TI - Block of Glu-mp phagocytosis correlates with sustained signalling by Dectin-1 and MAPK activation . AB - Internalization of cell surface signalling receptors can result in two distinct outcomes . Some receptors , such as TGF-beta receptor , nerve growth factor receptor and epidermal growth factor receptor continue to signal from endosomes , amplifying downstream responses before being eventually degraded 21-26 . In contrast , for the BCR , uptake is associated with diminished Ca2+ fluxes and decreased activation of RelA , Akt and ERK , suggesting that productive signalling occurs before receptor translocation to endosomes 27-30 . To determine the effect of internalization on Dectin-1 signalling , we evaluated MAP kinase ( ERK , p38 , JNK ) activation in BMDC stimulated with Glu-mp in the presence or absence of actin polymerization inhibitors . Cells stimulated with Glu-mp in the absence of latrunculin A displayed weak and transient PHOSphorylation of ERK and no detectable activation of p38 or JNK (Fig 3A) . In contrast , in cells stimulated in the presence of latrunculin A , we observed a strong and sustained activation of all three MAP kinases (Fig 3A) , mimicking the action of curdlan ( Fig 3B and Ref 7 ) . To prove that the actual lack of internalization rather than a general actin polymerization block is responsible for enhancing Dectin-1 -mediated inflammatory responses , we analyzed the effect of a dynamin inhibitor , dynasore 31-33 . Because this reagent needs to be used in serum-free conditions , which are not compatible with BMDC survival ( data not shown ) , we utilized HEK293 cells stably transfected with Dectin-1 . Like DC , such transfectants avidly internalized Glu-mp and this was fully blocked in the presence of dynasore ( Fig 3C ) . Notably , the same cells stimulated with Glu-mp in the presence of dynasore showed strong and sustained PHOSphorylation of ERK (Fig 3D) . In contrast to latrunculin A (Fig 3A) , dynasore did not induce ERK PHOSphorylation by itself ( Fig 3D ) , supporting the notion that the strong MAPK activation observed is selectively attributable to blockade of internalization . Finally , to diminish particle internalization while avoiding possible non-specific effects associated with chemical inhibitors , we tested cells with low phagocytic activity . In contrast to Dectin-1-expressing HEK293 cells , the B-cell hybridoma LK35.2 does not avidly internalize Glu-mp even after transfection with Dectin-1 ( data not shown ) . We therefore stimulated LK cells expressing Dectin-1 with Glu-mp and curdlan at different doses and evaluated their ability to produce IL-2 as a readout of Dectin-1/Syk activation 9 . Notably , in contrast to its low stimulatory activity on DC , Glu-mp acted as a significant inducer of cytokine production by Dectin-1-expressing LK cells , being only slightly weaker than curdlan ( Fig 3E ) . Thus , in poorly phagocytic cells , curdlan and Glu-mp act as comparable agonists for Dectin-1 -mediated inflammatory responses .