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. 1995 Jul 1;309(Pt 1):151–158. doi: 10.1042/bj3090151

Ligation of the alpha 2-macroglobulin signalling receptor on macrophages induces protein phosphorylation and an increase in cytosolic pH.

U K Misra 1, G Gawdi 1, S V Pizzo 1
PMCID: PMC1135813  PMID: 7542445

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aaronson S. A. Growth factors and cancer. Science. 1991 Nov 22;254(5035):1146–1153. doi: 10.1126/science.1659742. [DOI] [PubMed] [Google Scholar]
  2. Akiyama T., Ogawara H. Use and specificity of genistein as inhibitor of protein-tyrosine kinases. Methods Enzymol. 1991;201:362–370. doi: 10.1016/0076-6879(91)01032-w. [DOI] [PubMed] [Google Scholar]
  3. Banno Y., Yada Y., Nozawa Y. Purification and characterization of membrane-bound phospholipase C specific for phosphoinositides from human platelets. J Biol Chem. 1988 Aug 15;263(23):11459–11465. [PubMed] [Google Scholar]
  4. Bennett C. F., Crooke S. T. Purification and characterization of a phosphoinositide-specific phospholipase C from guinea pig uterus. Phosphorylation by protein kinase C in vivo. J Biol Chem. 1987 Oct 5;262(28):13789–13797. [PubMed] [Google Scholar]
  5. Benos D. J., McPherson S., Hahn B. H., Chaikin M. A., Benveniste E. N. Cytokines and HIV envelope glycoprotein gp120 stimulate Na+/H+ exchange in astrocytes. J Biol Chem. 1994 May 13;269(19):13811–13816. [PubMed] [Google Scholar]
  6. Bonner J. C., Badgett A., Osornio-Vargas A. R., Hoffman M., Brody A. R. PDGF-stimulated fibroblast proliferation is enhanced synergistically by receptor-recognized alpha 2-macroglobulin. J Cell Physiol. 1990 Oct;145(1):1–8. doi: 10.1002/jcp.1041450102. [DOI] [PubMed] [Google Scholar]
  7. Borth W. Alpha 2-macroglobulin, a multifunctional binding protein with targeting characteristics. FASEB J. 1992 Dec;6(15):3345–3353. doi: 10.1096/fasebj.6.15.1281457. [DOI] [PubMed] [Google Scholar]
  8. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  9. Cadena D. L., Gill G. N. Receptor tyrosine kinases. FASEB J. 1992 Mar;6(6):2332–2337. doi: 10.1096/fasebj.6.6.1312047. [DOI] [PubMed] [Google Scholar]
  10. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  11. Carpenter G., Cohen S. Epidermal growth factor. J Biol Chem. 1990 May 15;265(14):7709–7712. [PubMed] [Google Scholar]
  12. Chu C. T., Pizzo S. V. Receptor-mediated antigen delivery into macrophages. Complexing antigen to alpha 2-macroglobulin enhances presentation to T cells. J Immunol. 1993 Jan 1;150(1):48–58. [PubMed] [Google Scholar]
  13. Cross M., Dexter T. M. Growth factors in development, transformation, and tumorigenesis. Cell. 1991 Jan 25;64(2):271–280. doi: 10.1016/0092-8674(91)90638-f. [DOI] [PubMed] [Google Scholar]
  14. Delain E., Pochon F., Barray M., Van Leuven F. Ultrastructure of alpha 2-macroglobulins. Electron Microsc Rev. 1992;5(2):231–281. doi: 10.1016/0892-0354(92)90012-f. [DOI] [PubMed] [Google Scholar]
  15. Feldman S. R., Gonias S. L., Ney K. A., Pratt C. W., Pizzo S. V. Identification of "embryonin" as bovine alpha 2-macroglobulin. J Biol Chem. 1984 Apr 10;259(7):4458–4462. [PubMed] [Google Scholar]
  16. Gilman A. G. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615–649. doi: 10.1146/annurev.bi.56.070187.003151. [DOI] [PubMed] [Google Scholar]
  17. Graff J. M., Rajan R. R., Randall R. R., Nairn A. C., Blackshear P. J. Protein kinase C substrate and inhibitor characteristics of peptides derived from the myristoylated alanine-rich C kinase substrate (MARCKS) protein phosphorylation site domain. J Biol Chem. 1991 Aug 5;266(22):14390–14398. [PubMed] [Google Scholar]
  18. Harlan D. M., Graff J. M., Stumpo D. J., Eddy R. L., Jr, Shows T. B., Boyle J. M., Blackshear P. J. The human myristoylated alanine-rich C kinase substrate (MARCKS) gene (MACS). Analysis of its gene product, promoter, and chromosomal localization. J Biol Chem. 1991 Aug 5;266(22):14399–14405. [PubMed] [Google Scholar]
  19. Herskovits J. S., Burgess C. C., Obar R. A., Vallee R. B. Effects of mutant rat dynamin on endocytosis. J Cell Biol. 1993 Aug;122(3):565–578. doi: 10.1083/jcb.122.3.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hoffman M., Feldman S. R., Pizzo S. V. Alpha 2-macroglobulin 'fast' forms inhibit superoxide production by activated macrophages. Biochim Biophys Acta. 1983 Nov 8;760(3):421–423. doi: 10.1016/0304-4165(83)90384-7. [DOI] [PubMed] [Google Scholar]
  21. Hunter T., Karin M. The regulation of transcription by phosphorylation. Cell. 1992 Aug 7;70(3):375–387. doi: 10.1016/0092-8674(92)90162-6. [DOI] [PubMed] [Google Scholar]
  22. Imber M. J., Pizzo S. V. Clearance and binding of two electrophoretic "fast" forms of human alpha 2-macroglobulin. J Biol Chem. 1981 Aug 10;256(15):8134–8139. [PubMed] [Google Scholar]
  23. Johnson G. L., Vaillancourt R. R. Sequential protein kinase reactions controlling cell growth and differentiation. Curr Opin Cell Biol. 1994 Apr;6(2):230–238. doi: 10.1016/0955-0674(94)90141-4. [DOI] [PubMed] [Google Scholar]
  24. Johnson W. J., Pizzo S. V., Imber M. J., Adams D. O. Receptors for maleylated proteins regulate secretion of neutral proteases by murine macrophages. Science. 1982 Nov 5;218(4572):574–576. doi: 10.1126/science.6289443. [DOI] [PubMed] [Google Scholar]
  25. Katan M., Parker P. J. Purification of phosphoinositide-specific phospholipase C from a particulate fraction of bovine brain. Eur J Biochem. 1987 Oct 15;168(2):413–418. doi: 10.1111/j.1432-1033.1987.tb13435.x. [DOI] [PubMed] [Google Scholar]
  26. Kim H. K., Kim J. W., Zilberstein A., Margolis B., Kim J. G., Schlessinger J., Rhee S. G. PDGF stimulation of inositol phospholipid hydrolysis requires PLC-gamma 1 phosphorylation on tyrosine residues 783 and 1254. Cell. 1991 May 3;65(3):435–441. doi: 10.1016/0092-8674(91)90461-7. [DOI] [PubMed] [Google Scholar]
  27. Krieger M., Herz J. Structures and functions of multiligand lipoprotein receptors: macrophage scavenger receptors and LDL receptor-related protein (LRP). Annu Rev Biochem. 1994;63:601–637. doi: 10.1146/annurev.bi.63.070194.003125. [DOI] [PubMed] [Google Scholar]
  28. LaMarre J., Wollenberg G. K., Gonias S. L., Hayes M. A. Cytokine binding and clearance properties of proteinase-activated alpha 2-macroglobulins. Lab Invest. 1991 Jul;65(1):3–14. [PubMed] [Google Scholar]
  29. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  30. Lamy F., Wilkin F., Baptist M., Posada J., Roger P. P., Dumont J. E. Phosphorylation of mitogen-activated protein kinases is involved in the epidermal growth factor and phorbol ester, but not in the thyrotropin/cAMP, thyroid mitogenic pathway. J Biol Chem. 1993 Apr 25;268(12):8398–8401. [PubMed] [Google Scholar]
  31. Lee K. Y., Ryu S. H., Suh P. G., Choi W. C., Rhee S. G. Phospholipase C associated with particulate fractions of bovine brain. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5540–5544. doi: 10.1073/pnas.84.16.5540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Libby P., Raines E. W., Cullinane P. M., Ross R. Analysis of the mitogenic effect of fetuin preparations on arterial smooth muscle cells: the role of contaminant platelet-derived growth factor. J Cell Physiol. 1985 Dec;125(3):357–366. doi: 10.1002/jcp.1041250302. [DOI] [PubMed] [Google Scholar]
  33. Lukacs G. L., Kapus A., Nanda A., Romanek R., Grinstein S. Proton conductance of the plasma membrane: properties, regulation, and functional role. Am J Physiol. 1993 Jul;265(1 Pt 1):C3–14. doi: 10.1152/ajpcell.1993.265.1.C3. [DOI] [PubMed] [Google Scholar]
  34. Majerus P. W., Ross T. S., Cunningham T. W., Caldwell K. K., Jefferson A. B., Bansal V. S. Recent insights in phosphatidylinositol signaling. Cell. 1990 Nov 2;63(3):459–465. doi: 10.1016/0092-8674(90)90442-h. [DOI] [PubMed] [Google Scholar]
  35. Margolis B., Rhee S. G., Felder S., Mervic M., Lyall R., Levitzki A., Ullrich A., Zilberstein A., Schlessinger J. EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signaling. Cell. 1989 Jun 30;57(7):1101–1107. doi: 10.1016/0092-8674(89)90047-0. [DOI] [PubMed] [Google Scholar]
  36. Meisenhelder J., Suh P. G., Rhee S. G., Hunter T. Phospholipase C-gamma is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. Cell. 1989 Jun 30;57(7):1109–1122. doi: 10.1016/0092-8674(89)90048-2. [DOI] [PubMed] [Google Scholar]
  37. Misra U. K., Chu C. T., Gawdi G., Pizzo S. V. The relationship between low density lipoprotein-related protein/alpha 2-macroglobulin (alpha 2M) receptors and the newly described alpha 2M signaling receptor. J Biol Chem. 1994 Jul 15;269(28):18303–18306. [PubMed] [Google Scholar]
  38. Misra U. K., Chu C. T., Rubenstein D. S., Gawdi G., Pizzo S. V. Receptor-recognized alpha 2-macroglobulin-methylamine elevates intracellular calcium, inositol phosphates and cyclic AMP in murine peritoneal macrophages. Biochem J. 1993 Mar 15;290(Pt 3):885–891. doi: 10.1042/bj2900885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Misra U. K., Sahyoun N. Protein kinase C binding to isolated nuclei and its activation by a Ca2+/phospholipid-independent mechanism. Biochem Biophys Res Commun. 1987 Jun 15;145(2):760–768. doi: 10.1016/0006-291x(87)91030-8. [DOI] [PubMed] [Google Scholar]
  40. Mohammadi M., Dionne C. A., Li W., Li N., Spivak T., Honegger A. M., Jaye M., Schlessinger J. Point mutation in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenesis. Nature. 1992 Aug 20;358(6388):681–684. doi: 10.1038/358681a0. [DOI] [PubMed] [Google Scholar]
  41. Moolenaar W. H. Effects of growth factors on intracellular pH regulation. Annu Rev Physiol. 1986;48:363–376. doi: 10.1146/annurev.ph.48.030186.002051. [DOI] [PubMed] [Google Scholar]
  42. Nakanishi O., Shibasaki F., Hidaka M., Homma Y., Takenawa T. Phospholipase C-gamma 1 associates with viral and cellular src kinases. J Biol Chem. 1993 May 25;268(15):10754–10759. [PubMed] [Google Scholar]
  43. Park D. J., Min H. K., Rhee S. G. Inhibition of CD3-linked phospholipase C by phorbol ester and by cAMP is associated with decreased phosphotyrosine and increased phosphoserine contents of PLC-gamma 1. J Biol Chem. 1992 Jan 25;267(3):1496–1501. [PubMed] [Google Scholar]
  44. Pawelczyk T., Lowenstein J. M. Binding of phospholipase C delta 1 to phospholipid vesicles. Biochem J. 1993 May 1;291(Pt 3):693–696. doi: 10.1042/bj2910693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Peters K. G., Marie J., Wilson E., Ives H. E., Escobedo J., Del Rosario M., Mirda D., Williams L. T. Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca2+ flux but not mitogenesis. Nature. 1992 Aug 20;358(6388):678–681. doi: 10.1038/358678a0. [DOI] [PubMed] [Google Scholar]
  46. Prpic V., Yu S. F., Figueiredo F., Hollenbach P. W., Gawdi G., Herman B., Uhing R. J., Adams D. O. Role of Na+/H+ exchange by interferon-gamma in enhanced expression of JE and I-A beta genes. Science. 1989 Apr 28;244(4903):469–471. doi: 10.1126/science.2541500. [DOI] [PubMed] [Google Scholar]
  47. Rhee S. G. Inositol phospholipids-specific phospholipase C: interaction of the gamma 1 isoform with tyrosine kinase. Trends Biochem Sci. 1991 Aug;16(8):297–301. doi: 10.1016/0968-0004(91)90122-c. [DOI] [PubMed] [Google Scholar]
  48. Rozengurt E. Early signals in the mitogenic response. Science. 1986 Oct 10;234(4773):161–166. doi: 10.1126/science.3018928. [DOI] [PubMed] [Google Scholar]
  49. Rubin R. A., O'Keefe E. J., Earp H. S. Alteration of epidermal growth factor-dependent phosphorylation during rat liver regeneration. Proc Natl Acad Sci U S A. 1982 Feb;79(3):776–780. doi: 10.1073/pnas.79.3.776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sahyoun N., LeVine H., 3rd, Bronson D., Cuatrecasas P. Ca2+-calmodulin-dependent protein kinase in neuronal nuclei. J Biol Chem. 1984 Aug 10;259(15):9341–9344. [PubMed] [Google Scholar]
  51. Salomon D. S., Bano M., Smith K. B., Kidwell W. R. Isolation and characterization of a growth factor (embryonin) from bovine fetuin which resembles alpha 2-macroglobulin. J Biol Chem. 1982 Dec 10;257(23):14093–14101. [PubMed] [Google Scholar]
  52. Salvesen G., Quan L. T., Enghild J. J., Snipas S., Fey G. H., Pizzo S. V. Expression of a functional alpha-macroglobulin receptor binding domain in Escherichia coli. FEBS Lett. 1992 Nov 23;313(2):198–202. doi: 10.1016/0014-5793(92)81443-p. [DOI] [PubMed] [Google Scholar]
  53. Simchowitz L. Chemotactic factor-induced activation of Na+/H+ exchange in human neutrophils. II. Intracellular pH changes. J Biol Chem. 1985 Oct 25;260(24):13248–13255. [PubMed] [Google Scholar]
  54. Sorokin A., Mohammadi M., Huang J., Schlessinger J. Internalization of fibroblast growth factor receptor is inhibited by a point mutation at tyrosine 766. J Biol Chem. 1994 Jun 24;269(25):17056–17061. [PubMed] [Google Scholar]
  55. Strickland D. K., Ashcom J. D., Williams S., Burgess W. H., Migliorini M., Argraves W. S. Sequence identity between the alpha 2-macroglobulin receptor and low density lipoprotein receptor-related protein suggests that this molecule is a multifunctional receptor. J Biol Chem. 1990 Oct 15;265(29):17401–17404. [PubMed] [Google Scholar]
  56. Swallow C. J., Grinstein S., Rotstein O. D. A vacuolar type H(+)-ATPase regulates cytoplasmic pH in murine macrophages. J Biol Chem. 1990 May 5;265(13):7645–7654. [PubMed] [Google Scholar]
  57. Uhing R. J., Martenson C. H., Rubenstein D. S., Hollenbach P. W., Pizzo S. V. The exposure of murine macrophages to alpha 2-macroglobulin 'fast' forms results in the rapid secretion of eicosanoids. Biochim Biophys Acta. 1991 Jul 10;1093(2-3):115–120. doi: 10.1016/0167-4889(91)90111-a. [DOI] [PubMed] [Google Scholar]
  58. Ullrich A., Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990 Apr 20;61(2):203–212. doi: 10.1016/0092-8674(90)90801-k. [DOI] [PubMed] [Google Scholar]
  59. Wahl M. I., Jones G. A., Nishibe S., Rhee S. G., Carpenter G. Growth factor stimulation of phospholipase C-gamma 1 activity. Comparative properties of control and activated enzymes. J Biol Chem. 1992 May 25;267(15):10447–10456. [PubMed] [Google Scholar]
  60. Wise B. C., Glass D. B., Chou C. H., Raynor R. L., Katoh N., Schatzman R. C., Turner R. S., Kibler R. F., Kuo J. F. Phospholipid-sensitive Ca2+-dependent protein kinase from heart. II. Substrate specificity and inhibition by various agents. J Biol Chem. 1982 Jul 25;257(14):8489–8495. [PubMed] [Google Scholar]
  61. Yang L. J., Baffy G., Rhee S. G., Manning D., Hansen C. A., Williamson J. R. Pertussis toxin-sensitive Gi protein involvement in epidermal growth factor-induced activation of phospholipase C-gamma in rat hepatocytes. J Biol Chem. 1991 Nov 25;266(33):22451–22458. [PubMed] [Google Scholar]
  62. Yang L. J., Rhee S. G., Williamson J. R. Epidermal growth factor-induced activation and translocation of phospholipase C-gamma 1 to the cytoskeleton in rat hepatocytes. J Biol Chem. 1994 Mar 11;269(10):7156–7162. [PubMed] [Google Scholar]

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