Interaction of the RAGE Cytoplasmic Domain with Diaphanous-1 Is Required for Ligand-stimulated Cellular Migration through Activation of Rac1 and Cdc42
Cellular migration is a fundamental process linked to diverse pathological states such as diabetes and its complications, atherosclerosis, inflammation, and cancer. The receptor for advanced glycation end products (RAGE) is a multiligand cell surface macromolecule which binds distinct ligands that accumulate in these settings. RAGE-ligand interaction evokes central changes in key biological properties of cells, including proliferation, generation of inflammatory mediators, and migration. Although RAGE-dependent signal transduction is critically dependent on its short cytoplasmic domain, to date the proximate mechanism by which this RAGE domain engages and stimulates cytoplasmic signaling pathways has yet to be identified. Here we show that the RAGE cytoplasmic domain interacts with Diaphanous-1 (Dia-1) both in vitro and in vivo. We employed the human RAGE cytoplasmic domain as “bait” in the yeast two-hybrid assay and identified the formin homology (FH1) domain of Dia-1 as a potential binding partner of this RAGE domain. Immunoprecipitation studies revealed that the RAGE cytoplasmic domain interacts with the FH1 domain of Dia-1. Down-regulation of Dia-1 expression by RNA interference blocks RAGE-mediated activation of Rac-1 and Cdc42 and, in parallel, RAGE ligand-stimulated cellular migration. Taken together, these findings indicate that the interaction of the RAGE cytoplasmic domain with Dia-1 is required to transduce extracellular environmental cues evoked by binding of RAGE ligands to their cell surface receptor, a chief consequence of which is Rac-1 and Cdc42 activation and cellular migration. Because RAGE and Dia-1 are implicated in the regulation of inflammatory, vascular, and transformed cell migration, these findings highlight this interaction as a novel target for therapeutic intervention in inflammation, atherosclerosis, diabetes, and cancer. Cellular migration is a fundamental process linked to diverse pathological states such as diabetes and its complications, atherosclerosis, inflammation, and cancer. The receptor for advanced glycation end products (RAGE) is a multiligand cell surface macromolecule which binds distinct ligands that accumulate in these settings. RAGE-ligand interaction evokes central changes in key biological properties of cells, including proliferation, generation of inflammatory mediators, and migration. Although RAGE-dependent signal transduction is critically dependent on its short cytoplasmic domain, to date the proximate mechanism by which this RAGE domain engages and stimulates cytoplasmic signaling pathways has yet to be identified. Here we show that the RAGE cytoplasmic domain interacts with Diaphanous-1 (Dia-1) both in vitro and in vivo. We employed the human RAGE cytoplasmic domain as “bait” in the yeast two-hybrid assay and identified the formin homology (FH1) domain of Dia-1 as a potential binding partner of this RAGE domain. Immunoprecipitation studies revealed that the RAGE cytoplasmic domain interacts with the FH1 domain of Dia-1. Down-regulation of Dia-1 expression by RNA interference blocks RAGE-mediated activation of Rac-1 and Cdc42 and, in parallel, RAGE ligand-stimulated cellular migration. Taken together, these findings indicate that the interaction of the RAGE cytoplasmic domain with Dia-1 is required to transduce extracellular environmental cues evoked by binding of RAGE ligands to their cell surface receptor, a chief consequence of which is Rac-1 and Cdc42 activation and cellular migration. Because RAGE and Dia-1 are implicated in the regulation of inflammatory, vascular, and transformed cell migration, these findings highlight this interaction as a novel target for therapeutic intervention in inflammation, atherosclerosis, diabetes, and cancer. The receptor for advanced glycation end products (RAGE) 5The abbreviations used are: RAGE, receptor for advanced glycation end products; DN, dominant negative; CML, carboxyl methyl lysine; HSA, human serum albumin; GST, glutathione S-transferase; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl) propane-1,3-diol; MOPS, 4-morpholinepropanesulfonic acid; GFP, green fluorescent protein; siRNA, small interfering RNA; hDia-1, human Diaphanous-1. is a multiligand cell surface macromolecule of the immunoglobulin superfamily which binds diverse ligands, including advanced glycation end products (1Neeper M. Schmidt A.M. Brett J. Yan S.D. Wang F. Pan Y.C.P. Elliston K. Stern D. Shaw A. J. Biol. 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Nature. 2000; 405: 354-360Crossref PubMed Scopus (1109) Google Scholar). Extensive evidence suggests that pharmacological antagonism or genetic modulation of RAGE exerts protection against disease states characterized by up-regulation and accumulation of RAGE ligands, such as the complications of diabetes, atherosclerosis, inflammation, and tumors (2Hofmann M.A. Drury S. Fu C.F. Qu W. Taguchi A. Lu Y. Avila C. Kambham N. Bierhaus A. Nawroth P. Neurath M.F. Slattery T. Beach D. McClary J. Nagashima M. Morser J. Stern D. Schmidt A.M. Cell. 1999; 97: 889-901Abstract Full Text Full Text PDF PubMed Scopus (1617) Google Scholar, 7Taguchi A. Blood D.C. del Toro G. Canet A. Lee D.C. Qu W. Tanji N. Lu Y. Lalla E. Fu C. Hofmann M.A. Kislinger T. Ingram M. Lu A. Tanaka H. Hori O. Ogawa S. Stern D. Schmidt A.M. Nature. 2000; 405: 354-360Crossref PubMed Scopus (1109) Google Scholar, 8Park L. Raman K.G. Lee K.J. Lu Y. Ferran L.J. Chow W.S. Stern D. Schmidt A.M. Nat. 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Biol. 2001; 21: 905-910Crossref PubMed Scopus (257) Google Scholar, 10Kislinger T. Fu C. Huber C. Qu W. Taguchi A. Yan S.D. Hofmann M.A. Yan S.F. Pischetsrieder M. Stern D. Schmidt A.M. J. Biol. Chem. 1999; 274: 31740-31749Abstract Full Text Full Text PDF PubMed Scopus (794) Google Scholar). Both in vitro and in vivo experiments reveal that deletion of the short cytoplasmic domain of RAGE exerts a “dominant negative” (DN) effect in which the signal transduction response to RAGE ligand is blunted (5Huttunen H.J. Fages C. Rauvala H. J. Biol. Chem. 1999; 274: 19919-19924Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 6Sakaguchi T. Yan S.F. Yan S.D. Belov D. Rong L.L. Sousa M. Andrassy M. Marso S.P. Duda S. Arnold B. Liliensiek B. Nawroth P.P. Stern D.M. Schmidt A.M. Naka Y. J. Clin. Investig. 2003; 111: 959-972Crossref PubMed Scopus (331) Google Scholar, 7Taguchi A. Blood D.C. del Toro G. Canet A. Lee D.C. Qu W. Tanji N. Lu Y. Lalla E. Fu C. Hofmann M.A. Kislinger T. 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Full Text Full Text PDF PubMed Scopus Google Scholar, Chen J. 2001; PubMed Scopus Google Scholar, L. J. Zhang O. M.T. L. 2001; PubMed Scopus Google Scholar). the the diverse of RAGE signaling has yet to be identified. the domain of RAGE or in receptor we that the binding of the RAGE cytoplasmic domain a novel mechanism to signal transduction and changes in cellular properties linked to disease the by which the RAGE cytoplasmic domain stimulates cell we employed the yeast two-hybrid and that this domain of RAGE interacts with which we by in vitro and in vivo RAGE-mediated activation of Rac-1 and Cdc42 and cellular migration is dependent on Dia-1. studies a novel signaling by which extracellular cues by RAGE ligand binding are the cytoplasmic domain of the receptor Dia-1 to fundamental signaling and cell migration. and and in with serum and in with serum RAGE ligand carboxyl methyl serum by the of in and for by characterized and by assay of to of protein in the used to a human with the cytoplasmic domain of RAGE used as to the domain number the the RAGE cytoplasmic domain with the of the The yeast with the the activation domain of the and the domain that on the for expression by the and transformed to and and identified by identified a of the Dia-1 to number The yeast two-hybrid to number the of the and of the human by RNA of the for with to of to the RNA of the the end of on the to the the yeast two-hybrid to the The identified by the and a by a the and a the a and the by a with and the with a products the and the by the and and on a the used in RNA of and and the the Dia-1 in to the of the The Dia-1 in as number Studies and in RAGE cytoplasmic domain used for yeast with the in the with the in the to that RAGE and Dia-1 with the transformed E. and expression Because of of expression in the to the by the to the by or by with with or signal and to Studies and of the RAGE and RAGE cytoplasmic domain and to studies to be with of RAGE and The RAGE cytoplasmic domain used for yeast with the in the Dia-1 with the in the by to the RAGE and Dia-1 with the in and with domain and to for interaction by of with and with for with and and cellular and with of cell and of for by the of protein and by in and for as or Immunoprecipitation of and to for RAGE (1Neeper M. Schmidt A.M. Brett J. Yan S.D. Wang F. Pan Y.C.P. Elliston K. Stern D. Shaw A. J. Biol. Chem. 1992; 267: 14998-15004Abstract Full Text PDF PubMed Google by as A.M. E. M. H. Schmidt A.M. J. PubMed Scopus Google Scholar). by a the of human RAGE the end of the domain number to the cytoplasmic as for RAGE the RAGE and a and with human cytoplasmic RAGE or and by with for expression by (1Neeper M. Schmidt A.M. Brett J. Yan S.D. Wang F. Pan Y.C.P. Elliston K. Stern D. Shaw A. J. Biol. Chem. 1992; 267: 14998-15004Abstract Full Text PDF PubMed Google Scholar). RAGE and in and for in with for and cell as Immunoprecipitation with of RAGE with the as as and with Dia-1 and of and to for and with for in in and with in by with by for with (1Neeper M. Schmidt A.M. Brett J. Yan S.D. Wang F. Pan Y.C.P. Elliston K. Stern D. Shaw A. J. Biol. Chem. 1992; 267: 14998-15004Abstract Full Text PDF PubMed Google Scholar). and for by with or with and with a a and the of including and the by and by the of the RAGE and domain and cells, and and as with Cellular the with a and the in the of a and or serum and for to the the with by of studies a distinct RAGE used to activation of GTPases and activation RAGE and and for for by with RAGE and for and to GTPases the and activation The of and and the of and in cell by with GTPases and with dominant of Cdc42 and by with the expression as to migration as and of by that the Dia-1 RNA interference to Dia-1 and RNA interference Dia-1 Dia-1 and RAGE cells, and Dia-1 by both and the of RNA and the for Dia-1 Dia-1 to the and on of Dia-1 by on cell by to Dia-1 and of cell migration, with and expression with of Dia-1 as as Dia-1 with siRNA, and Rac-1 activation the Rac-1 and Cdc42 activation as are as the S.D. in by and a or to of Dia-1 as a of the RAGE the the RAGE cytoplasmic domain interacts with the to signal we a yeast two-hybrid human the human RAGE cytoplasmic domain as this we identified a potential interaction of the RAGE cytoplasmic domain with human Diaphanous-1 a of the formin protein by key formin homology FH1 and Full Text Full Text PDF PubMed Scopus Google Scholar). The FH1 domain of Dia-1 interacts with of the and signal transduction such as and to cellular A. M. M. J. 2001; PubMed Google Scholar, T. E. P. F. Cell. 2000; Full Text Full Text PDF PubMed Scopus (331) Google Scholar, T. A. Y. Y. 2000; PubMed Scopus Google Scholar, S. T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of potential revealed the to the FH1 domain number a novel number which not identified in the human Dia-1 Lee PubMed Scopus Google by to the on revealed to be the and the to be to a deletion or We this not to a as in this we identified for to the or by of cell and with to this not the yeast two-hybrid not by or the and that this the novel in the FH1 domain. Taken together, these this to be the of Dia-1 with the RAGE in in the interaction the RAGE cytoplasmic domain and hDia-1, we the of domain to in vitro to by in vitro to the domain not to the yeast two-hybrid that the RAGE cytoplasmic domain interacts in vitro with Dia-1 with the RAGE in we the RAGE cytoplasmic domain interacts with in vivo and experiments in with the RAGE cytoplasmic domain and revealed that the RAGE cytoplasmic domain not with the that Dia-1 with the cytoplasmic domain of RAGE in we experiments to the interaction with the of RAGE not to the cytoplasmic RAGE and cells, we studies against RAGE, which revealed that cells, Dia-1 with RAGE Dia-1 not with RAGE cytoplasmic in not Dia-1 Taken together, these in vivo that Dia-1 with RAGE and, with its cytoplasmic domain. Dia-1 and RAGE in the we the and cellular interaction RAGE and Dia-1 by RAGE and with RAGE cells, RAGE and Dia-1 a cytoplasmic in the which to the cell Extensive in the a of the signal in the cell Because RAGE, we as a the RAGE and Dia-1 in and shown in RAGE and Dia-1 a to the with a cytoplasmic in the to the cell with in the The RAGE with the FH1 of on these we of to the that for interaction with the RAGE cytoplasmic domain in with RAGE cytoplasmic domain and or and with revealed that both and the FH1 domain with the cytoplasmic domain a the this FH1 domain to the cytoplasmic domain. that are in that the interaction RAGE and Dia-1 the RAGE cytoplasmic domain and the FH1 domain of The RAGE for Cellular and key of these the interaction of the RAGE cytoplasmic domain with required to effect signal transduction and changes in cellular properties upon RAGE-ligand this we studies with RAGE to the RAGE-dependent pathways and, with RNA interference to Dia-1 to its in RAGE-ligand cellular studies have central for ligand-RAGE signaling and Dia-1 in modulation of cellular migration in such as cells, or of (6Sakaguchi T. Yan S.F. Yan S.D. Belov D. Rong L.L. Sousa M. Andrassy M. Marso S.P. Duda S. Arnold B. Liliensiek B. Nawroth P.P. Stern D.M. Schmidt A.M. Naka Y. J. Clin. Investig. 2003; 111: 959-972Crossref PubMed Scopus (331) Google Scholar, 7Taguchi A. Blood D.C. del Toro G. Canet A. Lee D.C. Qu W. Tanji N. Lu Y. Lalla E. Fu C. Hofmann M.A. Kislinger T. Ingram M. Lu A. Tanaka H. Hori O. Ogawa S. Stern D. Schmidt A.M. Nature. 2000; 405: 354-360Crossref PubMed Scopus (1109) Google Scholar, T. Hori O. Zhang J. Yan S.D. Ferran L. Y. Schmidt A.M. J. Clin. Investig. 1996; PubMed Scopus Google Scholar, Zhang G. Chen X. Yan S.F. Schmidt A.M. C. Stern A. J. L. Stern D.M. H. Nat. 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Scopus Google Scholar). of RAGE ligand activation of and in and a and effect on of these GTPases cells, both and Cdc42 activated ligand in activation or signal for or for with a Cdc42 a of and a of in response to RAGE-ligand stimulation and not by RAGE-ligand stimulation in or on these we the that RAGE Rac-1 not mediates cellular of Cdc42 and with cells, and RAGE migration Both and RAGE cellular migration, not RAGE cellular migration that RAGE-mediated cellular migration signaling by the GTPases Rac-1 and Cdc42 not Dia-1 for RAGE and we the of Dia-1 in RAGE-dependent signaling and cellular migration. cells, Dia-1 expression of Dia-1 both the and protein by with the migration Dia-1 with GFP, and for of Dia-1. and Dia-1 by we that of for signal for Dia-1 with siRNA, Dia-1 not as a for we that RAGE ligand-stimulated migration in Dia-1 with to the of RAGE ligand these Dia-1 not migration of in response to serum for Dia-1 in the effects of RAGE ligand these serum a or effect of Dia-1 on the of these to we to Dia-1 required for RAGE ligand-stimulated signal transduction pathways linked to cellular migration. of not RAGE ligand-stimulated Rac-1 and Cdc42 in Dia-1 cells, RAGE protein not studies in vitro and in vivo have that the RAGE cytoplasmic domain is the required for RAGE ligand-stimulated cellular effects (5Huttunen H.J. 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The signal transduction and cellular migration Dia-1 to a cell surface receptor. have with Dia-1 and formin Dia-1 has linked to interaction with the and the P. H. R. Biol. Cell. PubMed Scopus Google and to the receptor potential in signaling S. L. J. PubMed Scopus Google Scholar). with the that the interaction of formin in with the short cytoplasmic domain of the receptor J. M. N. M. J. PubMed Scopus Google Scholar), that the of formin in to their to signal transduction cell surface Taken together, we have identified a novel in the of RAGE in which extracellular cues by RAGE ligand binding are the cytoplasmic domain of RAGE to fundamental signaling activation of Rac-1 and and cellular migration in a Dia-1. Because cellular migration critically to diabetes and its complications, and inflammatory in RAGE findings highlight novel to RAGE-dependent We are to for the and We and for with