Receptor-recognized α2-macroglobulin-methylamine elevates intracellular calcium, inositol phosphates and cyclic AMP in murine peritoneal macrophages

Abstract

Human plasma alpha 2-macroglobulin (alpha 2M) is a tetrameric proteinase inhibitor, which undergoes a conformational change upon reaction with either a proteinase or methylamine. As a result, a receptor recognition site is exposed on each subunit of the molecule enabling it to bind to its receptors on macrophages. We have used Fura-2-loaded murine peritoneal macrophages and digital video fluorescence microscopy to examine the effects of receptor binding on second messenger levels. alpha 2M-methylamine caused a rapid 2-4-fold increase in intracellular Ca2+ concentration ([Ca2+]i) within 5 s of binding to receptors. The agonists induced a focal increase in [Ca2+]i that spread out to other areas of the cell. The increase in [Ca2+]i was dependent on the alpha 2M-methylamine concentration and on the extracellular [Ca2+]. Both sinusoidal and transitory oscillations were observed, which varied from cell to cell. Neither alpha 2M nor boiled alpha 2M-methylamine, forms that are not recognized by the receptor, affected [Ca2+]i in peritoneal macrophages under identical conditions of incubation. The alpha 2M-methylamine-induced rise in [Ca2+]i was accompanied by a rapid and transient increase in macrophage inositol phosphates, including inositol tris- and tetrakis-phosphates. Native alpha 2M did not stimulate a rise in inositol phosphates. Finally, binding of alpha 2M-methylamine to macrophages increased cyclic AMP transiently. Thus receptor-recognized alpha-macroglobulins behave as agonists whose receptor binding causes stimulation of signal transduction pathways.

The Conformation-Dependent Interaction of alpha sub2 -Macroglobulin with Vascular Endothelial Growth Factor: A Novel Mechanism of alpha sub2 -Macroglobulin/Growth Factor Binding

“…Excess Internalization assays. Peritoneal macrophages were obtained from thioglycolatestimulated C57BL/6J mice as previously described (39). Macrophages then were plated in 24-well plates at 2x10 5 cells/well and incubated at 37°C.…”

Regulation of Cytosolic Phospholipase A2 Activity in Macrophages Stimulated with Receptor-recognized Forms of α2-Macroglobulin

“…These receptor-recognized forms of ␣ 2 M, termed ␣ 2 M*, bind to the low density lipoprotein receptorrelated protein (LRP) present on a variety of cells including macrophages (1)(2)(3). In 1993, we demonstrated that the binding of ␣ 2 M* to macrophages activated signaling cascades characterized by an inositol 1,4,5-trisphosphate (IP 3 )-dependent increase in [Ca 2ϩ ] i (4). Subsequent studies demonstrated that the binding to macrophages activates a pertussis toxin-insensitive phospholipase C, which hydrolyzes membrane phosphoinositides generating both IP 3 and diacylglycerol (5,6).…”

Binding of Activated α2-Macroglobulin to Its Cell Surface Receptor GRP78 in 1-LN Prostate Cancer Cells Regulates PAK-2-dependent Activation of LIMK

“…The activated form of ␣ 2 M (␣ 2 M*) is also produced by direct reaction of internal thiol esters present in each of its four identical subunits with small amines or ammonia (13). Binding of ␣ 2 M* to macrophages (14,15), rheumatoid synovial fibroblasts (16), and 1-LN prostate cancer cells triggers increases in the levels of intracellular inositol 1,3,4-trisphosphate and cytosolic-free calcium [Ca 2ϩ ] i and is followed by activation of components of the Ras/MAPK and PI 3-kinase signaling cascades (17)(18)(19)(20). As a consequence of these events, ␣ 2 M* up-regulates DNA synthesis and cellular proliferation (17)(18)(19)(20).…”