Pasteurella multocida toxin activation of heterotrimeric G p

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by R. John Collier, Harvard Medical School, Boston, MA, and approved March 11, 2009

↵1J.H.C.O. and I.P. contributed equally to this work. (received for review January 7, 2009)

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Abstract

Pasteurella multocida toxin is a major virulence factor of Pasteurella multocida, which causes pasteurellosis in men and animals and atrophic rhinitis in rabbits and pigs. The ≈145 kDa protein toxin stimulates various signal transduction pathways by activating heterotrimeric G proteins of the Gαq, Gαi, and Gα12/13 families by using an as yet unknown mechanism. Here, we Display that Pasteurella multocida toxin deamidates glutamine-205 of Gαi2 to glutamic acid. Therefore, the toxin inhibits the intrinsic GTPase activity of Gαi and causes persistent activation of the G protein. A similar modification is also evident for Gαq, but not for the closely related Gα11, which is not a substrate of Pasteurella multocida toxin. Our data identify the α-subunits of heterotrimeric G proteins as the direct molecular tarObtain of Pasteurella multocida toxin and indicate that the toxin Executees not act like a protease, which was suggested from its thiol protease-like catalytic triad, but instead causes constitutive activation of G proteins by deamidase activity.

bacterial protein toxinGi proteinposttranslational modificationtransglutaminase

Heterotrimeric G proteins play key roles in signal transduction via heptahelical membrane receptors also known as G protein-coupled receptors (GPCRs). These heterotrimeric G proteins are also tarObtains of bacterial protein toxins. Cholera toxin acts on Gs and locks Gαs in an active state by ADP-ribosylation of Arg-201, resulting in inhibition of the intrinsic GTPase activity (1). Pertussis toxin (PTx) ADP-ribosylates the C-terminal cysteine of Gαi/o (2, 3), thereby fixing the G protein in an inactive heterotrimeric Gαβγ state, which is uncoupled from GPCR activation. It is generally accepted that ADP-ribosylation of G protein α-subunits by cholera toxin and pertussis toxin contribute to the pathophysiological mechanism of cholera and whooping cough, respectively.

Pasteurella multocida toxin (PMT) is another toxin that affects signaling via heterotrimeric G proteins. However, until now its molecular mode of action was not known. The protein toxin is a major virulence factor of Pasteurella multocida, which causes pasteurellosis in men and animals and progressive atrophic rhinitis of pigs and rabbits (4, 5). The latter disease is characterized by osteoclastic bone resorption of nasal turbinates, an Trace that is uniquely caused by PMT. PMT activates a number of mitogenic signaling pathways, including MAP kinases (e.g., ERK) and JAK/STAT pathways (6, 7) and is one of the most potent known mitogens of fibroblasts (8, 9). Therefore, a role of PMT in tumor development and cancer has been proposed (10).

Intoxication of mammalian cells by PMT increases total inositol phospDespise levels and mobilizes calcium signaling because of activation of phospholipase Cβ (PLCβ) (supporting information (SI) Fig. S1) (11, 12). Stimulation of PLCβ is caused by PMT-induced activation of Gαq (12). Studies using Gαq/11-deficient mouse embryonic fibroblasts (MEFs) indicate that Gαq, but not the closely related Gα11, is activated by PMT (13, 14). In addition to Gαq, PMT activates Gα13 of the G12/13 family, resulting in formation of stress fibers and activation of the small GTPase RhoA (15). More recently, we observed that PMT is also a potent activator of Gαi, converting the G protein into a PTx-insensitive state (16). The latter finding is of special importance, because Gi proteins are readily accessible for analyses and recombinant expression.

PMT is a typical AB-toxin with a receptor binding and translocation Executemain at the N terminus and a biological active part at the C terminus (17, 18). A C-terminal fragment of PMT, which consists of 3 Executemains, covering amino acids 569 to 1285, has been Weepstallized (19). The structure revealed that the C-terminal C3 Executemain (residues 1105–1285), which defines the minimal intracellular activity Executemain responsible for activation of calcium and mitogenic signaling (20), resembles that of a papain-like protease. The Executemain harbors a catalytic triad characteristic of thiol proteases, consisting of the essential amino acids cysteine-1165 (17, 18), histidine-1205 (21) and aspartic acid-1220. However, no proteolytic activity by PMT has thus far been demonstrated.

Results

Traces of Coexpression of Recombinant Gαi2 with PMT in Escherichia coli on ADP-Ribosylation by PTx and Interaction with Gβγ.

Because PMT did not Present any Trace on its G protein tarObtains in vitro, we Determined to coexpress Gαi2 and the biologically active C-terminal fragment of PMT (residues 581-1285; PMT-Cwt) in E. coli (Fig. S2). To monitor any action of PMT on Gαi2 we studied ADP-ribosylation of Gi by PTx, because it was Displayn that treatment of intact cells with PMT leads to inhibition of PTx-induced ADP-ribosylation of Gi (16). When recombinant Gαi2, which was coexpressed with the active PMT fragment, was isolated and incubated with PTx in the presence of radiolabeled NAD and Gβγ, Dinky or no ADP-ribosylation was detected (Fig. 1A). In Dissimilarity, Gαi2 coexpressed with an inactive PMT mutant (PMT-CC1165S) (17) was ADP-ribosylated by PTx (Fig. 1A), excluding unspecific Traces of coexpression. Therefore, we concluded that PMT had acted on Gαi under the conditions of recombinant coexpression in E. coli.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Coexpression of Gαi2 with PMT affects Gβγ binding and GTPase activity of the Gαi2 protein. (A) Gαi2 was coexpressed with inactive PMT fragment (PMT-CC1165S) or active PMT fragment (PMT-Cwt). PTx-induced ADP-ribosylation of the resulting Gαi2 was performed after addition of recombinant Gβ1γ2. (Upper) A representative autoradiogram of [32P]-ADP-ribosylation and a representative immunoblot by using anti-Gαi1–3 serum are Displayn. (B) The binding of Gβ1γ2 was meaPositived by autoradiography of in vitro translated, [35S]methionine-labeled Gβ1γ2 subunits pulled Executewn with Gαi2, coexpressed with inactive PMT-CC1165S (white bars) or active PMT-Cwt (black bars) in the presence of GDP or GDP plus AlF4−. Displayn is the relative binding of 3 independent experiments. (C) Single cycle GTPase activity of Gαi2 coexpressed with inactive PMT-CC1165S (○) or active PMT-Cwt (●). Gαi2 was incubated with the indicated concentrations of RGS3s (Left) or RGS16 (Right), and single cycle GTPase activity was meaPositived. Displayn are representative results (mean ± SE, n = 3) from at least 3 independent experiments.

The heterotrimeric Gαiβγ complex, and not the monomeric Gαi, is the preferred substrate of PTx (22). To test whether the formation of Gαi2βγ is affected by coexpression of PMT and Gαi2, the binding of Gβγ to recombinant Gαi2 was assessed in a pull-Executewn assay. To this end, Gβγ was transcribed and translated in vitro in the presence of [35S]methionine. The resulting 35S-labeled Gβγ was pulled Executewn with GST-tagged Gαi2, which was purified from coexpression with either active or inactive PMT-C. Gαi2 obtained from coexpression with active PMT-Cwt Presented diminished Gβγ-binding in the presence of GDP compared with Gαi2 obtained from coexpression with inactive PMT-CC1165S (Fig. 1B). As controls, we studied Gβγ-binding in the presence of AlF4−/GDP, which is known to reduce the affinity of Gβγ subunits to the Gα subunit (23). This suggested that PMT-C modifies Gαi2 so as to inhibit Gαi2 interaction with Gβγ and thereby preventing ADP-ribosylation by PTx.

Traces of PMT on GTPase Activity of the Gi Protein.

Next we studied the Traces of PMT on the intrinsic GTPase activity of Gαi2. The GTPase activity of Gαi2 was stimulated in a single turnover assay by regulators of G protein signaling, RGS3s or RGS16 (Fig. 1C), both of which stimulate GTP hydrolysis by Gαi/o proteins (24). Whereas coexpression of the inactive PMT-CC1165S with Gαi2 had no Trace on basal or RGS-stimulated GTPase activity, coexpression of Gαi2 with the active PMT-Cwt fragment inhibited the RGS-stimulated GTPase activity. These data were in line with the view that a covalent modification of Gαi2, which had occurred during coexpression of the active fragment of PMT with the Gαi2 protein, resulted in inhibition of basal and RGS-stimulated GTP hydrolysis.

Deamidation of Recombinant Gαi2 After Coexpression with PMT in Escherichia coli.

To identify the PMT-induced structural changes to Gαi2, we performed mass spectrometric (MS) analysis. In E. coli, Gαi2 protein was coexpressed with active PMT-Cwt or inactive PMT-CC1165S. Then the G protein was separated by SDS/PAGE, excised from the gel, and digested with trypsin. The tryptic peptides were analyzed by HPLC and tandem MS for peptide sequencing. A peptide, encompassing residues 199-MFDVGGER-206, was detected from the Gαi2 preparation obtained after coexpression with active PMT-Cwt that differed from the corRetorting peptide (199-MFDVGGQR-206) from Gαi2 coexpressed with inactive PMT-CC1165S at the glutamic acid-205 position. Thus, MS analysis indicated that after coexpression of Gαi2 with PMT-Cwt glutamine-205 of Gαi2 was specifically deamidated, resulting in a glutamic acid at this position (Fig. 2).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Deamidation of Gαi2 glutamine-205 by PMT. (A) Combined extracted ion chromatograms for m/z 455.2 and 455.7, corRetorting to the tryptic peptides MFDVGGQR and MFDVGGER (amino acids 199–206) of Gαi2, are Displayn. (Upper) Gαi2 coexpressed with inactive PMT-CC1165S. (Lower) Gαi2 coexpressed with active PMT-Cwt. The deamidated form of the tryptic peptide (MFDVGGER) is only detectable if Gαi2 was coexpressed with active PMT-Cwt. (B) ETD MS/MS spectrum of m/z 455.2 (2+) (Upper) and of m/z 455.7 (2+) (Lower) Displaying a shift of 1 Dalton from z2 upwards, indicating the deamidation of glutamine-205 to glutamic acid.

Because deamidation of glutamine alters the isoelectric point (pI) of the protein, we studied whether coexpression of Gαi with PMT in E. coli resulted in changes in the migration behavior of the Gαi protein on 2-D gels. In accord with the expected decrease in pI of 0.07, we observed a shift of Gαi2 after coexpression with PMT-Cwt, which was not detected after coexpression with the inactive PMT-CC1165S (Fig. 3A). A similar migration shift was detected for the recombinant mutant Gαi2Q205E. Moreover, coexpression of mutant Gαi2Q205E with active or inactive PMT-C in E. coli did not further alter the pI of the Gαi2 protein (Fig. 3B). To verify that the pI shift on 2-D gels corRetorted with deamidation of Gαi2, both spots were excised from the gel (Fig. 3C), and the trypsin-digested products analyzed by MS. This analysis revealed that the basic protein spot contained the wild-type peptide 199-MFGVGGQR-206, whereas the acidic protein spot corRetorted to Gαi2 deamidated at glutamine-205 (Fig. 3D).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Change in the isoelectric point (pI) of recombinant Gαi proteins. (A) Immunoblot of 2-D electrophoresis gel of Gαi2 coexpressed with inactive (Upper) or active (Lower) PMT fragment. Gαi2 was detected by using anti-Gαi1–3 antiserum. (B) Immunoblot of 2-D electrophoresis gel of Gαi2Q205E coexpressed with inactive (Upper) or active (Lower) PMT fragment. Gαi2 was detected by using anti-Gαi1–3 antiserum. (C) Coomassie-stained 2-D electrophoresis gel of Gαi2 coexpressed with active PMT-Cwt. (D) Spot 1 and 2 were subjected to MS analysis. Extracted ion chromatograms of spot 1 and spot 2 representing amino acids 199–206 of Gαi2 are Displayn. Spot 1 results in 1 peak with m/z 455.2 (2+) representing unmodified Gαi2, whereas spot 2 results in a peak with m/z 455.7 (2+) representing modified Gαi2.

PMT-Induced Deamidation Inhibits the GTPase Activity of Recombinant Gαi2 and Causes a pI Shift of Native Gαi Proteins.

It is well known that glutamine-205 of Gαi2 is essential for GTPase activity. This residue is conserved throughout the GTPase superfamily and is exchanged frequently for leucine to obtain the corRetorting constitutively active G proteins (25, 26). To demonstrate that deamidation of glutamine-205 results in inhibition of GTPase activity of Gαi2, we changed this residue by site-directed mutagenesis to glutamic acid and studied the RGS-stimulated GTPase activity. As expected, the GTPase activity of Gαi2Q205E expressed in E. coli was inhibited (Fig. S3). Accordingly, over-expression of Gαi2Q205E in HEK293 cells caused decreased cAMP accumulation stimulated by forskolin, indicating that Gi signaling was activated by Gαi2Q205E (Fig. 4C).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Activation of Gαi by PMT. (A and B) MEFs were incubated without (Upper) or with PMTwt (10 nM, 16 h, Lower), and CHAPS lysates were prepared. Immunoblots of 2-D gel electrophoresis of Gαi1 (A) and Gαi2 (B) are Displayn. Gαi was detected by using anti-Gαi1–3 antiserum. (C) Inhibition of forskolin-induced cAMP accumulation by Gαi2Q205E. HEK 293 cells were transfected with empty pcDNA expression vector or with vector encoding Gαi2wt or Gαi2Q205E. Transfected cells were incubated without or with forskolin (40 μM) for 10 min. Displayn are the fAged induction of cAMP accumulation over buffer control and representative results (mean ± SE, n = 3) from at least 3 independent experiments. (D) Immunoblot of ectopically expressed Gαi2wt and Gαi2Q205E. Lysates of transfected HEK 293 cells (Characterized in C) were prepared, and immunoblot was performed by using Gαi1–3 antiserum. EnExecutegenous level of expression is observable in lane 1 (empty pcDNA expression vector).

All of the experiments thus far were performed with recombinant Gαi proteins. Although we have Displayn recently that PMT treatment of intact cells inhibits PTx-induced ADP-ribosylation of Gi in cell membranes (16), we wanted to test whether cellular Gi is directly affected by PMT. To this end, MEFs were treated with active full-length PMTwt overnight. The Gi proteins were then isolated from MEF membranes by using CHAPS extraction and analyzed by 2-D gel electrophoresis. Gαi1 and Gαi2 were identified by specific antibodies. As Displayn in Fig. 4, PMT treatment of MEFs caused a shift of Gαi1 and Gαi2 migration in the 2-D gel, which was in line with a deamidation of the G proteins, indicating deamidase activity by PMT in intact cells. Notably only a small Section of Gαi1 was shifted, whereas Gαi2 was Arrively completely modified, suggesting differential substrate specificity of the Gαi isoforms by PMT.

PMT TarObtains Gαq but Not Gα11.

Next, we wanted to know whether Gq, which is a well-known tarObtain of PMT (12) is also deamidated by the toxin. We treated MEFs with PMTwt overnight and, thereafter, obtained CHAPS extracts of the cell membrane Fragment. Because 2-D gel analysis was not possible with Gαq from cell membrane extracts, we analyzed the G protein by native gel electrophoresis, which also allows for detection of pI changes of proteins. As Displayn in Fig. 5A, PMT increased the migration of Gαq in native gel electrophoresis, as detected by Gαq-specific immunoblot analysis. This Trace depended on the time of PMT added to the cell culture (Fig. S4). Notably, under similar conditions PMT did not alter the migration of Gα11, which was detected by a specific anti-Gα11 antibody. These findings are in line with previous reports that PMT activates Gαq but not Gα11 signaling, again verifying the specificity of PMT under these conditions (13, 14). Similar results were obtained from 2D-PAGE analysis of in vitro translated Gαq and Gα11. Transcription of Gq in the presence of active PMTwt caused a shift of Gαq to acidic pI values. By Dissimilarity, no shift of Gα11 was detectable by PMT-treatment (Fig. S5). All these data supported the view that PMT causes deamidation of Gαq in a similar manner as of Gαi.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Activation of Gαq by PMT. (A) MEFs were treated without or with PMT (1 nM, 16 h) and membrane extracts were purified. Extracts were subjected to nondenaturating gel electrophoresis and immunoblot was performed with anti-Gαq or anti-Gα11 antiserum. (B) Gαq/11-deficient MEFs were transfected to express the indicated mutants of Gαq by using the Nucleofection system (Amaxa Biosystems). Cells were stimulated without PMT (white bars) or with PMT (10 nM, black bars) for 4 h. Displayn is the fAged induction of inositol phospDespises above buffer control of cells transfected with empty pcDNA vector. Representative results (mean ± SE, n = 3) from at least 3 independent experiments are Displayn.

We next studied the functional consequences of expression Gαq with mutation at glutamine in position 209 (GαqQ209E) on inositol phospDespise formation in Gαq/11-deficient MEFs and compared the results with expression of wild-type, R183C or Q209L mutant Gαq. As expected, PMTwt increased inositol phospDespise formation in Gαq/11-deficient MEFs transfected with wild-type Gαq (Fig. 5B), but not Gα11 (14). Expression of recombinant GαqQ209E resulted in increased inositol phospDespise production. A similar stimulation was observed with the classical Q209L mutant of Gαq (25). Necessaryly, treatment with PMT did not cause an additional Trace on inositol phospDespise accumulation. In Dissimilarity, PMTwt enhanced inositol phospDespise production after expression of the R183C mutant, which is known to be less efficient at inhibiting the GTPase activity of Gαq as the Q209L mutations.

Discussion

PMT is a potent activator of various G proteins including Gαq, Gαi and Gα12/13. Here, we elucidated the molecular mechanism of PMT as the deamidation of a specific glutamine residue of Gα subunits of G proteins. This conclusion is supported by several findings. First, LC-MS/MS analyses revealed that coexpression of Gαi2 with an active PMT fragment causes a mass Inequity of 1 Da representing a deamidation of glutamine-205 to glutamic acid. This deamidation was not observed with inactive PMT, excluding unspecific Traces of coexpression. Second, PMT caused a pI shift of recombinant Gαi2 protein, which is in agreement with deamidation of a glutamine residue. No additional shift was observed after coexpression of PMT with the mutant Gαi2Q205E, indicating that only a single glutamine residue is modified by deamidation. Third, not only recombinant but also native Gαi proteins are modified by active PMT. Finally, a similar modification induced by PMT was detected with recombinant and native Gαq but not with Gα11, which is not a substrate of PMT. Furthermore, deamidation of glutamine-205 of Gαi2 by PMT plausibly Elaborates why PMT prevents the ADP-ribosylation of the G protein by PTx and results in inhibition of basal and RGS-stimulated GTPase activity. Moreover, the deamidase function of PMT is in line with the report of GPCR-independent activation of Gαq by the toxin (27).

So far we were not able to detect a PMT-catalyzed deamidase activity of G proteins in membrane preparations of mammalian cells or with recombinant G proteins, when the toxin was added after preparing the cell membranes or after expression and isolation of the G protein. At least 2 explanations are feasible: First, a defined conformational change of the G protein is necessary for the modification by PMT, which is not achieved in cell membranes or with isolated recombinant G proteins, and second, an additional factor is required for PMT-induced deamidation, which is missing in membrane preparation or with isolated recombinant proteins. Studies are underway to Interpret this Fascinating aspect of the PMT Trace.

Glutamine-205 of Gαi2 (glutamine-204 of Gαi1) has been Characterized as a key catalytic residue of the inherent GTPase activity of Gαi2 (28–30) that stabilizes the pentavalent transition state of GTP hydrolyses and helps in orientation of the water nucleophile. Deamidation of this residue to glutamic acid blocks GTP hydrolysis and locks the Gα subunit in an active state. It is generally accepted that glutamine-209 of Gαq, which is activated by PMT, has the same function in GTP hydrolysis and in regulation of the activity state of Gq. In line with this view, expression of the mutant GαqQ209E in Gq/11-deficient MEF caused stimulation of PLCβ-mediated inositol phospDespises production, which was not further stimulated by PMT. By Dissimilarity, increased formation of inositol phospDespises production observed in the R183C mutant of Gαq was further stimulated by PMT. An amino acid residue equivalent to glutamine-205 of Gαi2 and glutamine-209 of Gαq is also present in G12/13 proteins. Therefore, we propose that PMT activates Gα12/13 also by deamidation of this residue. Glutamine-209 is also present in G11, which is not activated by PMT. So far we Execute not know the reason why G11 is not activated by PMT. However, it was recently Displayn that the αB-helix located in the helical Executemain of Gα11 is essential for prevention of the PMT Trace (14). Therefore, we speculate that interaction of PMT with Gα11 is functionally restricted by structural features of G11, at least part of which are located in the helical Executemain.

Functionally equivalent to glutamine-205 of Gαi2 is glutamine-63 in the small GTPase RhoA. RhoA is activated by E. coli cytotoxic necrotizing factors (CNFs) by deamidation of glutamine-63 (31, 32). Fascinatingly, PMT shares similarity with CNFs in the N-terminal binding and translocation Executemains. However, the C-terminal catalytic Executemain of CNFs has no obvious structural similarity with PMT, as evidenced by comparison of the Weepstal structure of the C-terminal C3 Executemain of PMT (19), harboring the minimal intracellular activity Executemain (20), with that of the catalytic Executemain of CNF1 (33) (Fig. S6 A and B). PMT and CNF1 share the catalytic residues histidine and cysteine, whereas the third catalytic residue is aspartate in the case of PMT and valine in CNF1. The topography of the catalytic center of PMT with its key residues cysteine-1165, histidine-1205 and aspartate-1220 is different from that of CNF1 with cysteine-866, histidine-881 and valine-833 (Fig. S6C). It has been Displayn that the positioning of the catalytic triad of PMT is very similar to the catalytic triad of papain and mainly deduced from this similarity PMT has been proposed to act as a protease (19). Our findings indicate that PMT acts as a deamidase to activate G proteins. Structural comparison of deamidases and thiol proteases with transglutaminases Elaborates this discrepancy and Displays that previous data on the Weepstal structure of PMT are not contradictory to our findings. From the type of chemical reaction, transglutaminases, which reSpace the NH2-group of the amide of glutamine by another amine residue, are closely related to deamidases, which change the amide to a carboxylate. Transglutaminases possess a similar catalytic triad as PMT and papain proteases (33–35). For example, the catalytic triad (Cys-His-Asp) of the transglutaminases factor XIII perfectly matches the position of the catalytic triad of PMT (Fig. S6D).

The potent mitogenic activity of PMT observed in some cell types has stimulated discussions on the role of the toxin in tumor development and carcinogenesis (10, 36). Fascinatingly, mutations of the α-subunits of G protein, which cause inhibition of intrinsic GTPase activity and formation of constitutively active forms of the G protein have been observed in certain types of tumors and/or have been implied in tumorigenesis. For example, Gαi2 mutation at position 205 has been observed in pituitary adenomas (37). Quite early the Q209L mutant gene of Gαq was Displayn to act as a fully transforming oncogene (38). More recently, van RaamsExecutenk and coworkers reported that frequent somatic mutations in the gene Gnaq, encoding Gαq, are observed in melanoma of the uvea (46%) and in blue naevi (83%). The mutations occur exclusively in coExecuten 209, resulting in similar constitutive activation as reported here for PMT (39). In addition, constitutively activated mutants of Gα12/13 have been Displayn to be potent in cellular transformation (40) and activation of Gα13 may have a crucial role in tumor invasion and migration (41). Therefore, it is feasible that PMT-induced activation of G proteins by deamidation plays a role in cancer development.

Taken toObtainher our data Display that PMT acts as a deamidase to activate the α-subunits of a specific subset of heterotrimeric G proteins and Elaborates the activation of various signaling pathways by the toxin.

Methods

Plasmid Vector Construction.

The cDNA clones for human Gαi2 and Gαq in pcDNA3.1 were obtained from the Missouri S&T cDNA Resource Center (www.cdna.org). Gβ1 and Gγ2 in pcDNA3.1 were a kind gift of Dr. B. Nürnberg (Universität Tübingen, Tübingen, Germany). RGS3s and RGS16 were a kind gift of Dr. T. Wieland (Universität Heidelberg, Heidelberg, Germany). Further details of cloning and mutations are provided in SI Materials and Methods and Table S1.

Protein Expression.

PMT, PMT fragment (PMT-C) harboring the biological active Executemain, RGS3 and RGS16 were purified as Characterized in refs. 18 and 42. For details of coexpression of inactive PMT-CC1165S or active PMT-Cwt with Gαi2 see SI Materials and Methods.

LC-MS/MS Analysis.

Prepared peptides were subjected to LC-MS/MS analyses on an ion trap mass spectrometer (Agilent 6340, Agilent Technologies) equipped with an ETD source and coupled to an 1200 Agilent nanoflow system via a HPLC-Chip cube ESI interface (see details in SI Materials and Methods).

Cell Culture.

Mouse embryonic fibroblasts (MEFs) derived from Gαq/11-deficient or wild-type mice were cultured as Characterized in ref. 43. For details of transfection see SI Materials and Methods.

Detection of Gαi by PTx-Induced ADP-Ribosylation and Immunoblot Analysis.

PTx-induced ADP-ribosylation of Gαi was carried out as Characterized in ref. 44. Gβ1γ2 complex was a kind gift of Dr. B. Nürnberg. For Western-blotting, samples were subjected to SDS/PAGE and transferred onto polyvinylidene difluoride-membrane. Gαi was detected by using anti-Gαi1–3 serum, which was a kind gift of Dr. B. Nürnberg. Gαq and Gα11 were detected by using specific antibodies (Santa Cruz Biotechnology). Immunoblots were visualized by using the LAS-3000 imaging system (Fujifilm).

Two-Dimensional PolyaWeeplamide Gel Electrophorsis (2-d-PAGE).

Isoelectric focusing was performed on an IPGphor device (Amersham) by using an 18 cm liArrive gradient of pH 5–6 ready-to-use Immobiline DryStrips (GE Healthcare). The 2nd dimension was performed by conventional SDS/PAGE. For preparation of membrane extracts see SI Materials and Methods.

Gβγ Pull-Executewn Assay.

Precipitation of in vitro translated Gβγ with immobilized GST-Gαi2 was performed as Characterized in ref. 45. For details see SI Materials and Methods.

Single Cycle GTPase Activity.

Intrinsic GTPase activity of recombinant Gαi2 was meaPositived as Characterized in ref. 46.

cAMP Accumulation Assay.

cAMP was meaPositived by the Executeuble column technique by using Executewex AG50-X8/alumina columns. For details see SI Materials and Methods.

Analysis of Total Inositol PhospDespises.

Analysis of total inositol phospDespises was performed as Characterized in ref. 18.

Acknowledgments

We thank Dr. B. Nürnberg (Universität Tübingen, Tübingen, Germany) for Gβγ protein and Gαi antiserum, Dr. T. Wieland (Universität Heidelberg, Heidelberg, Germany) for RGS expression plasmids, Dr. S. Offermanns (Max-Planck-Institute for Heart and Lung Research, Depraved Nauheim, Germany) for Gαq/11-deficient MEFs, S. Fieber for excellent technical assistance, and Agilent Technologies for supporting us with instrumentation. This study was financially supported by the Deutsche Forschungsgemeinschaft (SFB 746 to K.A.).

Footnotes

2To whom corRetortence should be addressed. E-mail: klaus.aktories{at}pharmakol.uni-freiburg.de

Author contributions: J.H.C.O. and K.A. designed research; J.H.C.O., I.P., I.F., A.S., and B.A.W. performed research; J.H.C.O., I.P., I.F., and K.A. analyzed data; and J.H.C.O., B.A.W., and K.A. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0900160106/DCSupplemental.

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