Ggamma Subunit-selective G Protein beta 5 Mutant Defines Regulators of G Protein Signaling Protein Binding Requirement for Nuclear Localization*

Alexandra M. Rojkova, Geoffrey E. Woodard, Tzu-Chuan HuangDagger, Christian A. Combs§, Jian-Hua Zhang, and William F. Simonds

From the Metabolic Diseases Branch, NIDDK and § Confocal Microscopy Core Facility, Division of Intramural Research, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, July 20, 2002, and in revised form, January 15, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The signal transducing function of Gbeta 5 in brain is unknown. When studied in vitro Gbeta 5 is the only heterotrimeric Gbeta subunit known to interact with both Ggamma subunits and regulators of G protein signaling (RGS) proteins. When tested with Ggamma , Gbeta 5 interacts with other classical components of heterotrimeric G protein signaling pathways such as Galpha and phospholipase C-beta . We recently demonstrated nuclear expression of Gbeta 5 in neurons and brain (Zhang, J. H., Barr, V. A., Mo, Y., Rojkova, A. M., Liu, S., and Simonds, W. F. (2001) J. Biol. Chem. 276, 10284-10289). To gain further insight into the mechanism of Gbeta 5 nuclear localization, we generated a Gbeta 5 mutant deficient in its ability to interact with RGS7 while retaining its ability to bind Ggamma , and we compared its properties to the wild-type Gbeta 5. In HEK-293 cells co-transfection of RGS7 but not Ggamma 2 supported expression in the nuclear fraction of transfected wild-type Gbeta 5. In contrast the Ggamma -preferring Gbeta 5 mutant was not expressed in the HEK-293 cell nuclear fraction with either co-transfectant. The Ggamma -selective Gbeta 5 mutant was also excluded from the cell nucleus of transfected PC12 cells analyzed by laser confocal microscopy. These results define a requirement for RGS protein binding for Gbeta 5 nuclear expression.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In eukaryotic cells seven transmembrane-spanning receptors regulate intracellular processes in response to extracellular signals through their interaction with signal-transducing heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) (1). Complementary DNAs from five G protein beta  subunit genes (Gbeta 1-5) have been identified by molecular cloning. The Gbeta 5 isoform shares much less homology with other isoforms (~50%) and is preferentially expressed in brain (2). A splice variant of Gbeta 5, Gbeta 5-long (Gbeta 5L), is present in retina that contains a 42-amino acid N-terminal extension (3).

Gbeta 5 is the only Gbeta subunit known with the potential to assemble with either Ggamma subunits or regulators of G protein signaling (RGS)1 proteins. Gbeta 5 can heterodimerize with Ggamma (2, 4-6) and interact with other classical components of heterotrimeric G proteins signaling pathways such as Galpha (5, 6) and phospholipase C-beta (2, 4, 6-8) when tested in vitro. Other in vitro studies show that Gbeta 5 and Gbeta 5L, but not the other Gbeta isoforms, can form tight heterodimers with RGS proteins 6, 7, and 11, an interaction mediated by a Ggamma -like (GGL) domain present in a subfamily of RGS proteins (9-11). In native tissues, however, Gbeta 5 has been purified not as a heterodimer with Ggamma but instead bound to RGS6 (12) and RGS7 (12-15). The failure to demonstrate native Gbeta 5-Ggamma complexes is complicated by the known instability of such complexes in detergent solution (10, 16). Thus, purifications employing detergent may fail to identify potential Gbeta 5-Ggamma complexes that could dissociate in the process of isolation (16). Alternative approaches to assess the biologic importance of the Gbeta 5-Ggamma interaction demonstrated in vitro are needed.

We recently demonstrated the nuclear expression of Gbeta 5 in neurons and brain (17). In this study chimeric protein constructs containing green fluorescent protein (GFP) fused to wild-type Gbeta 5 demonstrated nuclear localization in transfected PC12 cells, but not GFP fusions with a mutant Gbeta 5 with proline substitutions in its putative coiled-coil domain. Introduction of prolines into the putative alpha -helical N-terminal region of Gbeta 5 would disrupt its ability to form a coiled-coil with both Ggamma subunits and RGS proteins containing GGL domains. Such a Gbeta 5 mutant therefore fails to provide mechanistic insight as to which of its two potential heterodimeric conformations is important for nuclear targeting. We describe here a Ggamma -selective Gbeta 5 mutant that fails to undergo nuclear localization in transfected PC12 and HEK-293 cells. In the latter cells the nuclear expression of wild-type Gbeta 5 is dependent on RGS7 co-transfection. These results suggest it is interaction with the GGL domain containing RGS proteins that directs nuclear localization of Gbeta 5. Additional studies of RGS7 do not support a role for a putative interaction with 14-3-3 proteins in Gbeta 5 nuclear localization.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of Gbeta 5-specific Residues in the Putative Coiled-coil Domain of Gbeta 5-- The Pileup algorithm of the University of Wisconsin Genetics Computer Group (18) was used to align the coding sequences of human (GenBankTM accession number NM_006578) (19), tiger salamander (GenBankTM accession number AF369757) (20), Drosophila melanogaster (GenBankTM accession number AAF46336), and Caenorhabditis elegans (GenBankTM accession number AF291847) (21-23) Gbeta 5 with mammalian Gbeta subunits 1-4. Amino acids in the line up that were identical among the four Gbeta 5 sequences, but not found in the corresponding positions of Gbeta 1-4, and belonging to a different amino acid homology group from the corresponding positions of Gbeta 1-4 were identified and considered to be conserved, Gbeta 5-specific residues. For the purposes of this analysis the amino acid homology groups were taken to be basic (His, Lys, and Arg), acidic (Asp and Glu), polar uncharged (Cys, Gly, Asn, Gln, Ser, Thr, and Tyr), and nonpolar (Ala, Phe, Ile, Leu, Met, Pro, Val, and Trp). The portion of this analysis including the N-terminal coiled-coil region of the Gbeta subunits is shown in Fig. 1.

cDNAs and Mutagenesis of Gbeta 5 and RGS7-- Expression constructs encoding full-length murine Gbeta 5 in pcDNA3 (4) and bovine RGS7 in pcDNA4/HisMax-C (17), which adds N-terminal His6 and Xpress epitope tags, have been described previously. N-terminally HA epitope-tagged Gbeta 5 in pcDNA3 (coding sequence inserted between HindIII and BamHI sites) was created by PCR to give the following sequence, MAYPYDVPDYAEFKAA ... , in which the starting methionine and the alanine corresponding to position 2 in the wild-type sequence are underlined, flanking 14 residues of inserted sequence including the nonapeptide HA epitope (24). The mutants HA-Gbeta 5-QAAC (Gbeta 5-Lys25 right-arrow Gln/Glu29 right-arrow Ala/Lys32 right-arrow Ala/Leu33 right-arrow Cys) and HA-Gbeta 5-YMIN (Gbeta 5-Phe93 right-arrow Tyr/Thr338 right-arrow Met/Val351 right-arrow Ile/Ala353 right-arrow Asn) were generated by PCR from a template of wild-type HA-Gbeta 5 in pcDNA3 employing the Pwo polymerase (Roche Molecular Biochemicals) by overlap extension as described previously (25). N-terminally HA epitope-tagged Gbeta 1 in pcDNA3 (coding sequence inserted between EcoRI and XbaI sites) was created by PCR to give the following sequence, MAYPYDVPDYAGS ... , in which the starting methionine and the serine corresponding to position 2 in the wild-type sequence are underlined, flanking 11 residues of inserted sequence including the nonapeptide HA epitope (24). The point mutant RGS7-Ser434 right-arrow Ala (S434A) was generated by PCR from a template of wild-type bovine RGS7 in pcDNA4/HisMax-C (17) employing the Pwo polymerase by overlap extension (25). Ggamma 2 in pcDNA4/HisMax-C vector (BamHI to NotI) was generated by Pwo polymerase PCR using bovine Ggamma 2 as a template (26). The DNA sequence of all inserts was verified by dideoxy sequencing (27).

Cell Culture-- Rat pheochromocytoma PC12 cells were grown in 75-cm2 flasks grown at 37 °C and 5% CO2 containing DMEM supplemented with 10% horse serum, 5% fetal bovine serum, 4 mM L-glutamine, 1× penicillin/streptomycin (BioFluids) (supplemented DMEM). HEK-293 cells were grown as above except that 10% fetal bovine serum and no horse serum were used in the medium.

Transient Transfection of PC12 and HEK-293 Cells-- One day prior to transient transfection, PC12 cells at 90% confluence were harvested and plated in 2 ml of supplemented DMEM into the chambers of poly-D-lysine-coated chamber slides. The number of cells was adjusted to 70% of their density at harvest. After overnight incubation of the cells, 1.5 µg of plasmid DNA and 4 µl of LipofectAMINE 2000 reagent (Invitrogen), diluted in 100 µl of Opti-MEMI medium, were mixed, incubated at room temperature for 20 min, and added directly to each chamber. After mixing gently, the cultures were incubated for 24-48 h prior to confocal microscopic analysis. HEK-293 cells were transfected in 75-cm2 culture flasks using either LipofectAMINE 2000 or Superfect transfection reagent (Qiagen) according to the manufacturer's recommendations. Typically 10 µg of total plasmid DNA and 60 µl of LipofectAMINE 2000 reagent or 15 µg of total plasmid DNA and 40 µl of Superfect reagent were used per 75-cm2 culture flask.

Gel Electrophoresis, Immunoblotting, and Immunoprecipitation-- Protein samples were separated on 4-20% gradient (for analysis of Gbeta 5, RGS7, or TFIID (TBP) protein expression) or uniform 14% (for analysis of Ggamma expression) polyacrylamide slab gels (NOVEX) by SDS-PAGE. Proteins were then transferred by electrophoresis to 0.45-µm nitrocellulose membranes (28), and after primary and secondary antibody incubation, chemiluminescent signal was detected using the SuperSignal® West Femtostable Peroxide Buffer (Pierce) according to the manufacturer's instructions. Primary antibodies used are as follows: affinity-purified rabbit ATDG polyclonal antibody against N terminus of Gbeta 5 (12), rabbit polyclonal antibody SGS against the C terminus of Gbeta 5 (4); goat anti-RGS7 C-19 IgG (sc-8139), rabbit polyclonal antibody against a GST fusion protein containing residues of 312-469 of bovine RGS7 (7RC-1); rabbit anti-TFIID (TBP) N-12 IgG (Santa Cruz Biotechnology); rabbit anti-Ggamma 2 A-16 IgG (sc-374) (Santa Cruz Biotechnology); and anti-Xpress epitope (Invitrogen) and HA.11 anti-hemagglutinin (HA) epitope (Covance) mouse monoclonal antibodies. Secondary antibodies employed were horseradish peroxidase-coupled donkey anti-rabbit (Amersham Biosciences) to detect rabbit polyclonal antibodies, horseradish peroxidase-coupled donkey anti-goat (Santa Cruz Biotechnology) for goat primary antibodies, and horseradish peroxidase-coupled goat anti-mouse IgG1 (Santa Cruz Biotechnology) for mouse monoclonal antibodies. Immunoprecipitation from detergent extracts of transfected HEK-293 cells employed HA.11 monoclonal antibodies and Protein A/G Plus-agarose beads (Santa Cruz Biotechnology) following the methodology described previously (12), except that buffer A was used. Buffer A consisted of 0.15% (w/v) polyoxyethylene (10) monolauryl ether (Genapol C-100®) detergent, 150 mM NaCl, 20 mM Na-HEPES, pH 7.4, 1× protease inhibitor mixture (Calbiochem Set III, number 539134).

Inositide-specific Phospholipase C Activity Determination-- To determine the Ggamma -dependent activation of inositide phospholipid-specific phospholipase C (PLC) by wild-type and mutant Gbeta 5 constructs, HEK-293 cells metabolically labeled with [3H]inositol and co-transfected with human PLC-beta 2 were employed, and the [3H]inositol phosphates isolated by the method of Berridge et al. (29) with modifications as described previously (4). The PLC activity in cells co-transfected with vector only was compared with that in cells transfected with Gbeta 5 construct without or with Ggamma 2.

Subcellular Fractionation of HEK-293 Cells-- 48 h following transfection, HEK-293 cells were harvested from a 75-cm2 flask, split into two fractions, and pelleted by centrifugation at 500 × g for 5 min at 4 °C. One cell pellet was used to isolate cell nuclei and generate nuclear extracts using a commercially available cell lysis kit according to the manufacturer's instructions (NE-PER Reagents, Pierce). The other cell pellet was resuspended in ice-cold lysis buffer (25 mM HEPES, pH 7.5, 0.3 M NaCl, 0.2 mM EDTA, 1.5 mM MgCl2, 1% Triton X-100, 0.1% SDS, and 0.5% sodium deoxycholate containing 1× protease inhibitor mixture (Calbiochem Set III)). After removal of the insoluble material by centrifugation, the supernatant fraction was employed as whole cell lysate.

Immunocytochemistry and Confocal Laser Microscopy-- PC12 cells were processed for immunofluorescent staining as described previously (30). Briefly, PC12 cells were plated onto poly-D-lysine pre-coated covered chamber slides (Lab-Tek II, Nalge) and grown in supplemented DMEM at 37 °C for 16 h. The medium was discarded, and the cells were washed and then fixed in 2% (v/v) formaldehyde in Dulbecco's phosphate-buffered saline (Biofluids, Rockville, MD). The slides were then incubated with one or more primary antibodies in Dulbecco's phosphate-buffered saline, 10% fetal calf serum (v/v), and 0.075% (w/v) saponin for 1 h, washed, and then incubated with appropriate labeled secondary antibody (fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, West Grove, PA)) and/or Rhodamine RedTM-X goat anti-mouse IgG (H + L) (Molecular Probes)) in the same buffer for 45 min. After staining, 1-2 drops of Permount (Vector Laboratories) was added to the sample surface. Confocal images were collected using a Zeiss LSM 510 laser scanning confocal microscope with a 100×/1.3 N.A. Plan-Neofluar objective. Scans were performed sequentially using 488 and 543 nm excitation and bandpass emission filters of 505-550 and 560-615 nm, respectively, for Rhodamine Red and fluorescein to eliminate spectral bleed through of fluorescence between the red and green channels. The pinhole in all cases was adjusted to produce a 1.5-µm optical slice thickness. Transmitted light differential interference contrast images were collected simultaneously with the green dye using 488 nm excitation. Final images were processed using Zeiss LSM software.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Design and Expression of Putative Ggamma -selective Gbeta 5 Mutants-- In order to assess the potential biologic importance of the Gbeta 5-Ggamma interaction for the nuclear localization of Gbeta 5, a Gbeta 5 mutant that retained demonstrable Ggamma interaction with selective loss of RGS protein binding was desired. By analogy with the crystal structures of G protein heterotrimers (31, 32) and free Gbeta 1gamma 1 complex (33), it was expected that the interaction of Ggamma or an RGS protein GGL domain with Gbeta 5 involved many of the interactions observed in Gbeta 1gamma . These interactions included a two-stranded parallel coiled-coil involving N-terminal portions of Gbeta 5 and Ggamma /GGL domain and hydrophobic interactions between more C-terminal regions of Ggamma /GGL domain and the beta -propeller structure encoded by WD-40 repeats of the Gbeta (10, 31-33).

Analysis of the coding sequences of Gbeta 5 of mammalian (2, 19), amphibian (20), insect (GenBankTM accession number AAF46336), or roundworm (21-23) origin in comparison to mammalian Gbeta subunits 1-4 reveals four conserved residues in the putative coiled-coil region of the Gbeta 5s that are Gbeta 5-specific (Fig. 1). These Gbeta 5-specific residues occupy the a, d, and g positions of the Gbeta 5 heptad repeat (Fig. 1), positions that may determine the specificity of coiled-coil dimerization (reviewed in Ref. 34) and that might contribute to specific interaction of Gbeta 5 with RGS protein GGL domains (9). In order to create a Ggamma -preferring Gbeta 5 mutant, these four residues in HA epitope-tagged mammalian Gbeta 5 were mutated to their counterparts in Gbeta 1 to produce mutant Gbeta 5-QAAC (Gbeta 5-K25Q/ E29A/K32A/L33C).


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Fig. 1.   Identification of Gbeta 5-specific residues in the putative coiled-coil domain of Gbeta 5. An alignment of the coding sequences of human, salamander (Sal), D. melanogaster (Dm), and C. elegans (Ce) Gbeta 5 with mammalian Gbeta subunits 1-4 was performed as described under "Experimental Procedures." The portion of this analysis that includes the N-terminal region coiled-coil region of the Gbeta subunits is shown. Amino acids that were identical among the 4 Gbeta 5 sequences, but not found in the corresponding positions of mammalian Gbeta 1-4 and belonging to a different amino acid homology group from the corresponding positions of Gbeta 1-4, were identified and considered to be conserved, Gbeta 5-specific residues (yellow highlights). The position of the residues of Gbeta 1 in the coiled-coil heptad repeats, taken from the crystal structures of Gbeta 1 complexes (31-33), are indicated as a-g above the Gbeta 1 sequence.

Modeling of a Gbeta 5-GGL domain heterodimer by Snow et al. (10) suggested that Gbeta 5 beta -propeller residues comprising a hydrophobic pocket able to accommodate a conserved Trp residue in the GGL domain (corresponding to Phe64 of Ggamma 1) might also contribute to the selectivity of Gbeta 5-RGS protein assembly. Based on the model by Snow et al. (10), four beta -propeller residues forming the hydrophobic pocket in HA-tagged Gbeta 5 were mutated to their more bulky Gbeta 1 counterparts. The aim of this mutagenesis was to reduce the size of the putative Gbeta 5 beta -propeller hydrophobic pocket to exclude the conserved Trp residue in the GGL domain while still accommodating the Phe residue present in Ggamma corresponding to residue 64 of Ggamma 1 (10). These alterations produced mutant Gbeta 5-YMIN (Gbeta 5-F93Y/T338M/V351I/A353N).

Both Gbeta 5 mutants could be expressed to levels comparable with wild-type Gbeta 5 when transiently transfected into HEK-293 cells (Fig. 2). The expression of the Gbeta 5-QAAC mutant, like that of wild-type Gbeta 5, was greater in cells co-transfected with RGS7 or Ggamma 2 than in cells co-transfected with vector only (Fig. 2A, lanes 2-5, cf. lanes 6 and 7). The ability of Ggamma or GGL domain-containing RGS protein co-expression to stabilize Gbeta 5 expression has been observed previously (15, 35) in several systems. Unlike the wild-type Gbeta 5, the expression of the Gbeta 5-YMIN mutant was greater in cells co-transfected with Ggamma 2 than in cells co-transfected with RGS7 (Fig. 2B, lanes 6 and 7, cf. lanes 2-5). Nevertheless, the expression of the Gbeta 5-YMIN mutant was greater in cells co-transfected with RGS7 than with vector only (Fig. 2B, cf. lanes 5 and 7). Also wild-type Gbeta 5 but not Gbeta 5-YMIN mutant co-transfection enhanced the expression of RGS7 (Fig. 2B, cf. lanes 2 and 7). As expected the C-terminally directed Gbeta 5 antibody SGS failed to recognize the Gbeta 5-YMIN mutant containing two mutations within the SGS epitope (4) (Fig. 2B, lane 6) but reacted with the Gbeta 5-QAAC mutant as well as with the wild type (data not shown).


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Fig. 2.   Expression of wild-type and mutant Gbeta 5 subunits in transfected HEK-293 cells. Immunoblots of lysates from transfected cells treated with vector (vec), wild-type (wt), or mutant Gbeta 5, RGS7, or Ggamma 2 cDNAs were prepared as described under "Experimental Procedures." The cDNA combinations used for each transfection are indicated below the immunoblots. The relative mobility of the immunoreactive bands on SDS-PAGE (in kDa) is shown to the left of each panel, and the primary antibodies used for the immunoblots are shown to the right. A, transfections employing mutant Gbeta 5-QAAC (Gbeta 5-K25Q/E29A/K32A/L33C). B, transfections employing mutant Gbeta 5-YMIN (Gbeta 5-F93Y/T338M/V351I/A353N). Shown are results from a single experiment representative of three similar experiments with comparable results.

Interactions of Putative Ggamma -selective Gbeta 5 Mutants with RGS7, Ggamma , and Ggamma -dependent Effectors-- The ability of the putative Ggamma -selective Gbeta 5 mutants to bind RGS7 and Ggamma subunits was tested in co-immunoprecipitation assays. In transiently transfected HEK-293 cells, immunoprecipitation of the Gbeta 5-QAAC mutant was able to co-immunoprecipitate RGS7 from cell lysates as well as wild-type Gbeta 5 (Fig. 3A, cf. lanes 2 and 3). In contrast only trace RGS7 was evident in immunoprecipitates of the Gbeta 5-YMIN mutant even though the mutant Gbeta 5 itself was precipitated and wild-type Gbeta 5 was able to fully co-precipitate RGS7 in the same experiment (Fig. 3B, cf. lanes 2 and 3). However, because the expression of RGS7 when co-transfected with the Gbeta 5-YMIN mutant is poor (Fig. 3B, lane 3 of lysates), the absence of RGS7 in the Gbeta 5-YMIN mutant immunoprecipitates cannot be interpreted.


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Fig. 3.   Selective co-immunoprecipitation of RGS7 with HA epitope-tagged wild-type and mutant Gbeta 5 subunits in transfected HEK-293 cells. Cell lysates prepared 2 days after transient transfection were incubated with anti-HA monoclonal antibody, and after precipitation, the starting lysates and washed immunoprecipitates (IP) were both analyzed for RGS7 (upper panels) and epitope-tagged Gbeta 5 (HA antibody, lower panels) immunoreactivity by immunoblotting as described under "Experimental Procedures." The relative mobility of the specific immunoreactive bands (in kDa) is indicated to the left of each panel. Cells were transfected with either vector alone (vec), or with cDNAs encoding RGS7 and wild-type (wt), or mutant HA-Gbeta 5 as indicated below the lanes. A, transfections employing mutant Gbeta 5-QAAC (Gbeta 5-K25Q/E29A/K32A/L33C). B, transfections employing mutant Gbeta 5-YMIN (Gbeta 5-F93Y/T338M/V351I/A353N). This experiment was repeated a total of three times with similar results.

The ability of the putative Ggamma -selective Gbeta 5 mutants to bind Ggamma subunits was separately tested in co-immunoprecipitation assays from lysates of transiently transfected HEK-293 cells and compared with wild-type Gbeta 5 and Gbeta 1 (Fig. 4). Analysis of the lysates demonstrated the co-expression of all transfected Gbeta constructs with Ggamma 2 (Fig. 4A). Neither the wild-type Gbeta 5 nor the Gbeta 5-QAAC mutant was able to co-immunoprecipitate Ggamma 2 from cell lysates, however (Fig. 4B, lanes 2 and 4), a finding consistent with the known instability of Gbeta 5-Ggamma 2 binding in detergent solution (10, 16). Unlike wild-type Gbeta 5 and Gbeta 5-QAAC, however, both the Gbeta 5-YMIN mutant and Gbeta 1 clearly co-immunoprecipitated with Ggamma 2 under the same conditions (Fig. 4B, lanes 3 and 5).


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Fig. 4.   Expression and selective co-immunoprecipitation of Ggamma 2 with HA epitope-tagged wild-type or mutant Gbeta subunits in transfected HEK-293 cells. Cell lysates prepared 2 days after transient transfection were incubated with anti-HA monoclonal antibody, and after precipitation, the starting lysates (A) and washed immunoprecipitates (IP) (B) were analyzed in parallel for epitope-tagged Gbeta (HA antibody, upper panels) and Ggamma 2 (lower panels) immunoreactivity by immunoblotting as described under "Experimental Procedures." The relative mobility of the specific immunoreactive bands (in kDa) is indicated to the right of each panel as is the mobility of the immunoglobulin G heavy chain (HC) in B. Cells were transfected with either vector alone (vec) or with cDNAs encoding Ggamma 2, wild-type (wt), mutant HA-Gbeta 5, or wild-type HA-Gbeta 1 as indicated below the lanes. The Gbeta 5 mutants employed were Gbeta 5-QAAC (Gbeta 5-K25Q/E29A/K32A/L33C) and Gbeta 5-YMIN (Gbeta 5-F93Y/T338M/V351I/A353N). A representative result is shown in an experiment repeated two more times with similar findings.

The ability of the putative Ggamma -selective Gbeta 5 mutants to activate inositide-specific PLC-beta in co-transfected HEK-293 cells in a Ggamma -dependent fashion was also tested. Wild-type Gbeta 5 activated PLC-beta 2 in a Ggamma -dependent fashion in COS-7 cells as described previously (2, 4). Both mutants Gbeta 5-QAAC and Gbeta 5-YMIN activated PLC-beta 2 in a Ggamma -dependent fashion indistinguishable from the wild-type Gbeta 5 (data not shown).

RGS7 Requirement for Nuclear Expression of Gbeta 5 in HEK-293 Cells-- Because neither endogenous Gbeta 5 (36) nor GGL domain-containing RGS proteins such as RGS7 is expressed in HEK-293 cells (Fig. 5, A and B, lane 1), they offer a useful model system to study the requirements for the expression and subcellular localization of these subunits upon transfection. When transfected alone, wild-type Gbeta 5 can be detected in the whole cell lysate but not in the 293 cell nuclear extract (Fig. 5A, lane 2). Co-transfection of Gbeta 5 with either RGS7 or Ggamma 2 greatly enhances the Gbeta 5 signal in the cell lysate (Fig. 5A, lanes 3 and 4, cf. lane 2), presumably due to subunit stabilization by heterodimer formation (15, 35), but only RGS7 co-transfection supported expression of Gbeta 5 in the nuclear extract (Fig. 5A, lane 3).


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Fig. 5.   Expression and nuclear targeting of wild-type and mutant Gbeta subunits in transfected HEK-293 cells. Cell harvested 2 days after transient transfection with either wild-type Gbeta 5 (A) or HA epitope-tagged Gbeta 5 and Gbeta 5 mutants (B) were processed for either nuclear isolation and extraction (upper panels) or whole cell lysates (lower panels) as described under "Experimental Procedures." The relative mobility of the specific immunoreactive bands (in kDa), including that of TBP, is indicated to the right of each panel. The antibody used for immunodetection is indicated to the left of each panel. Cells were transfected with either vector alone (vec) or with cDNAs encoding RGS7, Ggamma 2, wild-type untagged, or HA epitope-tagged Gbeta 5 or mutant HA-Gbeta 5, as indicated below the lanes. A representative result is shown in an experiment performed three times with similar findings.

The expression and subcellular localization of HA epitope-tagged Gbeta 5, Gbeta 5-QAAC, and Gbeta 5-YMIN were also studied in transfected HEK-293 cells in similar experiments (Fig. 5B). As expected HA epitope-tagged wild-type Gbeta 5 was expressed in nuclear extracts upon co-transfection with RGS7 (Fig. 5B, lane 2) but not with Ggamma 2 (data not shown). Like wild-type Gbeta 5, mutant Gbeta 5-QAAC also demonstrated RGS7 dependence for nuclear localization (Fig. 5B, cf. lanes 3 and 4). In contrast the Ggamma -selective mutant Gbeta 5-YMIN failed to appear in the nuclear extract with either RGS7 or Ggamma 2 co-transfection (Fig. 5B, lanes 5 and 6). The expression of RGS7 in Gbeta 5-YMIN co-transfected cells could be demonstrated in whole cell lysates with immunoblots employing the polyclonal antibody 7RC-1 such as that shown in Fig. 5B (anti-RGS7 panel). Nevertheless, the expression of RGS7 in lysates of Gbeta 5-YMIN co-transfected cells was much lower than in wild-type Gbeta 5 and Gbeta 5-QAAC co-transfected cells (Fig. 5B, lane 6, cf. lanes 2 and 4). Indeed, blots of lysates from RGS7 and Gbeta 5-YMIN co-transfected HEK-293 cells employing antibodies of lesser avidity, such as those in Figs. 2B (lane 7 of Xpress panel) and 3B (lane 3 of lysate RGS7 (C-19) panel), fail to show an RGS7 band at all. The relative instability of RGS7 when not partnered with Gbeta 5 as a heterodimeric complex has been well documented in transfected COS-7 cells (15).

Subcellular Localization of Gbeta 5 Mutants in Transfected PC12 Cells-- PC12 cells, unlike HEK-293 cells, express endogenous Gbeta 5 (36). Recent laser confocal microscopic analysis of naive and transfected PC12 cells demonstrated Gbeta 5 in the cell nuclei and cytosol (17). The subcellular localization of the Gbeta 5-QAAC and Gbeta 5-YMIN mutants in transfected PC12 cells was therefore examined by antibody staining and confocal microscopy and compared with wild-type Gbeta 5 (Fig. 6). In these experiments no GGL domain containing RGS proteins or Ggamma subunits were co-transfected in order to allow the transfected Gbeta 5 constructs to "choose" binding partners from the endogenous pool of RGS proteins and Ggamma subunits present in PC12 cells (17, 37). Antibodies to the Gbeta 5 N terminus and the HA epitope tag both demonstrated the distribution of transfected wild-type Gbeta 5 throughout the PC12 cell including the cell nucleus (Fig. 6) as described previously (17). The subcellular localization of the Gbeta 5-QAAC mutant closely resembled that of wild-type Gbeta 5 (Fig. 6). In contrast the Gbeta 5-YMIN mutant was excluded from the PC12 cell nucleus when probed by either antibodies to the Gbeta 5 N terminus or to the HA epitope tag. The Gbeta 5-YMIN mutant-transfected cells were still capable of Gbeta 5 nuclear localization, because when non-epitope-tagged Gbeta 5 was co-transfected with the Gbeta 5-YMIN mutant, nuclear localization of non-tagged Gbeta 5 but not the HA-tagged mutant was evident in the same cells (Fig. 6).


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Fig. 6.   Confocal dual immunofluorescence analysis of PC-12 cells transiently transfected with HA epitope-tagged and/or wild-type Gbeta 5. Two days following transfection with the cDNAs indicated above the columns, cells were analyzed by laser confocal microscopy after dual staining with affinity-purified ATDG anti-Gbeta 5 antibody (green signal) and anti-HA antibody (red signal) as described under "Experimental Procedures." Images of the cells illuminated by transmitted light only and the corresponding immunofluorescence signal (monitored singly or in combination (merge)) are indicated. Yellow-orange signal indicates co-localization of probes. Results of one experiment are shown as representative of four independent experiments with similar findings.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The unique ability of Gbeta 5 to interact selectively with Galpha - and Gbeta gamma -regulated effectors as a Gbeta 5-Ggamma complex and with RGS proteins 6, 7, 9, and 11 as a Gbeta 5-RGS heterodimer may be critical to the role of Gbeta 5 in signal transduction. New approaches are needed to assess the biological importance of the Gbeta 5-Ggamma interactions so well documented in vitro since detergent-based purifications might miss or underestimate the presence of native Gbeta 5-Ggamma complexes. Besides the mutational strategy employed here, other potential analytical tools such as conformation-dependent antibodies or selective proteolysis specific for the Ggamma -bound conformation of Gbeta 5 would be valuable in this regard. Although well documented purifications from defined subcellular fractions of adult tissues have demonstrated only the presence of Gbeta 5-RGS complexes and not Gbeta 5-Ggamma complexes (15), the potential importance of Gbeta 5-Ggamma complex formation during ontogeny or in highly localized brain regions remains to be addressed.

Several observations in this study suggest Gbeta 5 and its mutants are capable of a wider range of interactions with Ggamma 2 and/or RGS7 than the most stringent assays, such as co-immunoprecipitation, would seem to suggest. Both wild-type Gbeta 5 and the Gbeta 5-QAAC mutant stimulated PLC-beta in a Ggamma 2-dependent fashion, although neither co-immunoprecipitated Ggamma 2. Co-transfection of RGS7 was observed to enhance the expression of the Gbeta 5-YMIN mutant even though the pair could not be immunoprecipitated. Several laboratories (15, 35) have documented the ability of Gbeta 5-RGS and Gbeta 5-Ggamma protein interaction to mutually stabilize the heterodimeric subunits. Since the Gbeta 5-YMIN mutant was designed to impair interaction between a hydrophobic pocket on the side of the Gbeta 5 beta -propeller and a critical Trp residue in the RGS protein GGL domain (10), the ability of RGS7 to partially stabilize the Gbeta 5-YMIN mutant may reflect an RGS7-Gbeta 5 interaction outside this vicinity. The potential for such an interaction was recently demonstrated in C. elegans between an isolated N-terminal fragment of the RGS7 homolog EGL-10 and the Gbeta 5 homolog GPB-2 (38). The finding that only the Gbeta 5-YMIN mutant and Gbeta 1 were able to co-immunoprecipitate Ggamma 2 from detergent lysates complements an experiment performed by Snow et al. (10) in which the strength of Gbeta 5-Ggamma 2 interaction in detergent solution was greatly enhanced by replacing the conserved Phe-61 in Ggamma 2 with a corresponding Trp residue characteristic of GGL domains, and further supports their model of the Gbeta 5-GGL/Ggamma interface.

In previous studies of Gbeta 5 nuclear localization the possible requirement for Gbeta 5-Ggamma interaction for nuclear targeting was not fully assessed (17). Nuclear localization of G protein heterotrimers including apparent Gbeta gamma complexes has been reported in rat liver (39) and thrombin- and phorbol ester-treated Swiss 3T3 cells (40). In the previous study of Gbeta 5, a mutant GFP-Gbeta 5 fusion protein that failed to undergo nuclear localization was likely deficient in both Ggamma and GGL domain interaction due to double proline insertions in the Gbeta 5 alpha -helical N-terminal region (17). This approach thus provided little insight into the heterodimerization requirements for Gbeta 5 nuclear targeting.

The studies described herein strongly suggest Ggamma interaction is insufficient for Gbeta 5 nuclear targeting and that heterodimerization with a GGL domain-containing RGS protein such as RGS6 or -7 is critical. In HEK-293 cells, which do not express endogenous Gbeta 5 or RGS7, the nuclear targeting of wild-type Gbeta 5 (or the RGS7-interacting Gbeta 5-QAAC mutant) could only be reconstituted by RGS7 and not Ggamma 2 co-transfection. Next, because the Gbeta 5-YMIN mutant failed to localize to the nucleus in either 293 or PC12 cells, despite its tight binding to Ggamma demonstrated in vitro, the key molecular determinants of nuclear targeting appear to be intrinsic to the RGS protein, not to the Gbeta 5 or Ggamma subunits. However, the approach utilized here cannot exclude that mutation of the 4 residues in the Gbeta 5-YMIN mutant abrogated a nuclear localization signal (NLS), but this is unlikely for two reasons. No recognizable NLS motifs could be discerned in the native Gbeta 5 sequence (41). Furthermore, when tested in HEK-293 cells neither untagged or HA epitope-tagged wild-type Gbeta 5 demonstrated nuclear expression when co-transfected with Ggamma 2 making the presence of an occult NLS on Gbeta 5 unlikely.

Demonstration of a requirement for RGS protein binding is consistent with several reports demonstrating the nuclear targeting of RGS2 (42-44), RGS10 (42, 45), splice variants of RGS3 (46), and RGS12 (47). Mutation of a key serine residue in a conserved 14-3-3-binding site in the core RGS domain of RGS7 (Ser434 right-arrow Ala), a site present in many RGS proteins including RGS2 and RGS3 (48), made no difference in the steady-state distribution of RGS7 in resting PC12 cells (data not shown). This would seem to argue against a major role for 14-3-3 binding in the regulation of nucleocytoplasmic distribution of Gbeta 5-RGS complexes in resting cells. Whether phosphorylation or other post-translational modification of Gbeta 5-RGS complexes might govern their nuclear localization under stimulated conditions, and how potential nucleocytoplasmic shuttling of Gbeta 5-RGS heterodimers might mediate information transfer in the cell in response to extracellular signals remain two central questions for future study.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Medical College of Georgia, School of Medicine, 1120 15th St., Augusta, GA 30912.

To whom correspondence should be addressed: NIDDK, Metabolic Diseases Branch, Bldg. 10, Rm. 8C-101, 10 Center Dr., MSC 1752, National Institutes of Health, Bethesda, MD 20892-1752. Tel.: 301-496-9299; Fax: 301-402-0374; E-mail: wfs@helix.nih.gov.

Published, JBC Papers in Press, January 24, 2003, DOI 10.1074/jbc.M207302200

    ABBREVIATIONS

The abbreviations used are: RGS, regulators of G protein signaling; GFP, green fluorescent protein; GGL, Ggamma -like; GCG, Genetics Computer Group; DMEM, Dulbecco's modified Eagle's medium; HEK, human embryonic kidney; TBP, TATA-binding protein; PLC, phospholipase C; NLS, nuclear localization signal; HA, hemagglutinin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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