LIGATION OF THE ALPHA(2)-MACROGLOBULIN SIGNALING RECEPTOR ON MACROPHAGES INDUCES PROTEIN-PHOSPHORYLATION AND AN INCREASE IN CYTOSOLIC PH

We have recently described an alpha(2)-macroglobulin (alpha(2)M) signa lling receptor which is distinct from the low-density lipoprotein-rela ted protein/alpha(2)M receptor (LRP/alpha(2)MR). Ligation of the macro phage signalling receptor by alpha(2)M-methyiamine stimulates producti on of several second messengers and involves a pertussis toxin-insensi tive G-protein. We now report that binding of alpha(2)M-methylamine, o r the cloned M(r) = 20000 receptor-binding fragment from rat alpha(1)M , to macrophage alpha(2)M signalling receptors induces protein phospho rylation. By use of a monoclonal antibody to phospholipase C gamma 1 ( PLC gamma 1) we were able to identify it as one target for protein pho sphorylation. Phosphorylation was time and concentration dependent, be ing optimal at about 60 s of incubation and a 100-200 nM ligand concen tration. By use of a second monoclonal antibody directed against phosp hotyrosine, we were able to demonstrate that at least a portion of the label was incorporated into one or more tyrosine residues. PLC gamma 1 phosphorylation was then studied in membrane preparations at 4 degre es C in order to minimize serine or threonine modification. Preincubat ion of macrophage membranes with genistein, a tyrosine kinase inhibito r, drastically reduced phosphorylation of PLC gamma 1. Receptor-associ ated protein, which blocks alpha(2)M-binding to LRP/alpha(2)MR but not to the alpha(2)M signalling receptor, had no effect on alpha(2)M-meth ylamine-induced tyrosine phosphorylation of PLC gamma 1. Binding of la ctoferrin to LRP/alpha(2)MR failed to induce phosphorylation of PLC ga mma 1, further supporting the hypothesis that the alpha(2)M signalling receptor and LRI/alpha(2)MR are distinct entities. Growth factors whi ch induce tyrosine phosphorylation typically cause a rise in cytosolic pH. Binding of alpha(2)M-methylamine to macrophages also gradually in creased the intracellular pH in a concentration-dependent manner, bein g optimal at a 200 nM ligand concentration. The increase in pH was ami loride sensitive. We propose that receptor-recognized forms of alpha(2 )M may function like growth factors with regard to macrophage regulati on.