Ligation of the alpha 2-macroglobulin signalling receptor on macrophages induces protein phosphorylation and an increase in cytosolic pH.
We have recently described an alpha 2-macroglobulin (alpha 2M) signalling receptor which is distinct from the low-density lipoprotein-related protein/alpha 2M receptor (LRP/alpha 2MR). Ligation of the macrophage signalling receptor by alpha 2M-methylamine stimulates production of several second messengers and involves a pertussis toxin-insensitive G-protein. We now report that binding of alpha 2M-methylamine, or the cloned M(r) = 20,000 receptor-binding fragment from rat alpha 1M, to macrophage alpha 2M signalling receptors induces protein phosphorylation. By use of a monoclonal antibody to phospholipase C gamma l (PLC gamma l) we were able to identify it as one target for protein phosphorylation. Phosphorylation was time and concentration dependent, being optimal at about 60 s of incubation and a 100-200 nM ligand concentration. By use of a second monoclonal antibody directed against phosphotyrosine, we were able to demonstrate that at least a portion of the label was incorporated into one or more tyrosine residues. PLC gamma l phosphorylation was then studied in membrane preparations at 4 degrees C in order to minimize serine or threonine modification. Preincubation of macrophage membranes with genistein, a tyrosine kinase inhibitor, drastically reduced phosphorylation of PLC gamma l. Receptor-associated protein, which blocks alpha 2M binding to LRP/alpha 2MR but not to the alpha 2M signalling receptor, had no effect on alpha 2M-methylamine-induced tyrosine phosphorylation of PLC gamma l. Binding of lactoferrin to LRP/alpha 2MR failed to induce phosphorylation of PLC gamma l, further supporting the hypothesis that the alpha 2M signalling receptor and LRP/alpha 2MR are distinct entities. Growth factors which induce tyrosine phosphorylation typically cause a rise in cytosolic pH. Binding of a2M-methylamine to macrophages also gradually increased the intracellular pH in a concentration-dependent manner, being optimal at a 200 nM ligand concentration. The increase in pH was amiloride sensitive. We propose that receptor-recognized forms of a2M may function like growth factors with regard to macrophage regulation.
Misra
U K
UK
Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA.
Gawdi
G
G
Pizzo
S V
SV
D000818:Animals; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism; D019833:Genistein; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D007527:Isoenzymes / Q000378:metabolism*; D007529:Isoflavones / Q000494:pharmacology; D007781:Lactoferrin / Q000378:metabolism; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D051966:Phospholipase C gamma; D010766:Phosphorylation; D051381:Rats; D011971:Receptors, Immunologic / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000494:pharmacology
D007527:Isoenzymes; D007529:Isoflavones; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011971:Receptors, Immunologic; D000511:alpha-Macroglobulins; C065661:alpha2-macroglobulin-methylamine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D015544:Inositol 1,4,5-Trisphosphate; D019833:Genistein; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D007781:Lactoferrin