The Timing and Magnitude of CaSignaling by CD32 Depends on Its Redistribution on the Cell Surface

Ca²⁺signaling was correlated with microaggregation and capping of CD32 molecules on the myeloid cell line, U937. The cytosolic free Ca²⁺signal was related to the extent of CD32 cross-linking and arose asymmetrically within individual cells. Both the magnitude and the delay before Ca²⁺signaling via CD32 on U937 cells was dependent on the extent of CD32 cross-linking. The delay time was extended in cells in which lateral diffusion in the membrane was reduced by covalently cross-linking of surface proteins. Under these conditions, capping but not surface microaggregation of CD32 molecules was prevented. The delay time before Ca²⁺signaling but not the magnitude was also affected. At a higher density of covalent cross-linking of surface proteins, the magnitude of the Ca²⁺signal by CD32 was also reduced and could be completely inhibited. This evidence therefore shows that the formation of a CD32 “cap” was not required for Ca²⁺signaling by this route. However, the signaling delay time was a consequence of lateral diffusion of CD32 molecules in the membrane to form signaling-competent microaggregates, and the redistribution of CD32 molecules on the cell surface was required for Ca²⁺signal generation.