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High level production and purification of Gα0 using the baculovirus heterologous expression system
2172
The involvement of a pertussis toxin sensitive G-wotein in the activation of adenyinte cydase in jurkat cells Altiok, N., van der Ploeg, I., Nordstedt, C. a n d Fredholm, B.B. Depanmem of P
~
,
Karolinska Instituter, Box 60400, S.104 Ol Stockholm, Sweden
In the human T-cell line Jurkat cyclic AMP formation can be enhanced by adenosine, PGE 2, cholera toxin and forskolin. The effect of the adenosine analogue 5"-N-ethyl carboxamido adenosine (NECA) and of cholera toxin was enhanced by activation of protein kinase C with phorbol dibutyrate (PDBu), whereas the effect of forskolin was essentially unaffected and that of PG~,2 was reduced (Nordstedt et al., 1989). In order to examine the possible role of G-proteins the effect of pertussis toxin (PTX) pretreatment (200 ng/mi~ for 4 hours was examined. Surprisingly the PTX treatment, which caused an essentially total ADP-ribosylation of PTX-sensitive proteins in Jurk~*. cell membranes, did not enhance but actually reduced the cAMP accumulation induced by NECA, PGE2, cholera toxin and forskolin (by approximately 20, 35, 50 and 45~, respectively). This effect was not mimicked by incubating with an equivalent amount of purified PTX B-subunit for the same amount of time. This indicates that the effect of PTX is related to the A-subunit posst :sing ADP-ribosylating activity. It is, however, not known whether the results indicate the presence in Jurkat cells of a PTX-sensitive G-protein that has the ability to stimulate adenylate cyclase or if the results indicate an effect of the A-subunit that is unrelated to G-proteins and is exerted on the adenylate cyclase in some other fashion. The PTX h-~tment did not eliminate the ability of PDBu to enhance the cAMP accumulation stimulated by NECA or by cholera toxin. By contrast, the ability of PDBu to reduce the effect of P G E 2 w a s eliminated. In the presence of PTX PDBu in fact caused a slight enhancement (about 30~) of the PC}E 2 stimulated cAMP accumulation. This indicates that the effect of protein kinase C stimulation o n P G E 2 responses depends on the presence of PTX-sensitive G-protein~ whereas the effect on adenosine- or cholera toxin responses does not.
Refemnces Norastedt, C., Kvanta, A., Van der Ploeg, I. and F.,~dholm, B.B., Dual effects of Frotein kinase C on receptor-stimulated cAMP accumulation in a human T-cell leukemia line. Europ. J. Pharmacol. Molec. Pharm. Sect. 172. 51-60, 1989. P.fr.0631
High |eve| production and purification of Ga e using the baculovirus heterologous expression system Torossian, K., Labrecque~ J., Caron, M., Plamondon, J. and Dennis, M. Biotechnology Research Institute 6100 Royalmoum Ave, Montreal, Quebec H4P 2R2, Canada
The heterotrimeric (3 protein G o has been impficated in signal transduction between numerous receptors and effectors in mammalian cells. In order to examine the structure and function of this protein, we have established a high-level expression system for the a subunit (ao) using a baculovirus vector and insect host cells. The coding segment of rat a o cDNA was inserted into the transfer vector IpDc126, flanked by the promoter and 3' sequences of the viral polyhedrin gene. Following co-transfection into Sf9 insect cells of plasmid and wild-type viral DNA, recombinant ~,~-uses harhouring ao inserts were isolated by successive plaque purifications using hybridization and visual screening. Cultures of Sf9 cells infected in suspension with a 0 recombinant (5 moi) expressed a ca. 40 kDa protein reactive with polyclonal anti-a 0 antisera. Immunoreactive material was present in both membrane and soluble fractions following cell iysis. A high speed (100,000 × g) supernatant showed high levels ( > 0.15 nmol/mg protein) of
2173 GTPv3sS binding over wild-type controls. Both supernatant and precipitate showed high levels of G a 0 expression when probed with the anti-a o antibody. [35S]Methionine labeling experiments showed that maximum levels of expression were reached 72h post-infection when G a 0 represented approximatively 10~ of cellular protein. The high speed supernatant was chosen for further purification. It was loaded on a DEAE-Sephacel column equilibrated with 'Iris buffer containing 10 mM aluminum fluoride (AMF) to stabilize the G-protein and the column was eluted with a gradient of NaC! from 0 to 500 mM. Immunoreactive fractions (dot blots) eluting at 150 mM NaC! were pooled and assayed for protein content and, after dilution, for GTP3,3SS binding activity. Typically, the specific activity rose to 3 nmol/mg protein after this step. The pooled fractions were then loaded on a Sephacryl S-200 molecular exclusion column in a buffer devoid of AMF. lmmunoreactive fractions were pooled and assayed as described for DEAE-Sephacel, the spec;.'fic activity was increased to 10 nmol/mg protein after this purification step° SDS-PAGE after each purification step showed progressive enrichment of a ca. 40 kDa band. The purified material was stable at - 80 o C but lost its GTPy35S binding activity after 48-72 h at - 2 0 ° C. The pooled Sephacryl S-200 fractions will be fractionated further using hydroxyapatite. Preliminary experiments indicate that the G a 0 susbunit binds to, this matrix and is eluted at ca. 50 mM phosphate.
Phosphorylation of inhibitory GTP-binding protein by cyclic AMP-dependent protein kinase inhibits dissociation of the trimer into three subunits of the protein Watanabe, Y., Imaizumi, T. and Miki, N. Department of Pharmacology !, School of Medicine, Osaka University. 3,27-4 Nakanoshima Kita-ku, Osaka 530, Japan
We reported that inhibitory GTP binding (Oi) protein (41 K) partially purified from rat brains was phosi~horylated by activated cyclic AMP-dependent protein kinase (PICA) and that phosphorylation of the protein by PKA reduced ADP-ribosylation of the protein by pertussis toxin (islet-activating protein, IAP) in rat cardiac cell membranes (Watanabe, 1988, 1989). We examined whether the phosphorylation of the protein by PKA also reduced the ADP-ribosylation of the protein by lAP using purified Gi protein. The pretreatment caused the decrease of amounts of ADP-ribosylation of the protein by lAP (Table 1). Next we examined whether the reduction of ADP-.~:.bosylation of the protein by IAP comes from a conformational change of the trimer of Gi protein or from the dissociation into three ,,-subunits of Gi protein induced by the phosphorylation of Gi protein by PKA, because only the trimer of Gi protein was ADP-ribosylated by IAP. According to the described method (Mattera, 1987), we examined the effects of the phosphorylation of Gi protein by PKA on the dissociation of the trimer into the three subunits of Gi protein. Purified Gi protein treated or untreated with PKA and MgATP was placed on top of 5-20~ (w/w) sucrose gradients in buffer containing either 50 M GDP, or 50 M GDP, 500 M GTPS end 50 mM MgCI 2. The gradients were centrifuged at room temperature and fractionated at 4 into aliquots. The position of Gi protein and its -subunit was determined by subjecting aliquots of the fractions to ADP-ribosylation by IAP and then analyzing them by SDS-PAGE followed by autoradiography. Pretreatment of Gi protein with PEA inhibited the dissociation induced by Mg 2+ and GTPS of its trimer into three subunits of the Gi protein, while the pretreatment did not dissociate the trimer into three subunits. The findings suggest that phosphorylation modified the function of the Gi protein and changed its ADP-ribosylation by IAP. Thus the adenylate cyclase activating pathway can cross-talk with other coupling pathways through phosphorylation of their coupled GTP-binding proteins by activated PICA and this can be one of the mechesiis~ causing "heterologous desensitization". Table 1 Effects of PKA pretreatment on ADP-ribosylation of purified Gi by IAP. Expt. 1
Expt. 2
Expt. 3
Percentage of control value
49333 31146
5576 2735
2576 1700
59.3 :!:7.3 (3)
Area of absorbance
Control PKA pretreatment
The involvement of a pertussis toxin sensitive G-wotein in the activation of adenyinte cydase in jurkat cells Altiok, N., van der Ploeg, I., Nordstedt, C. a n d Fredholm, B.B. Depanmem of P
~
,
Karolinska Instituter, Box 60400, S.104 Ol Stockholm, Sweden
In the human T-cell line Jurkat cyclic AMP formation can be enhanced by adenosine, PGE 2, cholera toxin and forskolin. The effect of the adenosine analogue 5"-N-ethyl carboxamido adenosine (NECA) and of cholera toxin was enhanced by activation of protein kinase C with phorbol dibutyrate (PDBu), whereas the effect of forskolin was essentially unaffected and that of PG~,2 was reduced (Nordstedt et al., 1989). In order to examine the possible role of G-proteins the effect of pertussis toxin (PTX) pretreatment (200 ng/mi~ for 4 hours was examined. Surprisingly the PTX treatment, which caused an essentially total ADP-ribosylation of PTX-sensitive proteins in Jurk~*. cell membranes, did not enhance but actually reduced the cAMP accumulation induced by NECA, PGE2, cholera toxin and forskolin (by approximately 20, 35, 50 and 45~, respectively). This effect was not mimicked by incubating with an equivalent amount of purified PTX B-subunit for the same amount of time. This indicates that the effect of PTX is related to the A-subunit posst :sing ADP-ribosylating activity. It is, however, not known whether the results indicate the presence in Jurkat cells of a PTX-sensitive G-protein that has the ability to stimulate adenylate cyclase or if the results indicate an effect of the A-subunit that is unrelated to G-proteins and is exerted on the adenylate cyclase in some other fashion. The PTX h-~tment did not eliminate the ability of PDBu to enhance the cAMP accumulation stimulated by NECA or by cholera toxin. By contrast, the ability of PDBu to reduce the effect of P G E 2 w a s eliminated. In the presence of PTX PDBu in fact caused a slight enhancement (about 30~) of the PC}E 2 stimulated cAMP accumulation. This indicates that the effect of protein kinase C stimulation o n P G E 2 responses depends on the presence of PTX-sensitive G-protein~ whereas the effect on adenosine- or cholera toxin responses does not.
Refemnces Norastedt, C., Kvanta, A., Van der Ploeg, I. and F.,~dholm, B.B., Dual effects of Frotein kinase C on receptor-stimulated cAMP accumulation in a human T-cell leukemia line. Europ. J. Pharmacol. Molec. Pharm. Sect. 172. 51-60, 1989. P.fr.0631
High |eve| production and purification of Ga e using the baculovirus heterologous expression system Torossian, K., Labrecque~ J., Caron, M., Plamondon, J. and Dennis, M. Biotechnology Research Institute 6100 Royalmoum Ave, Montreal, Quebec H4P 2R2, Canada
The heterotrimeric (3 protein G o has been impficated in signal transduction between numerous receptors and effectors in mammalian cells. In order to examine the structure and function of this protein, we have established a high-level expression system for the a subunit (ao) using a baculovirus vector and insect host cells. The coding segment of rat a o cDNA was inserted into the transfer vector IpDc126, flanked by the promoter and 3' sequences of the viral polyhedrin gene. Following co-transfection into Sf9 insect cells of plasmid and wild-type viral DNA, recombinant ~,~-uses harhouring ao inserts were isolated by successive plaque purifications using hybridization and visual screening. Cultures of Sf9 cells infected in suspension with a 0 recombinant (5 moi) expressed a ca. 40 kDa protein reactive with polyclonal anti-a 0 antisera. Immunoreactive material was present in both membrane and soluble fractions following cell iysis. A high speed (100,000 × g) supernatant showed high levels ( > 0.15 nmol/mg protein) of
2173 GTPv3sS binding over wild-type controls. Both supernatant and precipitate showed high levels of G a 0 expression when probed with the anti-a o antibody. [35S]Methionine labeling experiments showed that maximum levels of expression were reached 72h post-infection when G a 0 represented approximatively 10~ of cellular protein. The high speed supernatant was chosen for further purification. It was loaded on a DEAE-Sephacel column equilibrated with 'Iris buffer containing 10 mM aluminum fluoride (AMF) to stabilize the G-protein and the column was eluted with a gradient of NaC! from 0 to 500 mM. Immunoreactive fractions (dot blots) eluting at 150 mM NaC! were pooled and assayed for protein content and, after dilution, for GTP3,3SS binding activity. Typically, the specific activity rose to 3 nmol/mg protein after this step. The pooled fractions were then loaded on a Sephacryl S-200 molecular exclusion column in a buffer devoid of AMF. lmmunoreactive fractions were pooled and assayed as described for DEAE-Sephacel, the spec;.'fic activity was increased to 10 nmol/mg protein after this purification step° SDS-PAGE after each purification step showed progressive enrichment of a ca. 40 kDa band. The purified material was stable at - 80 o C but lost its GTPy35S binding activity after 48-72 h at - 2 0 ° C. The pooled Sephacryl S-200 fractions will be fractionated further using hydroxyapatite. Preliminary experiments indicate that the G a 0 susbunit binds to, this matrix and is eluted at ca. 50 mM phosphate.
Phosphorylation of inhibitory GTP-binding protein by cyclic AMP-dependent protein kinase inhibits dissociation of the trimer into three subunits of the protein Watanabe, Y., Imaizumi, T. and Miki, N. Department of Pharmacology !, School of Medicine, Osaka University. 3,27-4 Nakanoshima Kita-ku, Osaka 530, Japan
We reported that inhibitory GTP binding (Oi) protein (41 K) partially purified from rat brains was phosi~horylated by activated cyclic AMP-dependent protein kinase (PICA) and that phosphorylation of the protein by PKA reduced ADP-ribosylation of the protein by pertussis toxin (islet-activating protein, IAP) in rat cardiac cell membranes (Watanabe, 1988, 1989). We examined whether the phosphorylation of the protein by PKA also reduced the ADP-ribosylation of the protein by lAP using purified Gi protein. The pretreatment caused the decrease of amounts of ADP-ribosylation of the protein by lAP (Table 1). Next we examined whether the reduction of ADP-.~:.bosylation of the protein by IAP comes from a conformational change of the trimer of Gi protein or from the dissociation into three ,,-subunits of Gi protein induced by the phosphorylation of Gi protein by PKA, because only the trimer of Gi protein was ADP-ribosylated by IAP. According to the described method (Mattera, 1987), we examined the effects of the phosphorylation of Gi protein by PKA on the dissociation of the trimer into the three subunits of Gi protein. Purified Gi protein treated or untreated with PKA and MgATP was placed on top of 5-20~ (w/w) sucrose gradients in buffer containing either 50 M GDP, or 50 M GDP, 500 M GTPS end 50 mM MgCI 2. The gradients were centrifuged at room temperature and fractionated at 4 into aliquots. The position of Gi protein and its -subunit was determined by subjecting aliquots of the fractions to ADP-ribosylation by IAP and then analyzing them by SDS-PAGE followed by autoradiography. Pretreatment of Gi protein with PEA inhibited the dissociation induced by Mg 2+ and GTPS of its trimer into three subunits of the Gi protein, while the pretreatment did not dissociate the trimer into three subunits. The findings suggest that phosphorylation modified the function of the Gi protein and changed its ADP-ribosylation by IAP. Thus the adenylate cyclase activating pathway can cross-talk with other coupling pathways through phosphorylation of their coupled GTP-binding proteins by activated PICA and this can be one of the mechesiis~ causing "heterologous desensitization". Table 1 Effects of PKA pretreatment on ADP-ribosylation of purified Gi by IAP. Expt. 1
Expt. 2
Expt. 3
Percentage of control value
49333 31146
5576 2735
2576 1700
59.3 :!:7.3 (3)
Area of absorbance
Control PKA pretreatment