The Inducibly Expressed GTPase Localizes to the Endoplasmic Reticulum, Independently of GTP Binding

The inducibly expressed GTPase (IGTP) is representative of a newly identified group of interferon γ-inducible GTPases, whose functions are currently unknown. We have begun to address the cellular function of IGTP by examining its subcellular distribution and its guanine nucleotide binding status. Using immunofluorescence, electron microscopy, and subcellular fractionation, IGTP was localized predominantly to the endoplasmic reticulum of both RAW 264.7 macrophages and C127 fibroblasts. In the immunostaining experiments, staining of discrete cytoplasmic structures on the periphery of the endoplasmic reticulum was also evident. Using polyethyleneimine-cellulose thin layer chromatography, the guanine nucleotides that complexed to immunoprecipitated IGTP, in both control and interferon γ-stimulated cells, were 90–95% GTP and 5–10% GDP, suggesting that the protein was in an active state. A mutant IGTP protein was created that had no detectable complexed GTP, and in both subcellular fractionation and IGTP-green fluorescent protein fusion studies, this mutant also localized to the endoplasmic reticulum. These results suggested that the GTP binding status of IGTP is independent of its capacity to localize to the endoplasmic reticulum. Given these results, we propose that IGTP is representative of a new family of endoplasmic reticulum GTPases that may be involved in protein processing or trafficking. The inducibly expressed GTPase (IGTP) is representative of a newly identified group of interferon γ-inducible GTPases, whose functions are currently unknown. We have begun to address the cellular function of IGTP by examining its subcellular distribution and its guanine nucleotide binding status. Using immunofluorescence, electron microscopy, and subcellular fractionation, IGTP was localized predominantly to the endoplasmic reticulum of both RAW 264.7 macrophages and C127 fibroblasts. In the immunostaining experiments, staining of discrete cytoplasmic structures on the periphery of the endoplasmic reticulum was also evident. Using polyethyleneimine-cellulose thin layer chromatography, the guanine nucleotides that complexed to immunoprecipitated IGTP, in both control and interferon γ-stimulated cells, were 90–95% GTP and 5–10% GDP, suggesting that the protein was in an active state. A mutant IGTP protein was created that had no detectable complexed GTP, and in both subcellular fractionation and IGTP-green fluorescent protein fusion studies, this mutant also localized to the endoplasmic reticulum. These results suggested that the GTP binding status of IGTP is independent of its capacity to localize to the endoplasmic reticulum. Given these results, we propose that IGTP is representative of a new family of endoplasmic reticulum GTPases that may be involved in protein processing or trafficking. Interferon γ (IFNγ) 1The abbreviations used are: IFNγ, interferon γ; ER, endoplasmic reticulum; GFP, green fluorescent protein; IGTP, inducibly expressed GTPase; PBS, phosphate-buffered saline; GTPγS, guanosine 5′-3-O-(thio)triphosphate; TLC, thin layer chromatography. is a pleiotropic cytokine that regulates a wide variety of immunological and inflammatory responses (for review see Ref. 1Farrar M.A. Screiber R.D. Annu. Rev. Immunol. 1993; 11: 571-611Crossref PubMed Scopus (1262) Google Scholar). While production of IFNγ is limited to T cells (2Vilcek J. Gray P.W. Rinderknecht E. Sevastopoulos C.G. Lymphokines. 1985; 11: 1-32Google Scholar) and natural killer cells (3Handa K. Suzuki R. Matsui H. Scimizu Y. Kumagi K. J. Immunol. 1983; 130: 988-992PubMed Google Scholar), its receptor is found on almost all cell types where it elicits diverse physiological responses. For example, in endothelial cells, IFNγ increases the expression of major histocompatibility class I molecules and the cell adhesion molecules, ICAM-1 and ELAM-1, thereby promoting recruitment of immune cells to areas of inflammation (4Munro J.M. Pober J.S. Cotran R.S. Am. J. Pathol. 1989; 135: 121-131PubMed Google Scholar). In macrophages, on the other hand, IFNγ induces expression of major histocompatibility class II molecules (5King D.P. Jones P.P. J. Immunol. 1983; 133: 313-318Google Scholar), thus increasing Fc receptor-mediated phagocytosis (6Warren M.K. Vogel S.N. J. Immunol. 1985; 134: 2462-2469PubMed Google Scholar) and mediating removal of neoplastic cells and parasitically and virally infected cells (7Liew F.Y. Li Y. Millott S. J. Immunol. 1990; 145: 4306-4310PubMed Google Scholar). In addition, IFNγ is thought to be involved in many pathological responses; in multiple sclerosis for instance, its production exacerbates disease symptoms (8Panitch H.S. Heraci R.I. Schindler J. Johnson K.P. Neurology. 1987; 37: 1097Crossref PubMed Google Scholar) and increases the relapse rate (9Paty D.W. Li D.K.B. the UBC MS/MRI Study Group the IFNβ Multiple Schlerosis Study Group Neurology. 1993; 43: 662-667Crossref PubMed Google Scholar). The potent immunomodulating properties of IFNγ have been utilized in a number of cancer treatment regimens that are currently used clinically or are under development. In particular, the interferons in combination with cytotoxic drugs have been quite effective in treating hairy cell leukemia (10Aulitzky W.E. Huber C. Peschel C. Barbacid M. Int. Arch. Allergy Immunol. 1993; 101: 221-226Crossref PubMed Scopus (21) Google Scholar) and chronic myelogenous leukemia (11Kurzrock R. Talpaz M. Kantarjian H.M. Walters R. Saks S. Trujillo J.M. Gutterman J.U. Blood. 1987; 70: 943-947Crossref PubMed Google Scholar). Preliminary results using IFNγ for treatment of renal cell carcinoma (12Brunda M.J. Luistro L. Hendrzak J.A. Fountoulakis M. Garotta G. Gately M.K. J. Immunother. Emphasis Tumor. Immunol. 1995; 17: 71-77Crossref PubMed Scopus (178) Google Scholar), breast carcinoma (13Ozello L. de Rosa C.M. Cantell K. Kauppinen H.L. Habiv D.V. J. Interferon Cytokine Res. 1995; 15: 839-848Crossref PubMed Scopus (11) Google Scholar), melanoma (14Kim T.S. Xu W.S. Sun T. Cohen E.P. Melanoma Res. 1995; 5: 217-227Crossref PubMed Scopus (26) Google Scholar), and glioma (15Lichtor T. Glick R.P. Kim T.S. Hand R. Cohen E.P. J. Neurosurg. 1995; 83: 1038-1044Crossref PubMed Scopus (54) Google Scholar) have also been promising. However, the molecular mechanisms that underlie these therapeutic effects as well as those underlying the normal physiological responses of IFNγ are poorly understood and are the subject of active investigation. The inducibly expressed GTPase (IGTP) is a recently identified 48-kDa protein whose expression is markedly up-regulated at the transcriptional level by IFNγ. In cultured macrophages and fibroblasts, IGTP mRNA accumulates within 1 h of exposure to IFNγ, reaching peak levels within 3 h and remaining at high levels for at least 48 h (16Taylor G.A. Jeffers M. Largaespada D.A. Jenkins N.A. Copeland N.G. Vande Woude G.F. J. Biol. Chem. 1996; 271: 20399-20405Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar); in the mouse, the mRNA is highly expressed in thymus, spleen, small intestine, and lung (16Taylor G.A. Jeffers M. Largaespada D.A. Jenkins N.A. Copeland N.G. Vande Woude G.F. J. Biol. Chem. 1996; 271: 20399-20405Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The IGTP protein, which contains three consensus GTP binding motifs, GXXXXGK(S/T), DXXG, and NTKXD (17Dever T.E. Glynias M.J. Merrick W.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1814-1818Crossref PubMed Scopus (467) Google Scholar), and inherent GTPase activity (16Taylor G.A. Jeffers M. Largaespada D.A. Jenkins N.A. Copeland N.G. Vande Woude G.F. J. Biol. Chem. 1996; 271: 20399-20405Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), is representative of an expanding group of closely related, IFNγ-regulated GTPases. Also included in this group of GTPases are the proteins encoded by the IRG-47 (18Gilly M. Wall R. J. Immunol. 1992; 148: 3275-3281PubMed Google Scholar), TGTP (19Carlow D.A. Marth J. Clark-Lewis I. Teh H.-S. J. Immunol. 1995; 154: 1734-1767Google Scholar)/Mg21 (20LaFuse W.P. Brown D. Castle L. Zwilling B.S. J Leukocyte Biol. 1995; 57: 477-483Crossref PubMed Scopus (78) Google Scholar), and LRG-47 (21Sorace J.M. Johnson R.J. Howard D.L. Drysdale B.E. J. Leukocyte Biol. 1995; 58: 477-484Crossref PubMed Scopus (62) Google Scholar) cDNAs. Although their expression patterns imply that they may be important mediators of IFNγ functions, the cellular and physiological functions of these proteins remain unknown. In the studies described here, we have begun to address the cellular function of IGTP by determining its subcellular localization, using a combination of immunostaining, electron microscopy, and subcellular fractionation procedures. We also examined the influence that the GTPase activity of the protein has on its subcellular localization, by first determining the guanine nucleotides complexed to the protein, and then examining the subcellular localization of a mutant protein that could no longer bind GTP. Based on our results, we propose that IGTP is representative of a family of IFNγ-regulated GTPases that localize to the endoplasmic reticulum (ER) and may be involved in protein processing or trafficking. The plasmid containing the 1.9-kilobase pair mouse IGTP cDNA in a Bluescript cloning vector (pBS) has been described previously (16Taylor G.A. Jeffers M. Largaespada D.A. Jenkins N.A. Copeland N.G. Vande Woude G.F. J. Biol. Chem. 1996; 271: 20399-20405Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). pBS/IGTP(S98N) was created by changing codon 98 from TCA to AAC, using a polymerase chain reaction overlapping primer mutagenesis technique (22Ho S.N. Hunt H.P. Horton D.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar). pBS/FLAG-IGTP and pBS/FLAG-IGTP(S98N) were created using the polymerase chain reaction to amplify a segment of pBS/IGTP or pBS/IGTP(S98N) so that the sequence encoding the FLAG amino acids DYKDDDDK would be inserted immediately downstream of the ATG start codon. This was accomplished using one primer that flanked the NcoI site at the ATG start codon (5′-GGCGCCATGGACTACAAGGACGACGACGACAAGGATTTAGTCACAAAGTTGCCACAAAATATCTGG-3′) and contained the sequence encoding the FLAG amino acids, and a second primer that flanked the PflMI site 460 bases downstream of the ATG (5′-CGGGCTAAAACAGGATATCAGG-3′); following amplification of this segment, theNcoI-PflMI-digested product was subcloned intoNcoI-PflMI-digested pBS/IGTP. The products were sequenced to verify the insertion. The FLAG-IGTP and FLAG-IGTP(S98N) SV40 promoter expression vectors were created by cloning the SpeI-EcoRI fragments of pBS/FLAG-IGTP and pBS/FLAG-IGTP(S98N) into theSpeI-EcoRI site of pSVZeo (Invitrogen, San Diego, CA). The plasmids encoding IGTP and IGTP(S98N) fused at their C termini to green fluorescent protein (GFP) were created using the polymerase chain reaction to amplify the coding regions of IGTP and IGTP(S98N) with primers (5′-AAAGCTAGCCGATTTAGTCACAAGTTGCCACAAAATATC-3′ and 5′-AAAGCTTAGCGTGAATTTCGGGAGGGAGGACAGAGTCCTT-3′) that containedNheI restriction sites. The NheI-digested products were then subcloned into the NheI site of pFRED25, 2G. Gaitanaris, R. Stauber, and G. Pavlakis, manuscript in preparation. a GFP expression plasmid containing a cytomegalovirus promoter and the bovine growth hormone polyadenylation signal. RAW 264.7 cells (ATCC TIB-71, American Type Culture Collection), C127 cells (ATCC CRL-1616), HeLa cells (ATCC CCL-2), and NIH/3T3 cells (ATCC were in with bovine and For expression plasmids were into cells using a G.A. M.J. W.S. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). For cells on of plasmid was in 1 of that was then to of for of the of used and the of the was C127 cells were on they cells were then for h to mouse interferon γ that was to the growth The cells were with in for with in for and with in for 1 The cells were then for with (16Taylor G.A. Jeffers M. Largaespada D.A. Jenkins N.A. Copeland N.G. Vande Woude G.F. J. Biol. Chem. 1996; 271: 20399-20405Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) to in by an with to in all the cells were with they were the cells were and the were using a the was with a with the for were using the green and were using a C127 and RAW cells were in and in for at with PBS, the cells were in a of and in and at 48 were then in and with The were three with for in bovine in for and then for h at with an previously with following with and one with bovine in PBS, the cells were for h at with the (16Taylor G.A. Jeffers M. Largaespada D.A. Jenkins N.A. Copeland N.G. Vande Woude G.F. J. Biol. Chem. 1996; 271: 20399-20405Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) previously with the second the cells were with for with for and then with for cells were with a the to that staining was were and were with a electron protein were from RAW 264.7 and C127 cells using a previously fractionation J. J. Biol. 1993; PubMed Scopus Google Scholar). of the were at and and were to all In the cells were three with PBS, from the by with cells and then by at and for The cells were in of a 1 and at for the cells were in 1 of and on for The of the cells were with of a the of the was by The was then of by at for The was in 1 of and to with a the were by at the was and the was with the other was to the to a of and this was at for The was on a of of and in the was at for h at in an CA). were then as described under and the of protein in was using the protein protein and were as described previously (16Taylor G.A. Jeffers M. Largaespada D.A. Jenkins N.A. Copeland N.G. Vande Woude G.F. J. Biol. Chem. 1996; 271: 20399-20405Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The (16Taylor G.A. Jeffers M. Largaespada D.A. Jenkins N.A. Copeland N.G. Vande Woude G.F. J. Biol. Chem. 1996; 271: 20399-20405Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) was used at a for and a for The mouse was used at for The was used at a for The was used for were protein was immunoprecipitated (16Taylor G.A. Jeffers M. Largaespada D.A. Jenkins N.A. Copeland N.G. Vande Woude G.F. J. Biol. Chem. 1996; 271: 20399-20405Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), and the complexed guanine nucleotides were by polyethyleneimine-cellulose thin layer to previously described Vogel J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). A of and was used for cell and a of the (16Taylor G.A. Jeffers M. Largaespada D.A. Jenkins N.A. Copeland N.G. Vande Woude G.F. J. Biol. Chem. 1996; 271: 20399-20405Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) was used for A control of was in with immunoprecipitated IGTP, as described previously (16Taylor G.A. Jeffers M. Largaespada D.A. Jenkins N.A. Copeland N.G. Vande Woude G.F. J. Biol. Chem. 1996; 271: 20399-20405Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), to and this was in to the fluorescent in the to the of GTP and on the GTP and were with a were and with and h the cells were using a in the with an at the subcellular distribution of IGTP, C127 mouse were to control or to IFNγ for h to expression of the protein, and the cells were used for immunostaining with an this had been the amino acids of IGTP and previously was to a 48-kDa in both and (16Taylor G.A. Jeffers M. Largaespada D.A. Jenkins N.A. Copeland N.G. Vande Woude G.F. J. Biol. Chem. 1996; 271: 20399-20405Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). In C127 cells, immunostaining was localized to the and was in the on structures a of The staining was in control and cells and following IFNγ as a of IGTP protein the staining was RAW 264.7 mouse macrophages were with the These results suggested that IGTP was localized to the in these cells in addition, that IFNγ the of IGTP and the subcellular The localization of IGTP in C127 and RAW cells was also examined by electron using the and an that a protein J.M. L. C. S. 1995; PubMed Scopus Google Scholar). In both RAW cells and C127 cells IGTP and on In the IGTP structures whose could be see also These structures to be of the it is that they were that had of the of and in HeLa HeLa cells were with an or expression h the cells were for protein using a in the with the at are representative of cells for expressed that the GFP fusion protein staining is to the staining of the protein in cells Also the in the The subcellular distribution of IGTP was also examined by and protein from RAW cells, using a previously J. J. Biol. 1993; PubMed Scopus Google Scholar). A cell was from RAW 264.7 cells in a containing and this was on of and and at were from the of the with 1 and from the 3 from the and from the and from the has been previously using that for in this 1 and and protein, 3 contains protein, and and protein J. J. Biol. 1993; PubMed Scopus Google Scholar). For the was to with the and control that the protein and the endoplasmic reticulum protein as was in and was in and IGTP was predominantly in with in a in and detectable in 1 and In using C127 fibroblasts, IGTP the distribution These results those of the immunostaining and suggested that the of IGTP in macrophages and was in the was to the endoplasmic reticulum. IGTP with the the that the protein could have been from these This the of a sequence at the IGTP (16Taylor G.A. Jeffers M. Largaespada D.A. Jenkins N.A. Copeland N.G. Vande Woude G.F. J. Biol. Chem. 1996; 271: 20399-20405Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), the small of IGTP in protein and the of IGTP the cell periphery However, the was by of protein from the of RAW cells to IFNγ for to h Although IGTP was in RAW cell no IGTP was in the cell IGTP was to GTP binding motifs, as well as GTPase activity (16Taylor G.A. Jeffers M. Largaespada D.A. Jenkins N.A. Copeland N.G. Vande Woude G.F. J. Biol. Chem. 1996; 271: 20399-20405Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar); the distribution of guanine nucleotides that were complexed to the protein in was address the guanine nucleotide binding status of IGTP, were used to protein from RAW cells to IFNγ or control and the nucleotides that were complexed to protein were then by thin layer control of the detectable guanine nucleotides complexed to IGTP were GTP, and the remaining were IFNγ the of guanine nucleotides with the of IGTP protein and the of GTP complexed to IGTP the at many as M. Annu. Rev. 1987; PubMed Scopus Google Scholar), are in an active they are in the these results suggested that the of IGTP in these cells was in an active that IFNγ influence the activity of the a control were from NIH/3T3 cells, and the guanine nucleotides to immunoprecipitated were also using thin layer of the guanine nucleotides were GDP, which was in with previously results Vogel J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). This that was under the and it suggested that of levels of IGTP was a of are in which the GTP binding status of IGTP could influence its one is that the of IGTP for the be by its GTP binding state. is of the GTPases, which are in the (for see Biol. PubMed Scopus Google Scholar). the of the GTP binding of IGTP, a mutant IGTP protein was created by 98 in the first GTP binding to an in the of the the is involved in the U. K. J. A. 1989; PubMed Scopus Google Scholar), and to an a protein with a for GTP Proc. Acad. Sci. U. S. A. 83: PubMed Scopus Google Scholar). A FLAG amino was also at the amino of the mutant IGTP(S98N) protein so that expressed protein could be from the the GTP binding properties of FLAG-IGTP and the proteins were expressed in NIH/3T3 cells, protein was immunoprecipitated with the and the complexed guanine nucleotides were by thin layer For the guanine nucleotide distribution that of the protein with of the guanine nucleotides GTP and the remaining and the small of were exposure of the to they were detectable with a small of were also complexed to FLAG-IGTP(S98N) no GTP levels was results were the FLAG-IGTP and FLAG-IGTP(S98N) were into HeLa cells and in all levels of expressed FLAG-IGTP and FLAG-IGTP(S98N) protein were the and were using an to protein, and in these studies, the of GTP and complexed to FLAG-IGTP and FLAG-IGTP(S98N) were the as those using the of FLAG-IGTP and FLAG-IGTP(S98N) in subcellular HeLa cells were with a FLAG-IGTP or FLAG-IGTP(S98N) expression vector and then used for of subcellular as described in the to of protein from were used for with an and with the The of the molecular is at the are described under the IGTP localization to the ER, FLAG-IGTP and FLAG-IGTP(S98N) were expressed in HeLa cells, protein were and the expressed proteins were by with an and with FLAG-IGTP and FLAG-IGTP(S98N) were almost in with small in These results that the GTP complexed to IGTP had no on the protein to the they that within the are by GTP. of the that FLAG-IGTP and FLAG-IGTP(S98N) were also in and The of these was they may have been IGTP that the FLAG and was with or they may have been that were by the The localization of IGTP(S98N) was also examined in cells by IGTP-green fluorescent protein fusion proteins in HeLa cells The and patterns were to the immunostaining of the IGTP protein with staining that into the to the cell the periphery of the ER, structures also staining for both and The of the structures could be they may have been of or of the that were These results suggested that localization of IGTP to the were that the of complexed guanine nucleotides on the binding of IGTP to In one in IGTP was with GDP, GTP, or and then with in the of so that the IGTP GTPase would be or in the of so that the GTPase would be were in which the of other proteins with as the of with J.A. S. J. Biol. 1993; PubMed Scopus Google Scholar), had been in However, under the of IGTP that to the the In a second RAW cells were used for of that were thus with the were then in containing GTP, GDP, or and or under of the was IGTP from the these results the that binding of IGTP to is by complexed GTP. Using a combination of and cellular fractionation we have that the of IGTP in macrophages and is to the endoplasmic and is in the protein In addition, the protein is predominantly is in an active state. However, it that with GTP regulates with the in our that could small of the protein within the Based on these we propose that IGTP is representative of a new family of GTPases that could be involved in protein processing or trafficking. The of the of IGTP with the endoplasmic reticulum to be as a sequence that is and for their in the Annu. Rev. Biol. 1989; 5: PubMed Scopus Google Scholar), IGTP this of IGTP to be with and with the of which that may the However, an is that IGTP is with a as a or that binding of the protein to the IGTP both a sequence 1989; 57: Full Text PDF PubMed Scopus Google Scholar) and an sequence D.A. E. L. J. Biol. Chem. Full Text PDF PubMed Google Scholar), the of a is In the fusion protein studies and also in the electron studies, we structures that were for IGTP, the of these structures could be they were on the periphery of the ER, one is that they were structures that had of the and were involved in from the to the or to other cellular this were the then the of of IGTP in the and that IGTP from these they their of these structures be important in determining the function of are many of GTPases that with the or and are involved in protein processing or trafficking. in these proteins are the a group of small GTPases that are and to their Biol. PubMed Scopus Google Scholar), and the a group of at least proteins that binding of proteins to (for see Ref. R.D. Biol. PubMed Scopus Google Scholar). These are IGTP and no sequence to GTPase that limited to IGTP is the of the that with a sequence to the Johnson Annu. Rev. Biol. PubMed Scopus Google Scholar); it is to IGTP at the amino a the studies on has a function to that of IGTP or IGTP and its a class of and GTPases. We for