular membranes throughout the cell body, predominantly at the perinuclear region with low levels present on the plasma membrane.…”

“…In addition to directly activating EPAC GEF activity, binding of cAMP also induces a conformational change in the DEP domain (607), which results in the exposure of a 17-amino acid polybasic PA binding motif within the DEP domain (FIGURE 4A) and enable EPAC1 binding to PA on the PM (198). In contrast, low intracellular cAMP levels favor distribution of EPAC1 towards the microtubule (MT) cytoskeleton (108,845). This is likely attributable to EPAC1's ability to directly interact with MT and to promote tubulin polymerization independent of Rap1 (686,930).…”

“…This loss of MTOC localization may lead to the suppression of phagocytosis through altered function of EPAC on microtubule dynamics, although the mechanism behind this EPAC1 function is not clear in phagocytizing cells. Also interesting is that after PGE 2 stimulation or direct activation of EPAC by 007, EPAC1 is found to visually and physically associate with maturing, late phagosomes, while only weak visual evidence of Rap1 with these structures can be defined (108). This could suggest that Rap1 is not a major target of EPAC in this signaling pathway, but rather EPAC may merely signal through Rap1 for other unidentified signaling cascades concurrent with inhibition of phagocytosis of the particle.…”

Purinergic A2b Receptor Activation by Extracellular Cues Affects Positioning of the Centrosome and Nucleus and Causes Reduced Cell Migration

“…Epac1 distribution in the cell varies with cell type and has been reported in the cytosol, at the plasma membrane, and in perinuclear regions, the nuclear envelope, mitochondria, and phagosomes (63)(64)(65). Its subcellular location changes with the cell cycle stage and with the state of its activation (63).…”