G-protein - cytoskeleton interactions in the human neutrophil during chemoattracant activation

Sammanfattning: The human body survives constant attacks by bacteria and other pathogens thanks to the surveillance of the neutrophil leukocytcs. At the site of an infection, several messenger molecules are generated that attract neutrophils from the blood and direct their migration toward the microbes. To fulfil its task in the host defence, the neutrophil must be able to sense and co-ordinate these extracellular signals and to translate them into intracellular signals which can then regulate the motile activity of the cell. The neutrophil senses most chemotactic agonists through GTP-binding protein-coupled receptors which dynamically interact with the locomotory machinery of the cell, i.e. the cytoskeleton. The aims of this thesis were to gain knowledge about and elucidate the mechanisms behind the interrelationship of GTPbinding proteins and the cytoskeleton during chemotactic activation.Stimulation of the chemotactic rcceptor for N-fonnyl-L-methionyl-L-leucyl-L-phenylalaninc (fMetLeu- Phe) results in the formation of a ligand-rcceptor complex that rapidly associates with a cytoskelelal fraction from human neutrophils. Upon stimulation with fMet-Lcu-Phe, there is also a concurrent release of the u-subunit of the heterotrimeric G-protcin (G;.2a) from the cytoskeleton. Inhibition of the G-protein, prevents both the G;_ 2 a-subunit from leaving the cytoskeleton and theN-formyl peptide rcceptor (FPR) to associate with the cytoskeleton. In other words, the association of FPR with the cytoskeleton seems to require the activation and release of Gi-2'1- from the cytoskeleton. Direct activation of the G-protein with AIF 4 - or G1PrS reveals that the association between the FPR and the cytoskeleton does not depend on the ligand-binding event and the subsequent conformational change of the FPR. A release of one or more unidentified low-molecular-weight G1P-binding proteins (LMWGs) from the cytoskeleton is also noted upon stimulation with fMet-Leu~Phe; this release seems to be direct, i.e. not mediated through a heterotrimeric G-protein. Direct activation of the G-protein with AlF4- or GTPrS causes a rapid and sustained polymerization of aclin. This polymerization is primarily mediated by a heterotrimeric aprotein and activated by a signal transduction pathway separate from phospholipase C. Actin filaments seem to be the major cytoskeleton component involved in the association between the FPR and the cytoskeleton, but actin polymerization per se is not required for the association process to lake place. Moreover, the release of LMWGs or G-protein is not an indirect effect of the prominent reorganization of the.actin network.The association of the FPR with the cytoskeleton is believed to be a mechanism by which the ligandinduced signaling is terminated. The present data confirm this hypothesis and expand it with the idea of a regulatory role of G-proteins in the termination/desensitization process.