Localization and Dynamics of Glucocorticoid Receptor at the Plasma Membrane of Activated Mast Cells

Abstract

In addition to their actions in the cell nucleus, glucocorticoids exhibit rapid non-nuclear responses that are mechanistically not well understood. To explain these effects, the localization of a glucocorticoid receptor (GR) expressed in mast cells as a GFP fusion was analyzed after activation of the cells on allergenic lipid arrays. These arrays were produced on glass slides by dip-pen nanolithography (DPN) and total internal reflection (TIRF) microscopy was used to visualize the GR. A rapid glucocorticoid-independent and -dependent recruitment of the GR-GFP to the plasma cell membrane was observed following contact of the cells with the allergenic array. In addition, the mobility of the GR at the membrane was monitored by fluorescence recovery after photobleaching (FRAP) and shown to follow binding kinetics demonstrating interactions of the receptor with membrane-bound factors. Furthermore the recruitment of the GR to the cell membrane was shown to result in a glucocorticoid-mediated increase in Erk phosphorylation. This is evidenced by findings that destruction of the membrane composition of the mast cells by cholesterol depletion impairs the membrane localization of the GR and subsequent glucocorticoid-mediated enhancement of Erk phosphorylation. These results demonstrate a membrane localization and function of the GR in mast cell signaling.

Effects of Glucocorticoids in the Immune System

“…Here, it has been shown that following allergen-mediated crosslinking of FcεRI of mast cells, the GR is rapidly recruited to the plasma membrane ( Fig. 9.1 ) and this process is further transiently enhanced by glucocorticoid treatment [ 52 ]. It therefore appears that the membrane-localized GR may serve as an early sensor for the hormone to amplify its main genomic action [ 52 ].…”

“…9.1 ) and this process is further transiently enhanced by glucocorticoid treatment [ 52 ]. It therefore appears that the membrane-localized GR may serve as an early sensor for the hormone to amplify its main genomic action [ 52 ]. In kinetic studies of hormonedependent nuclear translocation of the GR, a transient delay in the mobility of the GR was observed in allergen-activated compared to non-activated mast cells [ 52 ].…”

“…In yet another technique using mast cells, allergens were patterned on a glass surface, to immobilize and activate the mast cells [ 51 ]. This approach was used to visualize in real time the recruitment of a GR tagged with GFP to the site of interaction of the allergen with the cell [ 52 ]. Further analyses using photobleaching techniques revealed slower dynamics of the receptor at the membrane of the activated mast cells compared to non-activated cells, indicating possible interaction of the GR with membrane bound components in the activated cells [ 52 ].…”

“…This approach was used to visualize in real time the recruitment of a GR tagged with GFP to the site of interaction of the allergen with the cell [ 52 ]. Further analyses using photobleaching techniques revealed slower dynamics of the receptor at the membrane of the activated mast cells compared to non-activated cells, indicating possible interaction of the GR with membrane bound components in the activated cells [ 52 ]. With these brief descriptions of the pathways and methods used for studying glucocorticoid action in immune cells, we will now take a look at the effect of the hormone on individual immune cells.…”

Diamond Nanophotonic Circuits Functionalized by Dip‐pen Nanolithography

“…different inks written at the same time and in close vicinity on the surface) functionalization of surfaces with biomimetical membrane stacks used e.g. in 3 immunological assays [29][30][31][32]. This technique does not rely on a specific surface chemistry and can therefore be applied to various substrates, e.g.…”