Nm23-H2 Interacts with a G Protein-coupled Receptor to Regulate Its Endocytosis through an Rac1-dependent Mechanism

G protein-coupled receptors (GPCRs) represent a vast family of transmembrane proteins involved in the regulation of several physiological responses. The thromboxane A2 receptor (present as two isoforms: TPα and TPβ) is a GPCR displaying diverse pharmacological effects. As seen for many other GPCRs, TPβ is regulated by agonist-induced internalization. In the present study, we report the identification by yeast two-hybrid screening of Nm23-H2, a nucleoside diphosphate kinase, as a new interacting molecular partner with the C-terminal tail of TPβ. This interaction was confirmed in a cellular context when Nm23-H2 was co-immunoprecipitated with TPβ in HEK293 cells, a process dependent on agonist stimulation of the receptor. We observed that agonist-induced internalization of TPβ was regulated by Nm23-H2 through modulation of Rac1 signaling. Immunofluorescence microscopy in HEK293 cells revealed that Nm23-H2 had a cytoplasmic and nuclear localization but was induced to translocate to the plasma membrane upon stimulation of TPβ to show extensive co-localization with the receptor. Our findings represent the first demonstration of an interaction of an Nm23 protein with a membrane receptor and constitute a novel molecular regulatory mechanism of GPCR endocytosis. G protein-coupled receptors (GPCRs) represent a vast family of transmembrane proteins involved in the regulation of several physiological responses. The thromboxane A2 receptor (present as two isoforms: TPα and TPβ) is a GPCR displaying diverse pharmacological effects. As seen for many other GPCRs, TPβ is regulated by agonist-induced internalization. In the present study, we report the identification by yeast two-hybrid screening of Nm23-H2, a nucleoside diphosphate kinase, as a new interacting molecular partner with the C-terminal tail of TPβ. This interaction was confirmed in a cellular context when Nm23-H2 was co-immunoprecipitated with TPβ in HEK293 cells, a process dependent on agonist stimulation of the receptor. We observed that agonist-induced internalization of TPβ was regulated by Nm23-H2 through modulation of Rac1 signaling. Immunofluorescence microscopy in HEK293 cells revealed that Nm23-H2 had a cytoplasmic and nuclear localization but was induced to translocate to the plasma membrane upon stimulation of TPβ to show extensive co-localization with the receptor. Our findings represent the first demonstration of an interaction of an Nm23 protein with a membrane receptor and constitute a novel molecular regulatory mechanism of GPCR endocytosis. G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; TP, thromboxane A2 receptor; NDPK, nucleoside diphosphate kinase; GEF, guanine nucleotide exchange factor; TPβCT, TPβ C-terminal; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; BSA, bovine serum albumin; HA, hemagglutinin; GST, glutathione S-transferase; PBD, p21-binding domain; DP, prostaglandin in D2 receptor; IP, prostacyclin receptor. 1The abbreviations used are: GPCR, G protein-coupled receptor; TP, thromboxane A2 receptor; NDPK, nucleoside diphosphate kinase; GEF, guanine nucleotide exchange factor; TPβCT, TPβ C-terminal; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; BSA, bovine serum albumin; HA, hemagglutinin; GST, glutathione S-transferase; PBD, p21-binding domain; DP, prostaglandin in D2 receptor; IP, prostacyclin receptor. constitute the largest family of transmembrane proteins involved in signal transduction. They regulate a wide variety of physiological responses such as neurotransmission, inflammation, cell growth and differentiation, and smell and taste perception. For example, the thromboxane A2 receptor (TP) is implicated in the regulation of diverse pharmacological events, including platelet aggregation, constriction of vascular and bronchiolar smooth muscle cells, as well as mitogenesis and hypertrophy of vascular smooth muscle cells (1Narumiya S. Sugimoto Y. Ushikubi F. Physiol. Rev. 1999; 79: 1193-1226Google Scholar). Two TP receptor isoforms were identified that are generated by the alternative splicing of a single gene, TPα (343 amino acids) and TPβ (407 amino acids), which share the first 328 amino acids (2Hirata M. Hayashi Y. Ushikubi F. Yokota Y. Kageyama R. Nakanishi S. Narumiya S. Nature. 1991; 349: 617-620Google Scholar, 3Raychowdhury M.K. Yukawa M. Collins L.J. McGrail S.H. Kent K.C. Ware J.A. J. Biol. Chem. 1994; 269: 19256-19261Google Scholar). Previous experiments performed by Parent et al. (4Parent J.L. Labrecque P. Orsini M.J. Benovic J.L. J. Biol. Chem. 1999; 274: 8941-8948Google Scholar) demonstrated that only TPβ, but not TPα, undergoes agonist-induced and tonic (constitutive) internalization, which are dictated by distinct motifs in the C terminus of the TPβ receptor. Our efforts in the laboratory have been focused on understanding the molecular mechanisms involved in the regulation of TPβ (5Rochdi M.D. Watier V. La Madeleine C. Nakata H. Kozasa T. Parent J.L. J. Biol. Chem. 2002; 277: 40751-40759Google Scholar, 6Rochdi M.D. Parent J.L. J. Biol. Chem. 2003; 278: 17827-17837Google Scholar). The majority of GPCRs undergo internalization following agonist stimulation. This internalization can participate in receptor desensitization, resensitization, degradation, or activation of signaling cascades such as mitogen-activated protein kinase pathways (7Von Zastrow M. Biochem. Soc. Trans. 2001; 29: 500-504Google Scholar, 8Luttrell L.M. Lefkowitz R.J. J. Cell Sci. 2002; 115: 455-465Google Scholar, 9Cavalli V. Corti M. Gruenberg J. FEBS Lett. 2001; 498: 190-196Google Scholar). It has long been questioned if GPCRs signaling and endocytosis constitute two separate events, and if they are regulated by distinct molecular mechanisms (9Cavalli V. Corti M. Gruenberg J. FEBS Lett. 2001; 498: 190-196Google Scholar). In this regard, we have recently shown that Gαq signaling is directly involved in agonist-induced internalization of the TPβ receptor, suggesting that, in the case of some GPCRs, signaling and endocytosis are tightly connected (6Rochdi M.D. Parent J.L. J. Biol. Chem. 2003; 278: 17827-17837Google Scholar). However, the molecular mechanism by which Gαq protein regulates TPβ agonist-induced internalization is still unclear and is one of the major interests of our laboratory. Growing evidence has shown that endocytosis of GPCRs is governed by signaling molecules (6Rochdi M.D. Parent J.L. J. Biol. Chem. 2003; 278: 17827-17837Google Scholar, 9Cavalli V. Corti M. Gruenberg J. FEBS Lett. 2001; 498: 190-196Google Scholar). Indeed, some studies have demonstrated that members of the Rho family such as RhoA, Rac, and Cdc42 as well as a member of the ARF family, ARF6, play a crucial role in GPCR endocytosis (10Qualmann B. Mellor H. Biochem. J. 2003; 371: 233-241Google Scholar, 11Claing A. Chen W. Miller W.E. Vitale N. Moss J. Premont R.T. Lefkowitz R.J. J. Biol. Chem. 2001; 276: 42509-42513Google Scholar, 12Palacios F. Schweitzer J.K. Boshans R.L. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 929-936Google Scholar). Nevertheless, the regulation of these small G proteins and how they are involved in this process is not well understood. It has been suggested that the regulation of Rho/ARF-mediated actin cytoskeleton rearrangement is either directly or indirectly involved in the regulation of GPCR endocytosis (13Qualmann B. Kessels M.M. Kelly R.B. J. Cell Biol. 2000; 150: F111-F116Google Scholar, 14Machesky L.M. Nat. Cell Biol. 1999; 1: E29-E31Google Scholar, 15Song J.Z. Khachikian H. Radhakrishna J.G. Donaldson J. J. Cell Sci. 1998; 111: 2257-2267Google Scholar, 16Donaldson J.G. Jackson C.L. Curr. Opin. Cell Biol. 2000; 12: 475-482Google Scholar, 17Honda A. Nogami M. Yokozeki T. Yamazaki M. Nakamura H. Watanabe H. Kawamoto K. Nakayama K. Morris A.J. Frohman M.A. Kanaho Y. Cell. 1999; 99: 521-532Google Scholar, 18Malecz N. McCabe P.C. Spaargaren C. Qiu R. Chuang Y. Symons M. Curr. Biol. 2000; 10: 1383-1386Google Scholar, 19Murphy C. Saffrich R. Grummt M. Gournier H. Rybin V. Rubino M. Auvinen P. Lutcke A. Parton R.G. Zerial M. Nature. 1996; 384: 427-432Google Scholar, 20Gampel A. Parker P.J. Mellor H. Curr. Biol. 1999; 9: 955-958Google Scholar). In addition, several other signaling proteins participating in endocytosis have been identified. Recently, it has been shown that Nm23-H1 regulates dynamin-dependent endocytosis (12Palacios F. Schweitzer J.K. Boshans R.L. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 929-936Google Scholar, 21Deitcher D. Trends Neurosci. 1999; 24: 625-626Google Scholar, 22Krishnan K.S. Rikhy R. Rao S. Shivalkar M. Mosko M. Narayanan R. P. M. 2001; Scholar). Nm23 as NDPK, and was first identified as a and as an of N. N. Y. Y. M. N. J. 2003; Scholar). In Nm23 a family of nucleoside diphosphate by to and Nm23 were two is by the Nm23-H1 to the to isoforms N. N. Y. Y. M. N. J. 2003; Scholar). Nm23 proteins a kinase the other is of the kinase motifs for and only has been to kinase N. N. Y. Y. M. N. J. 2003; Scholar). Nm23 proteins are and were shown to involved in a variety of cellular including and N. N. M. Y. H. A. Y. N. J. 2003; Scholar). studies demonstrated that regulation of these by Nm23 proteins is the of to diverse transmembrane signaling including growth growth growth and growth as by et al. J. 2003; Scholar). was recently shown to activation of the of G proteins J. 2003; Scholar). Nm23 was suggested to as a nucleoside diphosphate kinase involved in the activation of by K.S. Rikhy R. Rao S. Shivalkar M. Mosko M. Narayanan R. P. M. 2001; Scholar). of the Nm23-H1 as in to the of endocytosis K.S. Rikhy R. Rao S. Shivalkar M. Mosko M. Narayanan R. P. M. 2001; Scholar). et al. (12Palacios F. Schweitzer J.K. Boshans R.L. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 929-936Google Scholar) that Nm23-H1 to endocytosis of and receptors of They that Nm23-H1 by the Rac1 signaling these studies demonstrated that Nm23-H1 Rac1 by a guanine nucleotide exchange for Rac1 (12Palacios F. Schweitzer J.K. Boshans R.L. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 929-936Google Scholar, J.G. FEBS Lett. 2003; Scholar). In addition, the that of a that the of involved in the and the of the of Rac1 it not (12Palacios F. Schweitzer J.K. Boshans R.L. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 929-936Google Scholar). our Nm23 proteins were demonstrated to with a transmembrane receptor and role in GPCR endocytosis to In our to involved in TPβ we performed yeast two-hybrid screening a cell we identified Nm23-H2 as a protein of interacting with the C-terminal tail of TPβ. we report a novel interaction Nm23-H2 and TPβ that regulates TPβ agonist-induced internalization through a Rac1 TPβ C-terminal acids were by the with the following and The was with and and the with the and used to yeast two-hybrid screening as and were by the by yeast two-hybrid and the following and yeast two-hybrid screening was performed following the two-hybrid 2002; Scholar). the was the yeast to the yeast 2002; Scholar) a yeast This was with a or with the and on and were and these were confirmed by growth on and the were and the were cells and yeast with either the or the and on and to the The were by The were by the Cell and cells were in with bovine serum in a of HEK293 cells to were performed to the was to the on were of Cell and internalization of TPβ and were by HEK293 cells as (4Parent J.L. Labrecque P. Orsini M.J. Benovic J.L. J. Biol. Chem. 1999; 274: 8941-8948Google Scholar). cells were in The cells were with or other receptor with or cells were as for cells were to with and for an the agonist-induced internalization, the cells were with phosphate-buffered by stimulation with for the in the cells were with for by with was with for The cells were with a or a of in for with and cells were for The cells were with a in of for The cells were with TBS, and a was following the the The were a with were to shown represent the of to experiments performed in of HEK293 cells with and in the cells were as for and for in the of to the cells were with and in of with and of the cells in with the were by for to of were to the of of protein in was by an were for in a and with of proteins were by of of by a and proteins were by and performed by and for the C-terminal of TPβ) was in the and the was used to a protein in by following the The was as by the The was by and was by a was with cellular in with and The were with was to the and the were for The were by and was performed with the in were performed as (4Parent J.L. Labrecque P. Orsini M.J. Benovic J.L. J. Biol. Chem. 1999; 274: 8941-8948Google Scholar). HEK293 cells were in The cells were as with the The cells were the following for with in The cells were in phosphate-buffered and in with bovine serum and for a stimulation with the was and the were by of of were in and a of a was were and for in a were on and with were by Immunofluorescence and Nm23-H2 localization was by HEK293 cells were in as The cells were with an or with and and as cells were on and The cells were with with for the cells were with and with for was with for The cells were with an and for in with cells were with PBS, by with a and a of for The cells were with were and by microscopy on a a were and with Rac1 activation was by the This on the of the of Rac1 to the regulatory of kinase to as the p21-binding D. Scholar, V. 2002; Scholar). HEK293 cells were in The cells were as with the cells were as for and for in the of to the cells were with and in of with and Cell were with of The La for The were for and the were with by two in of was to the The were by performed by the of Nm23-H2 with have recently shown that the C terminus of TPβ regulatory mechanisms to this of the thromboxane A2 receptor (4Parent J.L. Labrecque P. Orsini M.J. Benovic J.L. J. Biol. Chem. 1999; 274: 8941-8948Google Scholar, J.L. Labrecque P. M. Benovic J.L. J. Biol. Chem. 2001; 276: Scholar). that with the C terminus of TPβ, we performed yeast two-hybrid screening experiments the yeast with and a cell of were a growth on yeast and were and by and the a for the Nm23-H2 members of the Nm23 protein family were not in the that were In this we report the of Nm23-H2 as a which with the C-terminal tail of TPβ. As shown in growth on and was present only in yeast with and that Nm23-H2 is interacting with the C terminus of TPβ. and in the interaction of Nm23-H2 with TPβ in a cellular we performed experiments in HEK293 cells with and in the or of stimulation. Cell were with a and protein and were by with a Our that was co-immunoprecipitated with TPβ following agonist This that the interaction of Nm23-H2 with TPβ is by agonist stimulation. to the interaction of Nm23-H2 with the C-terminal tail of TPβ, we performed an in the C-terminal tail of TPβ to with HEK293 cell The that Nm23-H2 to and not to our that Nm23-H2 directly with the C-terminal tail of TPβ in an this the first a interaction of an Nm23 protein with a G protein-coupled receptor, and to our to transmembrane receptor, is Nm23-H2 TPβ were in the role of Nm23-H2 in TPβ Nm23-H1 and Nm23-H2 are Two of Nm23-H1 have been was as a of the was shown to to Rac1 it not and Rac1 (12Palacios F. Schweitzer J.K. Boshans R.L. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 929-936Google Scholar). The were We first how Nm23-H2 and two regulate agonist-induced internalization of TPβ. the of a TPβ internalization following a with performed by (4Parent J.L. Labrecque P. Orsini M.J. Benovic J.L. J. Biol. Chem. 1999; 274: 8941-8948Google Scholar) in HEK293 cells with and either or we observed that only the agonist-induced internalization of TPβ, of or had on the of receptor internalization was performed to the of in confirmed that only agonist-induced internalization of TPβ with that the kinase of Nm23-H2 is not involved in the process of agonist-induced internalization of TPβ. of to TPβ internalization that is not involved in modulation of this process by the other the of of Nm23-H2, in to for the of the agonist-induced internalization of TPβ, that Nm23-H2 is involved in this we that not agonist-induced internalization of the and as well as of the receptor of the receptor was by the In in a of agonist-induced endocytosis of two other the and the receptors This that the role of Nm23-H2 in endocytosis is to some are in with evidence and that endocytosis of GPCRs is regulated by distinct molecular of agonist-induced internalization of G protein-coupled receptors by internalization of the receptors was by in HEK293 cells with and either or with the of and for are in of when receptor were with are separate experiments with cells with receptor and Nm23-H2 Gαq was shown to a with the and G protein activation by the to F. F. S. F. T. J. Biol. Chem. 2003; 278: Scholar). et al. S. K. K. N. M. N. T. J. Biol. Chem. Scholar) that Nm23-H1 signaling. It is the kinase of Nm23-H1 that is involved in this S. K. K. N. M. N. T. J. Biol. Chem. Scholar). we were in if Nm23-H2 and two TPβ signaling. Our that of the or Gαq or signaling as by and not were with of Gαq and not We demonstrated that Gαq signaling was for TPβ internalization. The that is not Gαq signaling with of on TPβ agonist-induced internalization. this that of TPβ agonist-induced internalization is by a mechanism that is not the protein signaling. Rac1 TPβ the of involved in the of Rac1 as a the Nm23-H1 and as such with the of (12Palacios F. Schweitzer J.K. Boshans R.L. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 929-936Google Scholar). we that Nm23-H2 the agonist-induced internalization of TPβ by the of Rac1 signaling. However, to this we first had to on one the of Rac1 signaling on the agonist-induced internalization of TPβ on the other the modulation of activation following TPβ agonist stimulation. this we the of of a of on the agonist-induced internalization of TPβ. As seen in the agonist-induced internalization of TPβ. it that activation of the Rac1 signaling with agonist-induced internalization of TPβ. a of of TPβ agonist-induced internalization. This The activation of Rac1 was by on cell of cells following a stimulation of the receptor. The regulatory of kinase to as the p21-binding to the the V. 2002; Scholar) were by with a that TPβ stimulation a activation of Rac1 which by a in Rac1 and Rac1 the of Rac1 of stimulation with the the agonist-induced internalization of TPβ is observed and J.L. Labrecque P. Orsini M.J. Benovic J.L. J. Biol. Chem. 1999; 274: 8941-8948Google Scholar). of in a activation of Rac1 the that we This that with the of Rac1 by an Nm23 We that the of of the in was to the which the was is Rac1 not have of TPβ internalization in of of we observed Rac1 activation was As can seen in TPβ internalization the of Rac1 activation by TPβ, that Rac1 signaling internalization of the Rac1 activation by an Rac1 activation was by the as on cell of HEK293 cells TPβ either or in with with for the of stimulation of TPβ a activation of Rac1 a activation of Rac1 is observed in the of shown are of agonist-induced internalization of TPβ. The of on TPβ agonist-induced internalization was by following stimulation with for the of TPβ internalization and of receptor our findings show that activation of Rac1 signaling with TPβ agonist-induced internalization. receptor internalization when Rac1 is Our that TPβ agonist-induced internalization through of Rac1 Rac1 the of TPβ recently that Gαq signaling is in the agonist-induced internalization of TPβ. In this study, we have demonstrated that of was to the internalization of TPβ in of agonist stimulation (6Rochdi M.D. Parent J.L. J. Biol. Chem. 2003; 278: 17827-17837Google Scholar). we were in the of the on internalization of TPβ. In this internalization is by cell receptor by as we (6Rochdi M.D. Parent J.L. J. Biol. Chem. 2003; 278: 17827-17837Google Scholar). The show that induced a endocytosis of TPβ receptor as cell as (6Rochdi M.D. Parent J.L. J. Biol. Chem. 2003; 278: 17827-17837Google Scholar). However, with internalization of TPβ This that Rac1 signaling the of Gαq signaling in the process of TPβ endocytosis. of Nm23-H2 to the the localization of Nm23-H2 and regulation by TPβ we performed microscopy HEK293 cells with and and of cells was In the of TPβ Nm23-H2 has a cytoplasmic and nuclear TPβ has a cytoplasmic and membrane However, following Nm23-H2 undergoes to the plasma membrane in co-localization with TPβ of proteins is in In the the GPCRs signaling and endocytosis has been the of We have recently shown that the first of regulation of TPβ endocytosis the activation of Gαq signaling (6Rochdi M.D. Parent J.L. J. Biol. Chem. 2003; 278: 17827-17837Google Scholar). However, the molecular mechanisms and involved in this regulation are still such molecular we performed yeast two-hybrid screening the C-terminal tail of TPβ and a cell we report that Nm23-H2 with the C-terminal tail of TPβ. as kinase is a kinase involved in the regulation of a wide variety of cellular Nm23 was shown to regulate cell differentiation, kinase signal and recently endocytosis (12Palacios F. Schweitzer J.K. Boshans R.L. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 929-936Google Scholar, 22Krishnan K.S. Rikhy R. Rao S. Shivalkar M. Mosko M. Narayanan R. P. M. 2001; Scholar, N. N. M. Y. H. A. Y. N. J. 2003; Scholar). Nm23-H2 is one of the members of the Nm23 demonstrated that Nm23-H2 with TPβ in a cellular this interaction to by the activation of TPβ, suggesting that Nm23-H2 involved in the regulation of the molecular to TPβ agonist stimulation. we first the of Nm23-H2 on TPβ agonist-induced internalization. of TPβ and in HEK293 cells not the agonist-induced internalization of TPβ. Two of Nm23-H1 are in the (12Palacios F. Schweitzer J.K. Boshans R.L. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 929-936Google Scholar). is a shown to with endocytosis (12Palacios F. Schweitzer J.K. Boshans R.L. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 929-936Google Scholar). the other which the of is to Rac1 (12Palacios F. Schweitzer J.K. Boshans R.L. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 929-936Google Scholar). The were Nm23-H2, Nm23-H1 and Nm23-H2 are in the of the The of the two to TPβ agonist-induced internalization was had on the internalization of TPβ suggesting that the kinase of Nm23-H2 is not involved in this We that TPβ internalization is dynamin-dependent (4Parent J.L. Labrecque P. Orsini M.J. Benovic J.L. J. Biol. Chem. 1999; 274: 8941-8948Google Scholar). In this case the that Nm23-H2 regulate TPβ internalization through a mechanism not the internalization of TPβ the of of Nm23-H2 as a of this is of TPβ, this to internalization of the DP, and of the receptor was by which that the of Rac1 internalization of this receptor. by the endocytosis of the and the other internalization of the receptor was not by The receptor is a receptor but was to to Gαq K. H. 2003; Scholar). our that regulation of GPCR endocytosis by Nm23-H2 is a to but not in the of proteins or cell a of this for a receptor. studies to the of Nm23-H2 of Our that agonist-induced endocytosis of TPβ is not by of or in our not in the Gαq signaling by TPβ is for the activation of the for internalization of this receptor, and with this signaling in a in receptor internalization (6Rochdi M.D. Parent J.L. J. Biol. Chem. 2003; 278: 17827-17837Google Scholar). We observed that not Gαq as by This that was not the of Gαq et al. (12Palacios F. Schweitzer J.K. Boshans R.L. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 929-936Google Scholar) recently that ARF6, which is involved in membrane the process of membrane endocytosis by The that the of of Nm23-H1 the of Rac1 signaling by and with on our demonstrated that TPβ agonist-induced internalization, we that Nm23-H2 play a role in TPβ internalization by the of Rac1 signaling. This first that Rac1 signaling with TPβ agonist-induced internalization and that Rac1 signaling has to for the internalization process to on the first we the of Rac1 signaling on TPβ internalization with the of a of This revealed that to a of TPβ agonist-induced internalization. This suggested that the Rac1 signaling the agonist-induced internalization of TPβ. Rac1 activation was a well p21-binding V. 2002; Scholar) to the Rac1 the process of agonist-induced internalization of TPβ. We have seen that TPβ agonist stimulation a in the cellular of which and we demonstrated that, in the of a activation of Rac1 was observed TPβ stimulation. This that Nm23-H2 is involved in modulation of Rac1 signaling. findings are in with the It is to that TPβ internalization and that TPβ internalization with the when Rac1 was This that Rac1 activation by TPβ internalization, which Rac1 This is by the that a of a in the of agonist-induced internalization of TPβ and which with the activation of Rac1 signaling. had on TPβ agonist-induced internalization a that can by the of this activation of Rac1 signaling by either or to a in the agonist-induced internalization of TPβ. has been that Rac1 regulate receptor endocytosis by the plasma membrane (10Qualmann B. Mellor H. Biochem. J. 2003; 371: 233-241Google Scholar). In addition, et al. C. Chuang L.J. Nature. 1996; Scholar) demonstrated that the of Rac1 the internalization of the receptor. However, the molecular mechanism involved in this regulation is still an in the localization of Nm23-H2 to the plasma membrane was shown following TPβ receptor agonist stimulation by microscopy suggesting that TPβ Nm23-H2 to the plasma This of Nm23-H2 is for and of Rac1 proteins present the plasma membrane (12Palacios F. Schweitzer J.K. Boshans R.L. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 929-936Google Scholar, N. N. Y. Y. M. N. J. 2003; Scholar, N. N. M. Y. H. A. Y. N. J. 2003; Scholar). Growing evidence the crucial role by GPCRs signaling in endocytosis process (6Rochdi M.D. Parent J.L. J. Biol. Chem. 2003; 278: 17827-17837Google Scholar, 9Cavalli V. Corti M. Gruenberg J. FEBS Lett. 2001; 498: 190-196Google Scholar). We recently that Gαq signaling is involved in the regulation of agonist-induced internalization of GPCRs (6Rochdi M.D. Parent J.L. J. Biol. Chem. 2003; 278: 17827-17837Google Scholar). we have shown that, to Gαq Rac1 activation with the agonist internalization of TPβ. Gαq and Rac1 signaling to play an role in the internalization process of TPβ. Indeed, internalization of the receptor. Rac1 signaling has been shown to regulate the actin cytoskeleton and membrane It is that Rac1 signaling activation to an actin cytoskeleton or a plasma membrane not for TPβ endocytosis. In of this study, one can that agonist-induced internalization of TPβ and other GPCRs a of signaling we the first demonstration of an interaction of a member of the Nm23 proteins with a membrane receptor, in this a This interaction regulates internalization of the receptor through a was seen in the GPCRs that our findings constitute a distinct molecular regulatory mechanism of GPCR internalization.

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