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Binding of Receptor-recognized Forms of α2-Macroglobulin to the α2-Macroglobulin Signaling Receptor Activates Phosphatidylinositol 3-Kinase*

  • Uma Kant Misra
    Affiliations
    Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710
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  • Salvatore Vincent Pizzo
    Correspondence
    To whom correspondence should be addressed: Dept. of Pathology, Box 3712, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-3528; Fax: 919- 684-8689
    Affiliations
    Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710
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  • Author Footnotes
    * The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:May 29, 1998DOI:https://doi.org/10.1074/jbc.273.22.13399
      Ligation of the α2-macroglobulin (α2M) signaling receptor by receptor-recognized forms of α2M (α2M*) initiates mitogenesis secondary to increased intracellular Ca2+. We report here that ligation of the α2M 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 α2M* concentration-dependent with a maximal response at ~50 pm ligand concentration. The peak formation of PIP3 occurred at 10 min of incubation. The α2M receptor binding fragment mutant K1370R which binds to the α2M signaling receptor activating the signaling cascade, increased PIP3 formation by 2-fold. The mutant K1374A, which binds very poorly to the α2M signaling receptor, did not cause any increase in PIP3 formation. α2M*-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 α2M*-induced increases in PIP3 formation. We conclude that PI3K is involved in α2M*-induced mitogenesis in macrophages and intracellular Ca2+ plays a role in PI3K activation.
      The α-macroglobulins are part of a large super family including human α2-macroglobulin (α2M)
      The abbreviations used are: α2M, α2-macroglobulin; α2M*, α2M activated by proteinase or methylamine; α2MSR, α2M signaling receptor; LRP/α2MR, low density lipoprotein receptor-related protein/α2M receptor; BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acetoxymethylester; [Ca2+]i, intracellular free Ca2+; Fura-2/AM, 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxyl-2)-2′-amino-5-methyl-phenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethylester; HHBSS, Hanks' balanced salt solution containing 10 mm HEPES and 3.5 mmNaHCO3; IP3, inositol 1,4,5-triphosphate; PtdIns, phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate; PI, phosphatidylinositol; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5- triphosphate; PI3K, phosphatidylinositol 3-kinase; RBF, receptor binding factor; BSA, bovine serum albumin; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; MAPK, mitogen-activated protein kinase; PKC, protein kinase C.
      1The abbreviations used are: α2M, α2-macroglobulin; α2M*, α2M activated by proteinase or methylamine; α2MSR, α2M signaling receptor; LRP/α2MR, low density lipoprotein receptor-related protein/α2M receptor; BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acetoxymethylester; [Ca2+]i, intracellular free Ca2+; Fura-2/AM, 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxyl-2)-2′-amino-5-methyl-phenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethylester; HHBSS, Hanks' balanced salt solution containing 10 mm HEPES and 3.5 mmNaHCO3; IP3, inositol 1,4,5-triphosphate; PtdIns, phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate; PI, phosphatidylinositol; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5- triphosphate; PI3K, phosphatidylinositol 3-kinase; RBF, receptor binding factor; BSA, bovine serum albumin; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; MAPK, mitogen-activated protein kinase; PKC, protein kinase C.
      (
      • Sottrop-Jensen L.
      ,
      • Chu C.T.
      • Pizzo S.V.
      ). Proteolytic attack on the bait region or direct nucleophilic attack on the thiol ester bonds of human α2M subunits triggers a major conformational change that exposes receptor recognition sites present in each of the four α2M subunits (
      • Chu C.T.
      • Pizzo S.V.
      ,
      • Salvesen G.
      • Pizzo S.V.
      ). Two receptors bind α2M*, namely, LRP/α2MR and a recently discovered α2M signaling receptor (α2MSR) (
      • Krieger M.
      • Herz J.
      ,
      • Strickland D.K.
      • Ashcom J.D.
      • Williams S.
      • Burgess W.H.
      • Migliorini M.
      • Argraves W.S.
      ,
      • Misra U.K.
      • Chu C.T.
      • Rubenstein D.S.
      • Gawdi G.
      • Pizzo S.V.
      ,
      • Misra U.K.
      • Chu C.T.
      • Gawdi G.
      • Pizzo S.V.
      ,
      • Misra U.K.
      • Chu C.T.
      • Gawdi G.
      • Pizzo S.V.
      ,
      • Misra U.K.
      • Gawdi G.
      • Pizzo S.V.
      ,
      • Misra U.K.
      • Gonzalez-Gronow M.G.
      • Gawdi G.
      • Pizzo S.V.
      ,
      • Misra U.K.
      • Pizzo S.V.
      ,
      • Howard G.C.
      • Misra U.K.
      • DeCamp D.L.
      • Pizzo S.V.
      ,
      • Howard G.C.
      • Yamaguchi Y.
      • Misra U.K.
      • Gawdi G.
      • Nelson A.
      • DeCamp D.L.
      • Pizzo S.V.
      ). LRP/α2MR is a scavenger receptor that binds a wide variety of ligands. Binding of α2M* to LRP/α2MR is followed by uptake and degradation in lysosomes but not activation of a signaling cascade (
      • Misra U.K.
      • Chu C.T.
      • Gawdi G.
      • Pizzo S.V.
      ,
      • Misra U.K.
      • Chu C.T.
      • Gawdi G.
      • Pizzo S.V.
      ,
      • Howard G.C.
      • Misra U.K.
      • DeCamp D.L.
      • Pizzo S.V.
      ). By contrast, binding of α2M* or RBF to α2MSR triggers classical signaling cascades and regulates cell proliferation (
      • Misra U.K.
      • Chu C.T.
      • Rubenstein D.S.
      • Gawdi G.
      • Pizzo S.V.
      ,
      • Misra U.K.
      • Chu C.T.
      • Gawdi G.
      • Pizzo S.V.
      ,
      • Misra U.K.
      • Chu C.T.
      • Gawdi G.
      • Pizzo S.V.
      ,
      • Misra U.K.
      • Gawdi G.
      • Pizzo S.V.
      ,
      • Misra U.K.
      • Gonzalez-Gronow M.G.
      • Gawdi G.
      • Pizzo S.V.
      ,
      • Misra U.K.
      • Pizzo S.V.
      ,
      • Howard G.C.
      • Misra U.K.
      • DeCamp D.L.
      • Pizzo S.V.
      ,
      • Howard G.C.
      • Yamaguchi Y.
      • Misra U.K.
      • Gawdi G.
      • Nelson A.
      • DeCamp D.L.
      • Pizzo S.V.
      ,
      • Webb D.J.
      • Hussaini I.M.
      • Weaver A.M.
      • Atkins T.L.
      • Chu C.T.
      • Pizzo S.V.
      • Owens G.K.
      • Gonias S.L.
      ).
      The agonist-induced entry of Ca2+ from the extracellular medium is of major importance in the cytosolic Ca2+ signals that link activation of various receptors on the cell surface with the initiation and control of cell functions (
      • Berridge M.J.
      ,
      • Putney J.W.
      • Bird G.S.
      ,
      • Santella L.
      • Carafoli E.
      ). Elevated cytosolic Ca2+ modulates specific cell cycle events and DNA synthesis (
      • Ghosh T.K.
      • Bian J.
      • Short S.D.
      • Rybak S.L.
      • Gill D.L.
      ,
      • Short A.D.
      • Bian J.
      • Ghosh T.K.
      • Waldron R.T.
      • Rybak S.L.
      • Gill D.L.
      ,
      • Ufret-Vincerity C.A.
      • Short A.D.
      • Alfonso A.
      • Gill D.L.
      ,
      • Graber M.N.
      • Alfonso A.
      • Gill D.L.
      ,
      • Timmerman L.A.
      • Clipstone N.A.
      • Northrop J.P.
      • Crabtree G.R.
      ,
      • Charlesworth A.
      • Rozengurt E.
      ). Binding of α2M* to α2MSR raises p21RASGTP levels 2–3-fold in macrophages and pretreatment with wortmannin, a specific inhibitor of PI3K, does not affect α2M*-induced increases in p21RASGTP levels (

      Misra, U. K., and Pizzo, S. V. (1998) Cell. Signalling, in press

      ).
      Cellular 3-phosphoinositides are generated through the action of a family of PI3Ks (
      • Toker A.
      • Cantley L.C.
      ,
      • Carpenter C.L.
      • Cantley L.C.
      ). PI3K activity was first reported in association with v-SRC and v-RAS oncoproteins, where it catalyzes phosphorylation of inositol at the D-3 position of phosphatidylinositol (PtdIns), PtdIns 4-phosphate, and PtdIns 4,5-bisphosphate (
      • Toker A.
      • Cantley L.C.
      ,
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • Klarlund J.K.
      • Guilherme A.
      • Holik J.J.
      • Virbasins J.V.
      • Chawla A.
      • Czech M.P.
      ). Several down stream protein substrates for PI3K have been identified, which include certain protein kinase C isoforms (PKCδ, PKCε, PKCη, PKCξ) (
      • Toker A.
      • Cantley L.C.
      ,
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • Klarlund J.K.
      • Guilherme A.
      • Holik J.J.
      • Virbasins J.V.
      • Chawla A.
      • Czech M.P.
      ) and the plekstrin homology domain containing protein kinases cAKT and BTK (
      • Bos J.L.
      ). An increase in the intracellular concentration of PtdIns 3,4-bisphosphate and PtdIns 3,4,5-trisphosphate is observed in several cell types on stimulation with growth factors, cytokines, insulin, f-Met-Leu-Phe, agents that activate RAS, and viral transformation (
      • Toker A.
      • Cantley L.C.
      ,
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • Klarlund J.K.
      • Guilherme A.
      • Holik J.J.
      • Virbasins J.V.
      • Chawla A.
      • Czech M.P.
      ). Signaling by 3-phosphoinositides regulates diverse functions such as mitogenesis, cell growth, membrane ruffling, chemotaxis, oxidant production, secretory responses, insulin-mediated membrane translocation of the glucose transporter, membrane trafficking of growth factor receptors, cell adhesion, and Na/H+ exchange (
      • Toker A.
      • Cantley L.C.
      ,
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • Klarlund J.K.
      • Guilherme A.
      • Holik J.J.
      • Virbasins J.V.
      • Chawla A.
      • Czech M.P.
      ). Since many of the cellular responses elicited upon ligation of α2MSR with receptor-recognized forms of α2M 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 α2M*. Ligation of α2MSR increases the wortmannin-sensitive formation of PIP32–3-fold in a concentration-dependent manner and that the agonist-induced formation of PIP3 is influenced by [Ca2+]i levels.

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