Abstract
Ligation of the alpha2-macroglobulin (alpha2M) signaling receptor by receptor-recognized forms of alpha2M (alpha2M*) initiates mitogenesis secondary to increased intracellular Ca2+. We report here that ligation of the alpha2M signaling receptor also causes a 1. 5-2.5-fold increase in wortmannin-sensitive phosphatidylinositol 3-kinase (PI3K) activity as measured by the quantitation of phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 formation was alpha2M* concentration-dependent with a maximal response at approximately 50 pM ligand concentration. The peak formation of PIP3 occurred at 10 min of incubation. The alpha2M receptor binding fragment mutant K1370R which binds to the alpha2M signaling receptor activating the signaling cascade, increased PIP3 formation by 2-fold. The mutant K1374A, which binds very poorly to the alpha2M signaling receptor, did not cause any increase in PIP3 formation. alpha2M*-induced DNA synthesis was inhibited by wortmannin. 1, 2Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethylester a chelator of intracellular Ca2+, drastically reduced alpha2M*-induced increases in PIP3 formation. We conclude that PI3K is involved in alpha2M*-induced mitogenesis in macrophages and intracellular Ca2+ plays a role in PI3K activation.
Highlights
Bonds of human ␣2M subunits triggers a major conformational change that exposes receptor recognition sites present in each of the four ␣2M subunits [2, 3]
Since many of the cellular responses elicited upon ligation of ␣2MSR with receptor-recognized forms of ␣2M are similar to those elicited upon binding of growth factors to their receptors, we studied the activity of phosphatidylinositol 3-kinase (PI3K) by measuring the formation of PtdIns 3,4,5- trisphosphate (PIP3), in murine macrophages stimulated with ␣2M*
The maximum synthesis of PIP3 occurred at a ligand concentration of ϳ50 pM (Fig. 1A)
Summary
Bonds of human ␣2M subunits triggers a major conformational change that exposes receptor recognition sites present in each of the four ␣2M subunits [2, 3]. Since many of the cellular responses elicited upon ligation of ␣2MSR with receptor-recognized forms of ␣2M are similar to those elicited upon binding of growth factors to their receptors, we studied the activity of PI3K by measuring the formation of PtdIns 3,4,5- trisphosphate (PIP3), in murine macrophages stimulated with ␣2M*. In experiments where the effect of wortmannin on agonist-induced formation of PIP3 was studied, it was incubated (30 nM) with samples for 30 min at 37 °C prior to addition of agonists.
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