Chong-Shan Shi

Coronaviruses (CoV) have recently emerged as potentially serious pathogens that can cause significant human morbidity and death. The severe acute respiratory syndrome (SARS)-CoV was identified as the etiologic agent of the 2002-2003 international SARS outbreak. Yet, how SARS evades innate immune responses to cause human disease remains poorly understood. In this study, we show that a protein encoded by SARS-CoV designated as open reading frame-9b (ORF-9b) localizes to mitochondria and causes mitochondrial elongation by triggering ubiquitination and proteasomal degradation of dynamin-like protein 1, a host protein involved in mitochondrial fission. Also, acting on mitochondria, ORF-9b targets the mitochondrial-associated adaptor molecule MAVS signalosome by usurping PCBP2 and the HECT domain E3 ligase AIP4 to trigger the degradation of MAVS, TRAF3, and TRAF 6. This severely limits host cell IFN responses. Reducing either PCBP2 or AIP4 expression substantially reversed the ORF-9b-mediated reduction of MAVS and the suppression of antiviral transcriptional responses. Finally, transient ORF-9b expression led to a strong induction of autophagy in cells. The induction of autophagy depended upon ATG5, a critical autophagy regulator, but the inhibition of MAVS signaling did not. These results indicate that SARS-CoV ORF-9b manipulates host cell mitochondria and mitochondrial function to help evade host innate immunity. This study has uncovered an important clue to the pathogenesis of SARS-CoV infection and illustrates the havoc that a small ORF can cause in cells.

Regulator of G-protein signaling 3 (RGS3) enhances the intrinsic rate at which Gαi and Gαq hydrolyze GTP to GDP, thereby limiting the duration in which GTP-Gαi and GTP-Gαq can activate effectors. Since GDP-Gα subunits rapidly combine with free Gβγ subunits to reform inactive heterotrimeric G-proteins, RGS3 and other RGS proteins may also reduce the amount of Gβγ subunits available for effector interactions. Although RGS6, RGS7, and RGS11 bind Gβ5 in the absence of a Gγ subunit, RGS proteins are not known to directly influence Gβγ signaling. Here we show that RGS3 binds Gβ1γ2 subunits and limits their ability to trigger the production of inositol phosphates and the activation of Akt and mitogen-activated protein kinase. Co-expression of RGS3 with Gβ1γ2 inhibits Gβ1γ2-induced inositol phosphate production and Akt activation in COS-7 cells and mitogen-activated protein kinase activation in HEK 293 cells. The inhibition of Gβ1γ2 signaling does not require an intact RGS domain but depends upon two regions in RGS3 located between acids 313 and 390 and between 391 and 458. Several other RGS proteins do not affect Gβ1γ2 signaling in these assays. Consistent with the in vivo results, RGS3 inhibits Gβγ-mediated activation of phospholipase Cβ in vitro. Thus, RGS3 may limit Gβγ signaling not only by virtue of its GTPase-activating protein activity for Gα subunits, but also by directly interfering with the activation of effectors. Regulator of G-protein signaling 3 (RGS3) enhances the intrinsic rate at which Gαi and Gαq hydrolyze GTP to GDP, thereby limiting the duration in which GTP-Gαi and GTP-Gαq can activate effectors. Since GDP-Gα subunits rapidly combine with free Gβγ subunits to reform inactive heterotrimeric G-proteins, RGS3 and other RGS proteins may also reduce the amount of Gβγ subunits available for effector interactions. Although RGS6, RGS7, and RGS11 bind Gβ5 in the absence of a Gγ subunit, RGS proteins are not known to directly influence Gβγ signaling. Here we show that RGS3 binds Gβ1γ2 subunits and limits their ability to trigger the production of inositol phosphates and the activation of Akt and mitogen-activated protein kinase. Co-expression of RGS3 with Gβ1γ2 inhibits Gβ1γ2-induced inositol phosphate production and Akt activation in COS-7 cells and mitogen-activated protein kinase activation in HEK 293 cells. The inhibition of Gβ1γ2 signaling does not require an intact RGS domain but depends upon two regions in RGS3 located between acids 313 and 390 and between 391 and 458. Several other RGS proteins do not affect Gβ1γ2 signaling in these assays. Consistent with the in vivo results, RGS3 inhibits Gβγ-mediated activation of phospholipase Cβ in vitro. Thus, RGS3 may limit Gβγ signaling not only by virtue of its GTPase-activating protein activity for Gα subunits, but also by directly interfering with the activation of effectors. regulator of G-protein signaling GTPase-activating protein mitogen-activated protein kinase polymerase chain reaction hemagglutinin extracellular signal-regulated kinase phospholipase C glutathione S-transferase polyacrylamide gel electrophoresis Tween 20 Tris-buffered saline 4-morpholinepropanesulfonic acid inositol 1,4,5-trisphosphate Heterotrimeric G-proteins link seven transmembrane receptors to downstream signaling pathways. Receptor activation triggers the exchange of GTP for GDP by the Gα subunit of the heterotrimeric G-protein, causing a conformational change in the Gα subunit, which facilitates its dissociation from the receptor and Gβγ subunits. GTP-bound Gα and free Gβγ subunits then bind and activate downstream effectors. However, Gα subunits possess an intrinsic GTPase activity, which returns Gα to its GDP bound state and thereby limits the duration of Gα signaling. Because GDP-Gα possesses a high affinity for Gβγ subunits, the heterotrimeric G-protein rapidly reforms, effectively ending Gβγ-mediated signaling as well (reviewed in Refs. 1Hepler J.R. Gilman A.G. Trends Biochem. Sci. 1992; 17: 383-387Abstract Full Text PDF PubMed Scopus (928) Google Scholar and 2Neer E.J. Cell. 1995; 80: 249-257Abstract Full Text PDF PubMed Scopus (1289) Google Scholar). Cells possess another important mechanism that curtails the duration in which a Gα subunit remains GTP bound. Members of a family of proteins termed regulators of G-protein signaling (RGS)1 dramatically accelerate the intrinsic rate that certain Gα subunits hydrolyze GTP, a property that identifies them as GTPase-activating proteins (GAPs). The mammalian RGS proteins have a 120-amino acid region, RGS domain, or RGS box, which binds Gαi and Gαq subfamily members in a transition state of the GTP hydrolysis reaction, thereby lowering the free energy of the reaction (reviewed in Refs. 3Kehrl J.H. Immunity. 1998; 8: 1-10Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar and 4Hepler J.R. Trends Pharmacol. Sci. 1999; 20: 376-382Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). In addition, Rho guanine nucleotide exchange factors have a divergent RGS domain, which accelerates the intrinsic GTPase activity of Gα12 and Gα13 (5Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (741) Google Scholar, 6Mao J. Yuan H. Xie W. Wu D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12973-12976Crossref PubMed Scopus (114) Google Scholar). RGS proteins with GAP activity for members of the Gαs subfamily remain enigmatic. The mammalian RGS proteins can be broadly divided into two groups (7Cho H. Kehrl J.H. Curr. Top Biochem. Res. 2000; 2: 161-170Google Scholar): those composed predominantly of an RGS domain such as RGS1, RGS2, RGS4, and RGS5 and those that contain an RGS domain but also other domains. The second group includes RGS6, RGS7, RGS9, RGS11, RGS12, and RGS14. The smaller RGS proteins likely function solely as Gα GAPs, whereas some of the larger RGS proteins are undoubtedly Gα effectors such as p115 Rho guanine nucleotide exchange factors. RGS3 exists as two isoforms, thus falling into both groups (8Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (407) Google Scholar, 9Reif K. Cyster J.G. J. Immunol. 2000; 164: 4720-4729Crossref PubMed Scopus (124) Google Scholar): a shorter version that encodes largely an RGS domain (RGS3CT) and a larger isoform that has a strongly acidic region and an unusual region that contains a hexapeptide repeat enriched for proline, glutamine, and acidic residues (8Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (407) Google Scholar, 9Reif K. Cyster J.G. J. Immunol. 2000; 164: 4720-4729Crossref PubMed Scopus (124) Google Scholar). Both versions possess GAP activity for Gαi and Gαq and can impair signaling through Gαiand certain Gαq-linked signaling pathways (10Scheschonka A. Dessauer C.W. Sinnarajah S. Chidiac P. Shi C.-S. Kehrl J.H. Mol. Pharmacol. 2000; 58: 719-728Crossref PubMed Scopus (74) Google Scholar). The function of the N-terminal domain of RGS3 is unknown, although the N-terminal fragment shifts to membranes after a calcium signal (11Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J.H. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar). Transient expression of RGS3 potently inhibits the chemotaxis of a pre-B cell line, even better than does RGS1, which has excellent Gαi GAP activity (12Bowman E.P. Campbell J.J. Druey K.M. Scheschonka A. Kehrl J.H. Butcher E.C. J. Biol. Chem. 1998; 273: 28040-28048Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 13Moratz C. Kang V.H. Druey K.M. Shi C.S. Scheschonka A. Murphy P.M. Kozasa T. Kehrl J.H. J. Immunol. 2000; 164: 1829-1838Crossref PubMed Scopus (99) Google Scholar). The known importance of Gβγ signaling in chemokine-directed migration (14Arai H. Tsou C.L. Charo I.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14495-14499Crossref PubMed Scopus (150) Google Scholar, 15Neptune E.R. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14489-14494Crossref PubMed Scopus (245) Google Scholar) led us to test whether RGS3 employs another mechanism besides its Gα GAP activity to impair Gβγ signaling. In three in vivomodels of Gβ1γ2 signaling, inositol phosphate generation through the stimulation of phospholipase Cβ, the activation of mitogen-activated protein kinase (MAPK), and the activation of Akt, we find that RGS3 potently inhibits the activation of these signaling pathways even when it lacks a functional RGS domain. Supporting the in vivo data, purified RGS3 blocks Gβγ-induced inositol phosphate production by phospholipase Cβ2in vitro. To make pET15b His6-RGS3, PCR fragments generated from RcCMV-RGS3 (8Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (407) Google Scholar) were inserted into theNdeI/XhoI sites of the bacterial expression vector pET15b (Novagen, Madison, WI) in frame with the hexahistidine tag. To make FLAG-RGS3, FLAG-RGS3NT (RGS3 1–313), FLAG-RGS3CT (RGS3 314–520), FLAG-RGS3NT1(RGS3 1–390), and FLAG-RGS3NT2 (RGS3 1–458), the appropriate PCR products were subcloned into pFLAGCMV-2. FLAG-RGS3/4 was formed by fusing the coding region of the first 58 amino acids of RGS4 with the coding region of amino acids 390–519 of RGS3 via PCR using PCR primers that generate the appropriate overlapping fragments. The PCR products were denatured, annealed, and extended before subcloning into pFLAGCMV2. FLAG-RGS3 E419A/N420A (EN mutant) was created by site-directed mutagenesis of the FLAG-RGS3 construct (Stratagene, La Jolla, CA). To make GST-RGS3, GST-RGS3CT, and RGS3NT, PCR products encompassing the coding regions of RGS3, RGS3 (1), and RGS3 (314) were cloned in-frame with the GST-coding region using the GST expression vector pEBG. The veracity of all the DNA constructs was verified by nucleotide sequencing. The RGS2, RGS4, and RGS10 constructs have been previously described (8Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (407) Google Scholar, 16Heximer S.P. Watson N. Linder M.E. Blumer K.J. Hepler J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14389-14393Crossref PubMed Scopus (314) Google Scholar, 17Hunt T.W. Fields T.A. Casey P.J. Peralta E.G. Nature. 1996; 383: 175-177Crossref PubMed Scopus (311) Google Scholar). A schematic showing the expected RGS proteins from the constructs used is shown (Fig. 1). The constructs, which express RasV12, Gβ1, and Gγ2 were kindly provided by Dr. J. S. Gutkind (National Institutes of Health, Bethesda, Maryland). The construct that expresses Akt, pCMV6Akt-HA, was kindly provided by Dr. P. N. Tsichlis (Thomas The construct that expresses was kindly provided by Dr. J. The construct that expresses has been previously described Hepler J.R. Gilman A.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The purified Gβγ subunits were from HEK 293 cells and COS-7 cells were from the The cells were in with The cells were in the the cells were in and for the Akt were in for at which the was with The RGS3 and RGS4 were purified from with at for The proteins were using acid and with an The purified protein were the and at In some RGS3 was purified a The kinase was from CA). proteins were from of HEK 293 cells previously with the appropriate expression The proteins were purified as described by the The proteins were and using with the and The was using a to a that been with the in the high CA). were at or were at a rate of by of the for and an from to for were and 20 of were gel by The proteins were in by the proteins were were to by divided into and at The Gβ1γ2 was from cells with constructs Gβ1, and to the Gilman A.G. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). were using a and with a of for 20 The was by for at of protein from were by and to were with in for and then with an appropriate of the in and in The were with before the of a in a the was with and then with to The signal was by the of the The were using from COS-7 cells or HEK 293 cells Gβ1γ2 and RGS or was and the were with the appropriate were three in in with by and by with the appropriate phosphate production was as previously described K. S. Sternweis P.M. Gilman A.G. Kozasa T. E.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). COS-7 cells were using after the was with and for after which were The cells were for an before with saline by the of of 20 the was and a second was was to with and The were for at and to a that been previously with of of and of of the the was with of and of and The were with 3 of and of was to of and via of HEK 293 or COS-7 cells were at an of the before The HEK 293 or COS-7 cells were with or and RGS expression using a calcium phosphate or DNA were with after the were to in kinase using protein or as a The in kinase were as previously described Kehrl J.H. P. J. J.R. T. Nature. 1995; PubMed Scopus Google K. A. Tsichlis Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). the in kinase the were three with kinase and to which a was three with a and and three with kinase and The were by and The of and RGS protein expression were by cell The were using were by of proteins and Gβγ subunits in a for at reaction were and for the activity was as previously described Hepler J.R. Gilman A.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). was as of and of was to and was to the to free were in for at these was with to and Since RGS3 possesses GAP activity for both Gαi and Gαq whether RGS3 Gβγ signaling through or receptors is not However, the expression of or known to activate in the production of inositol phosphates D. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The Gβγ subunits to the Gα subunits to inactive The free Gβγ subunits can activate activity, which the second inositol 1,4,5-trisphosphate and by a construct that expresses RGS3, we test whether RGS3 the production of inositol phosphates by free Gβγ subunits. that expression of Gβ1γ2 inositol phosphate production by COS-7 The of RGS3 inositol phosphate production by whereas two other RGS RGS4 and not impair inositol phosphate production in (Fig. The expression of RGS3 not the of subunits or although the subunits with the subunits. Thus, although RGS4 has better GAP activity for Gαi than does RGS3 and (10Scheschonka A. Dessauer C.W. Sinnarajah S. Chidiac P. Shi C.-S. Kehrl J.H. Mol. Pharmacol. 2000; 58: 719-728Crossref PubMed Scopus (74) Google RGS4 does not impair Gβγ signaling to phospholipase Cβ RGS3 Gβγ subunits directly activate other effectors of the of the of subunits are to the of Gβγ for its effectors H. E. G. C.S. Science. 1998; 280: PubMed Scopus Google Scholar, E.J. Pharmacol. 1997; PubMed Scopus Google Scholar). effectors Gβγ subunits and Akt of Gβ1γ2 subunits in COS-7 cells enhances the activity of the P. N. Gutkind Nature. PubMed Scopus Google Scholar). by RGS3 we test whether RGS3 inhibits a second than COS-7 we used HEK 293 thereby both the signaling and the cell from the The of Gβ1γ2 into HEK 293 activation as by the amount of generated in an in kinase The of RGS3 activation to the in the absence of In RGS4 RGS10 Gβ1γ2 signaling in these cells (Fig. To test the importance of the N-terminal region of RGS3 and an intact RGS domain, we used three RGS3 The N-terminal of RGS3, by the not activation a The which the of RGS3, amino acids Gβ1γ2 activation as well as The which an RGS3 with at and of acid and Gβ1γ2-induced activation to that of A to in RGS4 its Gαi GAP activity and its chemotaxis (11Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J.H. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar, S.P. Watson N. Blumer K.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, K.M. Kehrl J.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar). that RGS3 inhibits in the it not the activation of by an of (Fig. Thus, to Gβ1γ2 activation of the RGS3 its an intact RGS domain. RGS3 also with activation of Akt is in pathways to cell in to and factors (reviewed in Tsichlis Biochem. 1999; PubMed Scopus Google and receptors Akt activation in by the of Gβγ subunits from and C. A. Gutkind J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). of Gβ1γ2 in COS-7 cells has been shown to the activity of a version of Akt in an in kinase C. A. Gutkind J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). in with constructs that express or RGS Gβ1γ2 expression Akt activity and the of RGS3, or it to acids also Akt it not express RGS3NT, acids 1–390), RGS2, and RGS4 (Fig. its inhibition of Gβ1γ2-induced Akt RGS3 not Akt activation by an of (Fig. also the regions of RGS3 for phospholipase Cβ COS-7 cells with constructs that Gβ1γ2 and in the or absence of constructs that RGS3, RGS3NT, or and inositol phosphate The construct a protein that the first 58 amino acids of RGS4 with amino acids 390–519 of to the inositol phosphate production by Gβ1γ2 even better than which the amino acids of RGS3, RGS3 of RGS3, RGS3NT, and that amino acids and both to the of RGS3 to phospholipase Cβ (Fig. from the with Akt the construct not impair amino acids 390–519 to the of RGS4 upon RGS4 the ability to Gβ1γ2 signaling to that of RGS3, although not to these show that RGS3 the residues between and to whether RGS3 with Gβ1γ2 in vivo by HEK cells with constructs the expression of RGS3 and Gβ1γ2 and for the The the RGS3 and in Gβγ subunits a subunits both Gβγ subunits and the Gβ1γ2 subunits and the RGS3 (Fig. and to FLAG-RGS3 or Gβγ subunits. we whether RGS3 directly bound Gβγ subunits by RGS3 with purified Gβγ subunits and then for RGS3 and Gβγ subunits. both RGS3 and Gβγ subunits In kinase to Gβγ subunits, and RGS3 Gβγ subunits with an that RGS3 can directly bind Gβγ subunits. RGS3 in cell predominantly in the and can be to membranes by G-protein signaling (11Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J.H. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar). A that RGS3 predominantly in a than the of cells Scheschonka A. Druey K.M. Kehrl J.H. 1997; PubMed Scopus Google Scholar). The RGS3 used in these RGS3, only with RGS3 and not with Here we used an a from the N-terminal of and RGS3 and a that with RGS3 in cells (10Scheschonka A. Dessauer C.W. Sinnarajah S. Chidiac P. Shi C.-S. Kehrl J.H. Mol. Pharmacol. 2000; 58: 719-728Crossref PubMed Scopus (74) Google Scholar). of COS-7 cells into a and that the the of the RGS3, although some RGS3 also in the not we using the broadly we that of RGS3 (Fig. RGS3 also of subunits. Thus, in COS-7 RGS3 predominantly at or and a is with Gβγ subunits. the signaling we expected to with Gβγ subunits but not In we that RGS3NT, and all with Gβγ subunits when we them with with a subunits but to when we used a the we Gβ1γ2 with RGS2, RGS3, or RGS4, subunits not with or RGS4, but with RGS3 (Fig. Thus, although both and with Gβγ subunits, only inhibits Gβγ signaling to and the and pathways. To test whether RGS3 can directly the activation of phospholipase Cβ by we to of and and then by Gβ1γ2 subunits that been with RGS3 or The that RGS3 may not directly affect the activation of by Gβγ subunits RGS3 it not However, when of using E. RGS3, we used RGS3, RGS3NT, and as GST proteins from HEK 293 we a The purified the Gβ1γ2-induced activity by and whereas the generation of Thus, the with proteins the in vivo we previously the purified was than the or RGS3 inhibits Akt activation and inositol phosphate production in COS-7 cells and activation in HEK 293 cells. The inhibition of these pathways by RGS3 does not upon an intact RGS domain or its N-terminal 390 amino acids but a region in RGS3 that the RGS domain. are by RGS3 Gβγ-mediated activation of its effectors. RGS3 directly the effectors or impair downstream in the signaling pathways. for RGS3 at the of Gβγ signaling to its RGS3 does not reduce activation (8Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (407) Google Scholar) or or Akt activation RGS3 Gβγ-mediated effector The of RGS4 and the of and the RGS3 proteins in Gβ1γ2 signaling that the Gαi and Gαq GAP activity of RGS3 its in the N-terminal of RGS3 does not in the inhibition of Gβ1γ2 signaling also potently Since RGS3 binds both in and in vivo and RGS3 in in the the RGS3 Gβ1γ2 from its thereby of Gβ1γ2 to its effectors. However, although both and bind Gβγ subunits, only potently inhibits Gβ1γ2 signaling. RGS3 and the activation of in whereas does that RGS3 can directly activation of Gβγ subunits. Thus, we the when in the of heterotrimeric G-proteins RGS3 can with GTP-bound Gα subunits, Gα GTP and with Gβγ subunits, their for effector Since the of Gβγ subunits does not impair RGS3 GAP activity, RGS3 can as a Gα GAP and a Gβγ the or sites in Gβ1γ2 that with RGS3 and the of RGS3 signaling by other Gβγ into the importance of RGS3 as a regulator of Gβγ signaling. the known RGS is RGS3 in its ability to Gβγ RGS4 RGS10 inhibits activation or the generation of inositol that not all RGS proteins Gα protein inhibits the of inositol phosphates after Gβ1γ2 expression in COS-7 cells. RGS only RGS3 signal that the inhibition of Gβγ signaling may be a property of RGS3 only by a of the RGS of with RGS2, RGS4, and Gα protein between the proteins in the regions N-terminal to the RGS domains. the RGS these proteins of their amino and RGS3 possesses residues not in the other To residues in RGS3 for its inhibition of signaling, we created constructs with in the region that encodes amino acids of or the proteins in COS-7 all of the RGS3 proteins their ability to Gβ1γ2 signaling. that we conformational is region than residues for the with RGS3 proteins a is in The of RGS3 with Gβ1γ2 is not by the amino acid of RGS3 to those of proteins known to directly with A in the Gβγ effectors and and may their with subunits S. T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar, T. 1997; 19: Full Text Full Text PDF PubMed Scopus (99) Google Scholar). However, such in a protein and all a acid region for Gβγ K. W. S. H. M.J. J. 1997; PubMed Scopus Google Scholar, J. X. X. 1998; PubMed Scopus Google Scholar, S. P. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). However, such of amino acids exists in RGS3, has two that bind The N-terminal domain of binds the of the overlapping the Gα whereas the N-terminal domain binds the of and first which may the of to the and receptor A. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). a for with for to T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the into the of the RGS3 does not possess a domain such as or receptor kinase through which it with Gβγ K. J. G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T. S. M.J. S. 1999; PubMed Scopus Google Scholar). the E. and RGS3 in their ability to Gβγ-mediated activation in purified Gβγ subunits in E. RGS3 the ability of Gβγ subunits to activate whereas the from mammalian cells the ability of Gβγ subunits to activate is that an in vivo of RGS3 whether it inhibits Gβγ effector such a and possess for with for to whereas does not T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Since RGS3 in the RGS3 is it to Gβγ-induced is that the RGS3 is Although to bind Gβγ in it is to Gβγ signaling, a of the is with proteins a of the regions in RGS3 for Gβγ-induced RGS3 with heterotrimeric Although we show that from COS-7 cell contain RGS3, Gαq Gαi from the cells The Gαq and Gαi contain as not than does the that RGS3 does not with or and is with RGS3 with or Gαi only after of the cell to N.O. Sorokin A. Reed E. Elliott S. Kehrl J.H. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google which the heterotrimeric G-proteins and the Gα subunits. these that RGS3 may with a of which is not bound Several the of an of Gβγ not with Gα Kozasa T. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar, J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). A of free Gβγ subunits has been to function in J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In the of RGS3 potently inhibits the activation of signaling pathways by Gβγ subunits. A of RGS3 located between amino acids which the RGS3 RGS domain, is to the RGS3 with Gβ1γ2 subunits, binds Gβγ and inhibits the activation of by Gβ1γ2 subunits. these that RGS3 can Gβγ signaling not only by virtue of its Gα GAP activity but also by directly interfering with Gβγ signaling. a mechanism may to the of RGS3 as an of chemokine-directed cell for and for and Dr. for

Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the Gα subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens.