Ribozyme Approach Identifies a Functional Association between the G Protein beta 1gamma 7 Subunits in the beta -Adrenergic Receptor Signaling Pathway*

Qin Wang, Bashar K. MullahDagger , and Janet D. Robishaw§

From the Henry Hood M.D. Research Program, Pennsylvania State University College of Medicine, Danville, Pennsylvania 17822 and Dagger  Applied Biosystems, Foster City, California 94404

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The complex role that the heterotrimeric G proteins play in signaling pathways has become increasingly apparent with the cloning of countless numbers of receptors, G proteins, and effectors. However, in most cases, the specific combinations of alpha  and beta gamma subunits comprising the G proteins that participate in the most common signaling pathways, such as beta -adrenergic regulation of adenylyl cyclase activity, are not known. The extent of this problem is evident in the fact that the identities of the beta gamma subunits that combine with the alpha  subunit of Gs are only now being elucidated almost 20 years after its initial purification. In a previous study, we described the first use of a ribozyme strategy to suppress specifically the expression of the gamma 7 subunit of the G proteins, thereby identifying a specific role of this protein in coupling the beta -adrenergic receptor to stimulation of adenylyl cyclase activity in HEK 293 cells. In the present study, we explored the potential utility of a ribozyme approach directed against the gamma 7 subunit to identify functional associations with a particular beta  and alpha s subunit of the G protein in this signaling pathway. Accordingly, HEK 293 cells were transfected with a ribozyme directed against the gamma 7 subunit, and the effects of this manipulation on levels of the beta  and alpha s subunits were determined by immunoblot analysis. Among the five beta  alpha s subunits detected in these cells, only the beta 1 subunit was coordinately reduced following treatment with the ribozyme directed against the gamma 7 subunit, thereby demonstrating a functional association between the beta 1 and gamma 7 subunits. The mechanism for coordinate suppression of the beta 1 subunit was due to a striking change in the half-life of the beta 1 monomer versus the beta 1 heterodimer complexed with the gamma 7 subunit. Neither the 52- nor 45-kDa subunits were suppressed following treatment with the ribozyme directed against the gamma 7 subunit, thereby providing insights into the assembly of the Gs heterotrimer. Taken together, these data show the utility of a ribozyme approach to identify the role of not only the gamma  subunits but also the beta  subunits of the G proteins in signaling pathways.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The most common type of signaling pathway requires the sequential interactions of a seven-transmembrane receptor, a G protein composed of alpha , beta , and gamma  subunits, and an effector. How the specificity of this signaling pathway is encoded in the protein-protein interactions of ever increasing numbers of these signaling partners is not known. A simple way to encode the specificity would be for each receptor to interact with a specific G protein alpha beta gamma heterotrimer. In this regard, the recent identification of 23 alpha  subunits (1), beta  subunits (2, 3), and 12 gamma  subunits (4-6) predicts the potential existence of several hundred G protein alpha beta gamma heterotrimers that could serve as intermediaries between a similarly high number of receptors and a somewhat smaller number of effectors. Supporting this scenario, there is increasing evidence that the subunit composition of G protein alpha beta gamma heterotrimers may provide the level of selectivity that is needed to interact with the multitude of receptors and effectors that are now known to exist. Antisense (7, 8) and ribozyme (9) strategies have proven to be most useful in identifying which of the potential G protein alpha beta gamma heterotrimers are physiologically relevant. These approaches have the advantage that they allow the selective suppression of individual G protein subunits and the subsequent identification of which signaling pathway(s) are impaired.

In a recent study, we described the first use of the ribozyme strategy to suppress specifically the expression of G protein gamma 7 subunit, thereby identifying a specific role of this subtype in coupling the beta -adrenergic receptor to stimulation of adenylyl cyclase activity in HEK 293 cells (9). In the present study, we have explored the use of the same ribozyme approach to identify functional associations of the G protein gamma 7 subunit with particular G protein beta  or alpha  subunits in this signaling pathway. To this end, a ribozyme directed against the gamma 7 subunit was transfected into HEK 293 cells to suppress specifically the expression of the gamma 7 protein, and then the effects of this manipulation on the levels of the beta  and alpha s subunits of the G proteins were determined. Of the beta  subunits, only the beta 1 subunit was coordinately reduced following treatment with the ribozyme directed against the gamma 7 subunit, thereby demonstrating a functional association between the beta 1 and gamma 7 subunits. By contrast, neither the 52- nor 45-kDa alpha s subunits were suppressed following treatment with the ribozyme directed against the gamma 7 subunit. Taken together, the results indicated that the gamma 7 and beta 1 subunits play a specific role in the beta -adrenergic receptor signaling pathway and that their role cannot be compensated for by other members of these large, multi-gene families.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ribozyme Design and Delivery to HEK 293 Cells-- A chimeric DNA-RNA hammerhead ribozyme targeted against the G protein gamma 7 subunit mRNA was chemically synthesized and modified by adding two phosphorothioate linkages at the 3'-end, as described previously (9). Also, HEK 293 cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and plated into 100-mm dishes, as described previously (9). The cells were then transfected at approximately 60-80% confluence with fresh serum-free medium containing premixed gamma 7 ribozyme (2 µM) and the cationic lipid, LipofectAMINE (20 µg/ml, Life Technologies, Inc.). At 5 h post-transfection, fetal bovine serum was added to a final concentration of 6%, and sequential addition of 0.5 µM ribozyme was supplemented to the total concentration of 4 µM at 48 h post-transfection. The control cells were treated identically but without added ribozyme.

Immunoblot Analysis-- To determine the effect of ribozyme treatment on G protein subunits, membranes from control and gamma 7 ribozyme-transfected cells were extracted with 1% sodium cholate overnight as described previously (10). The protein concentrations were determined by Amido Black assay, and equal amounts of proteins were resolved on 15% SDS-polyacrylamide gel and transferred to Nitro-plus nitrocellulose (0.45-µm pore size, Micron Separations Inc.) using a high temperature procedure described previously (11). Following transfer to nitrocellulose, the blots were probed with anti-G protein subtype-specific antibodies (9, 11-14). Briefly, after blocking, the nitrocellulose blots were incubated with the primary antibodies for 1 h in high detergent blotto at a dilution of 1:500 for B-69 (beta 1); a dilution of 1:150 for D-17-6 (beta 2); a dilution of 1:100 for B-24 (beta 3); a concentration of 1 µg/ml for C-16 (beta 4) (Santa Cruz Biotechnology Inc.); a dilution of 1:2000 for beta 5 (CytoSignal Research Products); and a dilution of 1:500 for 584 (Galpha s). After three successive washes, the blots were incubated for 1 h with 125I-labeled goat anti-rabbit F(ab')2 fragment (1 × 105 dpm/ml, NEN Life Science Products) in high detergent blotto. After washing, the blots were subjected to autoradiography by exposure to BioMax MS film (Eastman Kodak Co.), and the intensity of immunodetectable bands was quantified using the Molecular Dynamics PhosphorImager SI.

Subcellular Fractionation-- After transfection, cells were washed with ice-cold phosphate-buffered saline and then lysed with ice-cold lysis buffer which contains 2 mM MgCl2, 1 mM EDTA, 20 mM Hepes, 10 mM dithiothreitol, 1 mM aminoethylbenzenesulfonyl fluoride, 1 µg/ml pepstatin A, and 1 mM benzamidine. The separation of the cytosolic and particulate fractions was accomplished by centrifugation at 250,000 × g for 30 min. The particulate was extracted with 1% sodium cholate overnight at 4 °C. Equal percentages of the cytosolic and solubilized particulate fractions from control and ribozyme-transfected cells were resolved by SDS-polyacrylamide gel electrophoresis and subjected to immunoblot analysis.

Construction of FLAG-tagged Human beta 1 cDNA and Hemagglutinin-tagged Human gamma 7 cDNAs-- In order to monitor the half-life of beta 1 subunit in the intact cell setting, epitope tagging was used in this study. The FLAG mammalian transient expression system was used for constructing FLAG-tagged human beta 1 subunit, and the influenza virus hemagglutinin (HA)1 tag was used for constructing HA-tagged human gamma 7 subunit cDNA. The FLAG system is designed for intracellular expression of amino-terminal Met-FLAG fusion protein which relies on the fusion of the FLAG peptide of eight amino acids (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) to the coding sequence of a target gene in a pCMV5 expression vector. This FLAG epitope tag is recognized by an anti-FLAG M2 monoclonal antibody (Sigma). The human beta 1 subunit cDNA coding region was generated by polymerase chain reaction and then cloned into the pFLAG-CMV-2 vector. The human G protein gamma 7 subunit cDNA coding region was generated by polymerase chain reaction with 5' primer sequence coding for nine amino acids of HA tag epitope (Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) (15) and then cloned into the pCI-neo Mammalian Expression Vector (Promega Corp., Madison, WI). A mutant form of the HA-tagged gamma 7m cDNA was generated in the same way by mutating Cys to Ser in the carboxyl-terminal CAAX box, thereby preventing its modification by isoprenylation (16). This HA epitope tag is recognized by anti-HA monoclonal antibody (Roche Molecular Biochemicals).

Metabolic Labeling and Immunoprecipitation-- Following transfection, the FLAG-tagged beta 1 protein and HA-tagged gamma 7 protein were detected by immunoprecipitation with anti-FLAG M2 monoclonal antibody and anti-HA high affinity monoclonal antibody. Briefly, HEK 293 cells grown in 60-mm dishes were transfected with a plasmid encoding either FLAG-tagged hybrid protein beta 1 subunit or HA-tagged gamma 7 protein by LipofectAMINE transfection method. At 24 h post-transfection, cells were incubated for 45 min in methionine- and cysteine-free Dulbecco's modified Eagle's medium and then pulse-labeled with 70-100 µCi of [35S]methionine (NEN Life Science Products) either for 2.5 min for monomer detection or 1 h for dimer detection, and then chased for the time points indicated in the figure legends. The incorporation of label was stopped by addition of complete medium supplemented with non-radioactive methionine at a final concentration of 1 mM. Cells were lysed in the lysis buffer (50 mM Tris-HCl, pH 7.6, 5 mM MgCl2, 0.5% Nonidet P-40, 0.1% Lubrol, 1 mM EDTA, 10 µg/ml leupeptin, 10 mM benzamidine, 1 mM aminoethylbenzenesulfonyl fluoride, and 10 µg/ml pepstatin A) and then passed through 25-gauge 5/8 needle and freeze-thawed once in -80 °C freezer. The lysates were depleted of nuclei and cell debris by centrifugation for 10 min at 14,000 rpm in an Eppendorf centrifuge and then pre-cleared twice with 20 µl of protein A/G plus-agarose (Santa Cruz Biotechnology). Equal amounts of control and transfected cells were subjected to immunoprecipitation. For recovery of the beta 1 subunit in the monomer form, immunoprecipitation was performed by overnight incubation at 4 °C on a rocker with 15 µg/ml anti-FLAG M2 monoclonal antibody. For recovery of the beta 1 subunit in the dimer form, immunoprecipitation was carried out with 5 µg/ml anti-HA high affinity monoclonal antibody. The immune complexes were precipitated by adsorption to protein A/G plus-agarose for an additional 3 h at 4 °C followed by four washes in NET buffer (50 mM Tris-HCl, pH 7.6, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40). Finally, the immune complexes were dissociated by heating for 10 min in SDS sample buffer. Protein A/G plus-agarose beads were pelleted by centrifugation, and the supernatants were resolved by SDS-polyacrylamide gel electrophoresis. The gel was fixed and soaked for 30 min in Amplify reagent (Amersham Pharmacia Biotech) and then dried and exposed to Kodak BioMax MS film with Kodak Biomax Transcreen-LE Intensifying Screen at -80 °C. The intensity of the immunodetectable bands on the autoradiogram was quantified by densitometry analysis.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of Ribozyme-mediated Suppression of the gamma 7 Subunit on the Levels of beta  Subunits in HEK 293 Cells-- Under physiological conditions, the beta  and gamma  subunits exist as a dimer that functions as a single entity (17-20). Synthesis and assembly of the beta gamma dimer begins in the cytosol, with subsequent translocation to the plasma membrane being dependent on post-translational lipid modifications of the gamma  subunit (21, 22). Therefore, we reasoned that ribozyme-mediated loss of the gamma 7 protein might limit the formation and translocation of a specific beta gamma dimer to the plasma membrane. In this event, we expected that the content of one or more beta  subunits might be reduced in the plasma membrane. Accordingly, the expression of the known beta  subunits was examined following the treatment of HEK 293 cells with ribozyme specific for the gamma 7 subunit.

To date, six beta  subunits have been identified by molecular cloning (2, 3). To determine which of these are expressed in HEK 293 cells, it was first necessary to generate (for beta 1, beta 2, and beta 3) or obtain commercially available (for beta 4 and beta 5) antibodies specific for each beta  subtype. Then, the specificities of these antibodies were determined by immunoblot analysis of the recombinantly expressed beta  proteins. As shown in Fig. 1, the sizes of the recombinantly expressed beta 1, beta 2, beta 3/6, beta 4, and beta 5 proteins ranged from 35 to 39 kDa and the antibodies reacted only with their corresponding beta  proteins. To determine whether the ribozyme-mediated loss of the gamma 7 protein would affect the expression of one or more of these beta  subunits, membrane proteins from HEK 293 cells treated in the absence (C) or presence (RZ) of the ribozyme were immunoblotted with these beta  subtype-specific antibodies. As shown in Fig. 2A and quantified for three separate experiments in Fig. 2B, the beta 1, beta 2, beta 4, and beta 5 subunits were readily detected in membranes from HEK 293 cells treated in the absence (C) or presence (RZ) of the ribozyme. Under the same condition, the beta 3 subunit was not expressed at a detectable level, but whether this is due to the lack of expression or the relatively poor affinity of the beta 3 subtype-specific antibody is not known. When the relative amounts of the beta 1, beta 2, beta 4, and beta 5 subunits were quantified in the ribozyme-treated membranes and then expressed as a percentage of their levels in control membranes, the levels of the beta 2, beta 4, and beta 5 subunits showed no reduction. However, the level of the beta 1 subunit showed a striking reduction in ribozyme-treated membranes to 30.2 ± 6.9% of its level in control membranes. By way of comparison, the level of the gamma 7 subunit showed a similarly striking loss in ribozyme-treated membranes to 21 ± 8.4% of its level in control membranes (Fig. 2, A and B). These data showing coordinate suppression of the beta 1 subunit along with the gamma 7 subunit provide strong evidence for their functional association in the intact cell setting.


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Fig. 1.   Specificity of G protein beta  subtype-specific antibodies. The recombinantly expressed G protein beta 1, beta 2, beta 3, and beta 5 subunits were resolved on a 15% SDS-polyacrylamide gels, transferred to nitrocellulose, and then immunoblotted with beta  subtype-specific antibodies, as described previously (11, 12). Since recombinantly expressed G protein beta 4 subunit is not available yet, a bovine brain membrane preparation containing this protein was used as positive control. The sizes of the various beta  subtypes ranged from 35 to 39 kDa. Br, brain.


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Fig. 2.   Ribozyme suppression of G protein beta  subtypes in HEK 293 cells at the protein level. Membrane proteins from control (C) and gamma 7 ribozyme-transfected (RZ) cells were extracted, resolved on 15% SDS-polyacrylamide gels, transferred to nitrocellulose, and immunoblotted as described under "Experimental Procedures." Following transfer, the nitrocellulose was cut along the 30-kDa marker; the higher molecular blot was probed with one of the beta  subtype-specific antibodies, and the lower molecular blot was probed with the gamma 7 subtype-specific antibody (A-67). A, shows representative immunoblots demonstrating the selective loss of the beta 1 and gamma 7 subunits. B, quantifies the loss of the beta 1 and gamma 7 subunits in the gamma 7 ribozyme-treated cells. The intensities of the bands from three separate experimental sets of immunoblots were determined by PhosphorImager analysis. The relative amounts of proteins in the gamma 7 ribozyme-treated cells were expressed as a percentage of their levels in the control cells. The data shown are mean ± S.E.

Mechanism of Loss of the beta 1 Protein-- The resulting decrease in the expression of the beta 1 protein following ribozyme treatment indicated that the gamma 7 protein is required for the appearance of the beta 1 protein in the membrane. This requirement could reflect the need for the gamma 7 protein to allow the translocation of the beta 1 protein from its place of synthesis in the cytosol to the site of its function in the plasma membrane. To examine this possibility, subcellular fractionation experiments were performed. HEK 293 cells treated in the absence or presence of ribozyme directed against the gamma 7 subunit were fractionated and the resulting soluble and particulate fractions were subjected to immunoblot analysis with the beta 1 subtype-specific antibody (B-69). As shown in Fig. 3A and quantified for three separate experiments in Fig. 3B, the amount of beta 1 protein in the particulate fraction was reduced to 38 ± 2.98% (n = 3) in the ribozyme-treated cells (RZ) compared with the control cells (C). However, this loss in the particulate fraction did not result in detectable accumulation of the beta 1 protein in the cytosolic fraction in the ribozyme-treated cells (RZ, S) compared with the control cells (C, S). These data support the notion that the beta 1 protein, without benefit of association with gamma 7 protein, may be rapidly degraded.


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Fig. 3.   Subcellular fractionation of beta 1 subunit. After transfection, control (C) and gamma 7 ribozyme-treated cells (RZ) cells were lysed, separated into soluble (S) and particulate (P) fractions by centrifugation at 250,000 × g, and then equal percentages of these fractions were resolved on 15% SDS-polyacrylamide gels, transferred to nitrocellulose, and immunoblotted with the beta 1-specific antibody (B-69). A shows representative immunoblots demonstrating the loss of the beta 1 subunit from the particulate fraction of the gamma 7 ribozyme-treated cells (RZ, P) compared with the control (C, P) but no detectable accumulation of beta 1 subunit in the soluble fraction (RZ, S) compared with control (C, S). B quantifies the change in the beta 1 subunit. The intensities of the bands from three separate experimental sets of immunoblots were determined by densitometric analysis. The relative amounts of proteins in the particulate fraction from the gamma 7 ribozyme-treated cells (RZ) were expressed as a percentage of their levels in the control (C) cells. The data shown are mean ± S.E.

Change in Half-life of beta 1 Subunit in Monomeric Versus Heterodimeric State-- To test if the stability of the beta 1 subunit is different in the monomeric state versus heterodimeric state in complex with the gamma 7 subunit, pulse-chase labeling studies were performed for each condition. For this purpose, FLAG- and HA-tagged versions of the human beta 1 and gamma 7 subunits were constructed, respectively. Previous studies have shown that the use of these epitope tags does not interfere with assembly of the beta gamma dimer (23, 24). Moreover, these tagged proteins can be readily monitored with well characterized monoclonal antibodies whose immunoprecipitation capabilities and spectrums of cross-reactivity with non-tagged proteins are already known.

To determine the half-life of the beta 1 monomer, HEK 293 cells expressing the FLAG-tagged beta 1 subunit alone were labeled with [35S]methionine for 2.5 min and then chased for various time points. Subsequently, cells were lysed and immunoprecipitated with the anti-FLAG M2 antibody in the presence of 0.5% SDS for the recovery of beta 1 monomer. The immune complexes were precipitated, denatured in SDS sample buffer, and resolved on 15% SDS-polyacrylamide gels. As shown in Fig. 4A, the 35S-labeled beta 1 monomer was readily recovered at 0 min of chase. However, essentially no labeled beta 1 monomer was detected by 3 h of chase. To determine the half-life of the beta 1 monomer more accurately, shorter chase periods were employed. As shown in Fig. 4B and quantified for three separate experiments in Fig. 4C, there was progressive loss of the 35S-labeled beta 1 monomer from 0 to 3 h of chase. Based on densitometric and curve fitting analysis, the loss of the labeled beta 1 monomer was best fit to a single phase exponential decay, with an estimated half-life of only 20.8 min.


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Fig. 4.   Half-life of the beta 1 monomer. At 24 h post-transfection with the FLAG-tagged human beta 1 subunit, HEK 293 cells were pulse-labeled with 70 µCi of [35S]methionine for 2.5 min and chased for the indicated time points. The 35S-labeled beta 1 subunit was immunoprecipitated with anti-FLAG M2 monoclonal antibody for recovery of beta 1 monomer and then resolved on 15% SDS-polyacrylamide gels. Gels were fixed, dried, and exposed to Kodak BioMax MS film with a Kodak Biomax Transcreen-LE Intensifying Screen at -80 °C. The images were quantified by densitometric analysis. A and B show representative autoradiograms of the 35S-labeled beta 1 monomer over longer and shorter time points, respectively. C quantifies the loss of the 35S-labeled beta 1 monomer over the shorter time points (B). Curve fitting analysis of the results from three separate experimental sets revealed the loss of the 35S-labeled beta 1 monomer was best fit to a single phase exponential decay, with an estimated half-life of only 20.8 min.

To determine the half-life of beta 1 protein in a heterodimeric complex with the gamma 7 protein, HEK 293 cells expressing the beta 1 subunit alone or in combination with the HA-tagged gamma 7 subunit were labeled with [35S]methionine for 1 h. Then cells were lysed and immunoprecipitated with the anti-HA antibody in the absence or presence of 0.5% SDS. Subsequently, immune complexes were resolved on 15% SDS-polyacrylamide gels. As shown in Fig. 5, recovery of the 35S-labeled gamma 7 subunit was the same when immunoprecipitated with the anti-HA antibody in the presence (lane 1) or absence (lane 2) of SDS. By contrast, recovery of the labeled beta 1 subunit was very different between these two conditions. No labeled beta 1 subunit was detected when the gamma 7 subunit was immunoprecipitated in the presence of SDS (lane 1), but it was readily observed when the gamma 7 subunit was immunoprecipitated in the absence of SDS (lane 2). These results indicated that the beta 1 subunit is present as a complex with the gamma 7 subunit in the absence of SDS and, as a result, can be brought down with the anti-HA antibody directed against the gamma 7 subunit. Without transfection of gamma 7 subunit (lane 3) or without addition of anti-HA antibody (lane 4), neither the beta 1 subunit nor the gamma 7 subunit were immunoprecipitated, showing that the appearance of the beta 1 subunit is dependent on the presence of the gamma 7 subunit or the anti-HA antibody, respectively.


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Fig. 5.   Detection of the beta 1gamma 7 dimer by immunoprecipitation. HEK 293 cells expressing the beta 1 subunit alone or in combination with the HA-tagged gamma 7 subunit were labeled with [35S]methionine for 1 h. Cells were then lysed and immunoprecipitated with the anti-HA antibody in the absence or presence of 0.5% SDS. Subsequently, immune complexes were resolved on 15% SDS-polyacrylamide gels, and the gels were fixed, dried, and exposed to Kodak BioMax MS film with a Kodak Biomax Transcreen-LE Intensifying Screen at -80 °C.

By having validated this method for detection of labeled beta 1 subunit in the heterodimeric state, we sought to determine the half-life of the beta 1 subunit when complexed with the gamma 7 subunit. For this purpose, HEK 293 cells expressing the beta 1 subunit and the HA-tagged gamma 7 subunit together were labeled with [35S]methionine for 1 h, chased for the various time points, and then immunoprecipitated with the anti-HA antibody in the absence of SDS. As shown in Fig. 6A and quantified for three separate experiments in Fig. 6B, there was progressive loss of the 35S-labeled beta 1 protein from 0 to 54 h of chase. Based on densitometric and curve fitting analysis, the loss of the majority of the labeled beta 1 subunit when complexed with the wild type HA-tagged gamma 7 subunit was best fit to a single phase exponential decay, with an estimated half-life of 14.2 h. However, a small portion of the labeled beta 1 protein in the heterodimeric state was stable for at least 54 h. Taken together, these data indicated that the beta 1gamma 7 dimer is turned over >40-fold more slowly than the beta 1 monomer in the intact cell setting.


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Fig. 6.   Half-life of the beta 1 subunit complexed with the wild type HA-tagged gamma 7 subunit. HEK 293 cells co-expressing beta 1 subunit and HA-tagged gamma 7 subunit were labeled with [35S]methionine for 1 h, chased for the various time points, and then immunoprecipitated with the anti-HA antibody in the absence of SDS for recovery of the HA-gamma 7 subunit complexed with the beta 1 subunit. The immune complexes were resolved on 15% SDS-polyacrylamide gels, and the gels were fixed, dried, and exposed to Kodak BioMax MS film with a Kodak Biomax Transcreen-LE Intensifying Screen at -80 °C. The images were quantified by densitometric analysis. A shows a representative autoradiogram of the 35S-labeled beta 1 complexed with the HA-gamma 7 subunit. B quantifies the loss of the 35S-labeled beta 1 complexed with the HA-gamma 7 subunit. Curve fitting analysis of the results from three separate experimental sets revealed the loss of the 35S-labeled beta 1 in the heterodimeric state was best fit to a single phase exponential decay, with an estimated half-life of 14.2 h. For this analysis, the "0" time point was extrapolated.

To determine whether association with the gamma 7 subunit is sufficient to stabilize the half-life of the beta 1 subunit or whether translocation of the beta 1gamma 7 complex to the membrane is required for this stabilization, we generated a mutant form of the HA-tagged gamma 7 subunit by replacing the Cys residue with a Ser residue in the carboxyl-terminal "CAAX" motif. As a result of this substitution, the mutant HA-tagged gamma 7 subunit is not able to undergo prenylation or translocation to the membrane (16). HEK 293 cells expressing the beta 1 subunit and the mutant HA-tagged gamma 7 subunit together were labeled with [35S]methionine for 1 h, chased for the various time points, and then immunoprecipitated with the anti-HA antibody in the absence of SDS. As shown in Fig. 7A and quantified for two separate experiments in Fig. 7B, there was a progressive and similar loss of the 35S-labeled beta 1 protein whether complexed with mutant or wild type HA-tagged gamma 7 subunit (compare with Fig. 6A). Based on densitometric and curve fitting analysis, the loss of the majority of the labeled beta 1 subunit when complexed with the mutant HA-tagged gamma 7 subunit was best fit to a single phase exponential decay, with an estimated half-life of 16.3 h. These data demonstrated that association with the gamma 7 subunit is sufficient for stabilization of the beta 1 subunit.


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Fig. 7.   Half-life of the beta 1 subunit complexed with the mutant HA-tagged gamma 7 subunit. A mutant form of the HA-tagged gamma 7 subunit was generated by replacing the Cys residue with a Ser residue in the carboxyl-terminal CAAX motif. HEK 293 cells co-expressing the beta 1 subunit and the mutant HA-tagged gamma 7 subunit were labeled with [35S]methionine for 1 h, chased for the various time points, and then immunoprecipitated with the anti-HA antibody in the absence of SDS. The immune complexes were resolved on 15% SDS-polyacrylamide gels, and the gels were fixed, dried, and exposed to Kodak BioMax MS film with a Kodak Biomax Transcreen-LE Intensifying Screen at -80 °C. The images were quantified by densitometric analysis. A shows a representative autoradiogram of the 35S-labeled beta 1 complexed with the mutant HA-gamma 7 subunit. B quantifies the loss of the 35S-labeled beta 1 complexed with the mutant HA-gamma 7 subunit. Curve fitting analysis of the results from two separate experimental sets revealed the loss of the 35S-labeled beta 1 complexed with the mutant HA-gamma 7 subunit was best fit to a single phase exponential decay, with an estimated half-life of 16.3 h. For this analysis, the 0 time point was extrapolated.

Effect of Ribozyme-mediated Suppression of the gamma 7 Subunit on the Levels of 45 and 52 kDa alpha s Subunits in HEK 293 Cells-- To determine whether a ribozyme directed against the gamma 7 subunit would also affect the expression of one or more of the alpha s subunits, membrane proteins from HEK 293 cells treated in the absence or presence of the ribozyme against gamma 7 subunit were immunoblotted with the G protein alpha s subtype-specific antibody (antibody 584). As shown in Fig. 8A, both the 45- and 52-kDa alpha s subunits, which are derived from alternative splicing (25), were detected in control and ribozyme-treated cells. The relative amounts of the two forms of alpha s subunits were quantified in control and gamma 7 ribozyme-treated cells by densitometry and then expressed as percentages of their levels in control cells for three separate experiments in Fig. 8B. There were no differences in the intensities of the 45- and 52-kDa alpha s subunits between the control and ribozyme-treated cells even though the intensities of the beta 1 and gamma 7 subunits were markedly and coordinately suppressed in the ribozyme-treated cells.


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Fig. 8.   Lack of ribozyme suppression of G protein as subtypes in HEK 293 cells at the protein level. Membrane proteins from control (C) and gamma 7 ribozyme-transfected (RZ) cells were extracted, resolved on 15% SDS-polyacrylamide gels, transferred to nitrocellulose, and immunoblotted as described under "Experimental Procedures." A shows representative immunoblots demonstrating no loss of the alpha s subtypes even though selective loss of the beta 1 and gamma 7 subunits occurred. B quantifies the results in control (C) versus gamma 7 ribozyme-treated (RZ) cells. The intensities of the bands from three separate experimental sets of immunoblots were determined by PhosphorImager analysis. The relative amounts of proteins in the gamma 7 ribozyme-treated cells were expressed as a percentage of their levels in the control cells. The data shown are mean ± S.E.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The G protein beta gamma subunits exist as a tightly associated complex that plays a prominent role in transducing information from a receptor to an appropriate effector in a specific fashion. Thus, the beta gamma subunit complex binds directly to receptors (26-28), where it may act as a conformational switch to direct receptor-G protein coupling (29). Also, the beta gamma subunit complex interacts directly with a variety of effectors to regulate their activities (for reviews see Refs. 4 and 30). Finally, the beta gamma subunit complex regulates kinases involved in desensitization of receptors (31). Recently, in vitro (32-36) and in vivo (7-9) studies raise the strong possibility that the specific composition of the beta gamma subunit complex contributes to the recognition of these signaling components.

With the identification of 6 beta  subtypes (2, 3) and 12 gamma  subtypes (4-6), it has become increasingly important to decipher which beta gamma subunit complexes actually exist in intact cells and to identify their roles in particular signaling pathways. In vitro strategies have revealed that certain combinations are not possible, but most combinations are physically able to form beta gamma dimers (18, 37, 38). While providing valuable information on structure-function relationships, these reconstitution approaches fall critically short of establishing the roles of specific beta gamma subunits in particular signaling pathways in the intact cell setting. Increasingly, reverse genetics strategies are being employed to fill this gap (7-9). In a previous study, we used the ribozyme approach to identify a specific role of the gamma 7 subunit in the beta -adrenergic receptor signaling pathway (9). HEK 293 cells transfected with a ribozyme directed against the gamma 7 subunit showed a specific reduction of the gamma 7 protein that was associated with a significant attenuation of isoproterenol-stimulated adenylyl cyclase activity. In the present study, we asked whether loss of the gamma 7 protein would have any effect on the expression and/or membrane localization of the associated beta  and alpha s proteins that comprise the G protein heterotrimer in the beta -adrenergic receptor signaling pathway. Our results show the first successful use of a ribozyme approach directed against a specific gamma  subunit to identify a functional association with a particular beta  subunit.

Functional Association of beta 1 and gamma 7 Subunits-- The beta  subunit is synthesized in the cytosol and then translocated to the membrane upon association with the appropriately modified gamma  subunit (21, 22). This suggested the possibility that ribozyme-mediated loss of the gamma 7 protein might be a useful approach to obtain information on its functional association with a particular beta  subtype. Following treatment of HEK 293 cells with ribozyme specific for the gamma 7 subunit, only the beta 1 protein showed a coordinate reduction with the gamma 7 protein in the membranes of ribozyme-treated cells compared with control cells (Fig. 2). Next, the mechanism underlying the coordinate suppression of the beta 1 protein was explored. Subcellular fractionation studies revealed that loss of the beta 1 protein in the membrane did not lead to any detectable increase in the beta 1 protein in the cytosol of ribozyme-treated cells. However, pulse-chase labeling studies showed a dramatic difference in the half-life of the beta 1 monomer (20.8 min, Fig. 4) compared with the beta 1gamma 7 dimer (14.2 h, Fig. 6). These data indicate that the beta 1 protein is rapidly and specifically degraded when sufficient gamma 7 protein is not available for dimerization. The gamma 7 protein undergoes post-translational processing, including prenylation and carboxyl methylation (16, 20, 21). To explore the influence of processing, a mutant gamma 7 protein was produced by replacing the Cys residue four residues from the carboxyl terminus with a Ser residue. The effect of this substitution is to prevent prenylation and carboxyl methylation of the mutant gamma 7 protein. Pulse-chase labeling studies revealed no significant difference in the half-life of the beta 1 protein when complexed with the wild type versus the mutant gamma 7 protein (compare Figs. 6 and 7). This result indicates that dimerization rather than post-translational processing and translocation to the membrane is sufficient to increase the half-life of the beta 1 protein. This contrasts with a previous study showing prenylation and carboxyl methylation of RhoA leads to an increase in the half-life of this protein (39). While coordinate suppression of the beta 1 and gamma 7 subunits provides strong evidence for their functional association in the intact cell setting, these results also raise a number of questions. One question is whether the pairing of the beta 1gamma 7 subunits is specific to HEK 293 cells or is common to various types of cells. A second question revolves around what factors govern the preferential assembly of the beta 1gamma 7 dimer in cells expressing multiple beta  and gamma  subtypes. One possibility is a structural feature that favors certain beta gamma subunit combinations. However, in vitro studies show that the beta 1 and gamma 7 subunits have the potential to interact with a wide variety of subtypes (37, 38). Another possibility is a spatial factor that directs selective assembly of the beta 1gamma 7 dimer within a particular subcellular compartment. In this regard, there is evidence that certain mRNAs are localized within cells, resulting in proteins being synthesized within discrete subcellular compartments (40, 41). That the gamma  subtypes are localized within different subcellular domains is increasingly clear (42, 43).

Lack of Functional Association of alpha s and gamma 7 Subunits-- Ribozyme-mediated suppression of the gamma 7 protein is associated with significant attenuation of beta -adrenergic receptor-stimulated adenylyl cyclase activity (9). Since loss of the gamma 7 protein results in corresponding reduction of the beta 1 protein, the results of the present study provide strong evidence that the gamma 7 and beta 1 subunits, together with the alpha s subunit, comprise the Gs heterotrimer that couples the beta -adrenergic receptor to adenylyl cyclase (44). The alpha s subunit comes in multiple forms that are generated by alternative splicing of a single gene (45, 46). Interestingly, ribozyme-mediated suppression of the gamma 7 subunit has no effect on either the 52- or 45-kDa alpha s proteins (Fig. 8). This result indicates that the alpha s subunits associate with the membrane and are stable in the absence of the beta 1gamma 7 subunit complex. This is consistent with studies showing that the alpha s subunits contain their own membrane targeting signals (47, 48).

Assembly of G Protein Heterotrimers-- These and other studies (49) have begun to answer very basic questions regarding the synthesis and assembly of G protein heterotrimer. The G protein alpha , beta , and gamma  subunits are synthesized on ribosomes in the cytosolic compartment, and they are directed to the plasma membrane by the way of post-translational modification of both alpha  (48, 50) and gamma  (21, 22) subunits. Increasing evidence suggests that there is a specific order of addition proceeding from the individual monomers through the beta gamma dimer to the alpha beta gamma trimer (20, 22, 47, 51). Importantly, this order of assembly has not been examined for a specific combination of G protein alpha beta gamma subunits that is known to exist in the intact cell setting. The results of the present study showing a functional association of the G protein alpha sbeta 1gamma 7 subunits should allow examination of this question.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM39867 and GM58191 (to J. D. R.) and American Heart Association Affiliate Research Services of the Mid-Atlantic Grant-in-aid S98665P (to Q. W.).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.

§ To whom correspondence should be addressed: Henry Hood MD Research Program, Weis Center for Research, 100 North Academy Ave., Danville, PA 17822. Tel.: 570-271-6684; E-mail: jrobishaw{at}psghs.edu.

    ABBREVIATIONS

The abbreviations used are: HA, hemagglutinin; RZ, ribozyme.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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