β2 integrin-induced signal transduction in granulocytic cells
Sammanfattning: Neutrophil granulocytcs are highly mobile, phagocytic white blood cells whose motile functions are critically dependent on adhesion receptors belonging to the B2 integrin subfamily. These integrins are vital for neutrophil functions due to their ability to generate intracellular signals. The B2 integrin~rnediated phagocytosis of complement-opsonised particles is believed to be regulated by PKC. Particle engulfment has previously been found to be accompanied by accumulation of DG, a lipid known to activate PKC. In the present experiments, this DO originated mainly from PLD-mediated hydrolysis of phosphatidylcholinc. The subsequent activation of PKC was demonstrated by the ability of this kinase to phosphorylate the PKC~ specific substrate MARCKS. Moreover, PKC regulated the activity ofPLD in a positive way and thereby maintained its own activity. PKC has also been shown to phosphorylate the B2 subunit of the integrin. Although this phosphorylation does not regulate the avidity of the integrins, it may be needed for the induction of intracellular signals. However, antibody-induced ligation of B2 integrins did not alter the phosphorylation status of these integrins, indicating that this phosphorylation is not involved in the generation of second messengers. In contrast, pretreatment of cells with phorbol esters inhibited the tyrosine kinase activation and the subsequent rise in cytosolic free Ca2+ in a way that correlated with its ability to induce a phosphorylation of the B2 subunit of the integrin. Therefore, a PKC-mediated phosphorylation of this subunit may represent a mechanism for other receptors to regulate the signalling capacity of B2 integrins.The B2 integrin-induced ca2+ signal was shown to be due to both a release of ca2+ from intracellular stores and an influx across the plasma membrane, Moreover, integrin ligation induced a tyrosine phosphoryialion of PLCy2 accompanied by a small formation of Ins(1,4,5)P3, indicating that PLCy2 is responsible for the intracellular release of ca2+. Further evidence was provided by the findings that an activation tyrosine kinase(s) precedes, and is a prerequisite for, the rise in cytosolic free ca2+ concentration. The Src family tyrosine kinase Fgr is activated by B2 integrin-dependent neutrophil adhesion, and this occurs through mechanisms that are yet unidentified. The present results show that ligation of B2 integrins elicited a rapid and transient association of Fgr with B2 integrins. The tyrosine kinase inhibitor genistein did not affect this association, but it did prevent the subsequent dissociation. The interaction between these two proteins could represent an initial step in the B2 integrin-induced activation of Fgr, whereas the dissociation of Fgr from B2 integrins appears to be dependent on the activation of a tyrosinekinase, probably Fgr itself.
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