Abstract
The interplay between activated G proteins and intracellular calcium ([Ca(2+)](i)) in the regulation of secretion was studied in the macrophage, coupling membrane capacitance with calcium-sensitive microfluorimetry. Intracellular elevation of either the nonhydrolyzable analogue of GTP, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), or [Ca(2+)](i) enhanced the amplitude and shortened the time course of stimulus-induced secretion in a dose-dependent manner. Both the ionophore- and the stimulus-induced secretory response were abolished in the presence of guanosine-5'-O-(2-thiodiphosphate) (GDP beta S). The K(d) of Ca(2+)-driven secretion was independent of GTP gamma S concentration, whereas the K(d) of the GTP gamma S-driven response decreased from 63 to 31 microM in the presence of saturating concentrations of [Ca(2+)](i). The time course of stimulus-induced secretion was dependent upon the concentration of [Ca(2+)](i). The time course of GTP gamma S-driven secretion was concentration-independent at high levels of [Ca(2+)](i), suggesting that a calcium-dependent translocation/binding step was rate-limiting. Our data strongly support a model in which [Ca(2+)](i) and activated G proteins act independently of one another in the sequential regulation of macrophage secretion. [Ca(2+)](i) appears to play a role in the recruitment and priming of vesicles from reserve intracellular pools at a step that is upstream of G protein activation. While activated, G proteins appear to play a key role in fusion of docked vesicles. Thus, secretion can result either from activating more G proteins or from elevating [Ca(2+)](i) at basal levels of G protein activation.
Highlights
Lular signal transduction cascades from the receptors that eventuate secretion and/or phagocytosis are known, the Ca2ϩ and G protein dependence of the pathway(s) that links receptor binding and cross-linking to vesicle fusion is still unclear
The interplay between activated G proteins and intracellular calcium ([Ca2؉]i) in the regulation of secretion was studied in the macrophage, coupling membrane capacitance with calcium-sensitive microfluorimetry
We demonstrate that the pathway linking receptor binding and exocytosis induced by human heat-aggregated immunoglobulin G (HAIGG) involves both G protein activation and calcium signaling as independent yet synergistic secretory stimuli
Summary
Lular signal transduction cascades from the receptors that eventuate secretion and/or phagocytosis are known, the Ca2ϩ and G protein dependence of the pathway(s) that links receptor binding and cross-linking to vesicle fusion is still unclear. The resultant tyrosine-phosphorylated residues form anchoring sites for SH2 domain-containing proteins, the most important of which seems to the nonreceptor tyrosine kinase Syk, which can phosphorylate several downstream substrates involved in the phagocytic response. One such substrate is the Rho family guanine nucleotide exchange factor Vav, which activates the small G proteins Rac and Cdc.
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