INTRODUCTION

Both the heterotrimeric G protein  subunit and the complex transmit signals to effector molecules (1, 2). Multiple isoforms of  and  subunits have been identified by cDNA cloning (3, 4), and the formation of heterodimers from particular combinations of  and  subtypes may contribute to signaling specificity by the complex (5). G protein -regulated effector molecules in vertebrates include inwardly-rectifying potassium channels (6), certain isoforms of adenylyl cyclase (7, 8) and phospholipase C- (PLC-)¹ (9, 10, 11), and as yet unidentified upstream targets in the mitogen-activated protein kinase (MAPK) (12, 13, 14) and c-Jun N-terminal kinase (JNK) (15) pathways.

The effector specificity of the recently described brain-specific ₅ subunit(3) was examined by transient cotransfection with ₂ in COS cells and compared with ₁. We report here that while both ₁ and ₅ were found to activate PLC-₂ in a ₂-dependent fashion consistent with previous reports (3, 10), ₅, unlike ₁, did not stimulate the MAPK or JNK pathways. Cotransfection of different  isoforms failed to confer MAPK or JNK stimulatory ability to ₅. These results imply that the G subunit can define the pattern of downstream signaling mediated by the complex and suggest that distinct mechanisms mediate the activation of different -responsive effectors.

EXPERIMENTAL PROCEDURES

The cDNA for human phospholipase C-₂ (16) (GenBank^TM accession number M95678[GenBank]) (in pMT2) was a gift from Dr S. G. Rhee. Constructs encoding ₁, ₁, and ₂ in the vector pCDM8.1 (17) were described previously (18, 19). The expression constructs for hemagglutinin epitope-tagged (HA)-ERK2 and HA-JNK in pcDNA3 were described previously (12, 20).

The cDNA for ₅ was obtained by PCR of mouse brain cDNA (Clontech) using specific primers (3) and the thermostable DNA polymerase Pyrococcus furiosus (Pfu) (Stratagene). The final construct contained two silent base changes (Ser¹³⁰ TCt and Phe¹³⁶ TTc) relative to the published sequence (3) (GenBank^TM accession number L34290[GenBank]). The cDNA for ₄ was obtained by reverse transcription-PCR employing the Pyrococcus woesei (Pwo) (Boehringer Mannheim) thermostable DNA polymerase from human brain total RNA (Clontech) with primers based on the published sequence (4) (GenBank^TM accession number U31382[GenBank]). A silent nucleotide substitution in codon Ala³⁸ (GCt) was found. The construct for ₅ (GenBank^TM accession number M95779[GenBank]) was obtained by PCR employing Pwo DNA polymerase and bovine liver ₅ cDNA (kindly provided by Dr. Nathan N. Aronson) as a template in combination with specific primers (21). The cDNA for ₇ (GB M99393[GenBank]) was obtained by PCR employing Pfu DNA polymerase from bovine brain cDNA (Clontech) and specific primers (22). The finished ₅, ₄, ₅, and ₇ constructs all contained the sequence GAATTCAAG at their 5 ends (starting methionine codon underlined) and after the stop codon were followed by an XbaI site at their 3 end and were ligated between EcoRI and XbaI sites of pCDM8.1 (₅) or pcDNA3 (₄, ₅, and ₇). Constructs in pcDNA3 were amplified in Escherichia coli XL1Blue (Stratagene). The resulting plasmid preparations were purified by column chromatography (Qiagen Maxiprep kits). The DNA sequence of all inserts was verified by the chain termination method (23) using Sequenase 2.0 (U. S. Biochemical Corp.).

Growth, maintenance, transfection (24), and fractionation of COS-7 cells was as described previously (18). Protein was determined by the method of Bradford (25) using bovine serum albumin as a standard. Membrane proteins or crude lysates were separated on 11% slab gels by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (26) and electrotransferred onto polyvinylidene difluoride membranes in Dunn's buffer (27). For analysis of low molecular weight proteolytic fragments, the Tricine gel system of Schägger and von Jagow (28) was employed as indicated. Detection of G₅ subunits employed the primary antibody SGS generated in rabbits against the synthetic dodecapeptide SGSWDHTLRVWA (conjugated to keyhole limpet hemocyanin (29)) corresponding to residues 342-353 at the C terminus of ₅ (3). The antibody RA generated against a peptide corresponding to ₁ residues 256-265 has been described (30). Secondary detection employed ¹²⁵I-Protein A followed by autoradiography on film (19) or a storage phosphor screen (Molecular Dynamics PhosphorImager), or else enhanced chemiluminescence using goat anti-rabbit or anti-mouse antibodies coupled to horseradish peroxidase (Boehringer Mannheim).

Cleared cholate extracts of crude membrane preparations of transfected COS cells or BALB/c mouse brains were prepared by extraction with 1% (w/v) cholate, 10 mM EDTA, 50 mM Tris-HCl (pH 8.0) (buffer A) on ice for 30 min followed by centrifugation at 16,000 × g for 10 min. The preparation of bovine brain membrane cholate extract was as described (31, 32).

For limited proteolytic digestion of membrane detergent extracts, samples were diluted into buffer A and then incubated for 30 min at 37 °C at a 1:30 or 1:40 (w/w) ratio of enzyme:extract protein. Reactions were terminated by the addition of denaturing sample buffer and boiling. The enzymes employed were L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-trypsin (Sigma T-8642), endoproteinase Lys-C (Calbiochem 324715), and endoproteinase Glu-C (V8 protease) (Calbiochem 324713).

The PI-PLC activity of transfected cells was estimated by a modification of the procedure of Berridge et al. (33) as described previously (10, 34).

The assays for MAPK and JNK activity were essentially as described by Crespo et al. (12) and Coso et al. (20), respectively. Approximately 2.5 × 10⁶ COS-7 cells were plated into 75-cm² flasks and incubated at 37 °C overnight. On the following day, the cells were transfected by the DEAE-dextran method (24) using a total of 15 µg of DNA per cotransfection, typically including 5 µg of HA-ERK2 or HA-JNK (12, 20), 5 µg of G, and 5 µg of G. Vector DNA was added where necessary to keep the total amount of plasmid DNA per flask constant. The remainder of the assay was as described (12, 20) except that mouse monoclonal HA.11 (Berkeley Antibody Co.) was used for the immunoprecipitations (3 µl of HA.11 ascites fluid per 900 µl of detergent lysate).

RESULTS AND DISCUSSION

Functional Properties of ₅ Cotransfected with ₂

The low degree of sequence homology between the brain-specific ₅ subunit recently described by Simon and co-workers (3) and other G subunits (e.g. only 53% amino acid identity with ₁) suggests a possible specialization of ₅ for interactions with G, G, receptor, and/or effector molecules. The effector specificity of the ₅ subunit was examined by cotransfection with ₂ in COS cells and compared with ₁. As previously reported (3), transfection of the combination of ₅ and ₂ produced a robust stimulation of cotransfected PLC-₂ (Fig. 1A). This stimulation was comparable with that of the ₁/₂ combination and was not seen with transfection of ₅ or ₂ alone (Fig. 1A).

Functional properties and expression of G protein ₅ in COS cells.

G protein ₅ and ₁ were then compared in their ability to activate the MAPK pathway in a -dependent fashion using a cotransfection paradigm employing HA-epitope-tagged ERK2 (12). The complex of heterotrimeric G proteins activates the MAPK cascade in metazoans (12, 13, 14) as it does in yeast (35). Previous analysis by Hawes et al. (36) of G subtypes 1-4 and multiple  isoforms in transfected COS-7 cells showed a strong correlation between the ability of a specific combination to activate PLC-₂ and its ability to activate the MAPK pathway. In contrast, we found that ₅ was unable to stimulate MAPK activity in combination with ₂ despite the ability of ₁ to do so under the same conditions (Fig. 1B and see below) and despite the efficacy of ₅/₂ in the PLC assay (Fig. 1A). The inability of ₅ to activate the MAPK pathway was not due to failure of expression of the ₅ or HA-ERK2 proteins under the conditions of the MAPK assay as documented by immunoblots of the Triton lysates of transfected COS cells with specific antibodies (Fig. 1C and see below).

Analysis of Recombinant ₅ by Immunoblotting and Limited Proteolysis

The ability of ₅ to activate PLC-₂ in a -dependent fashion strongly implies its proper expression and assembly with G. To further exclude the possibility, however, that the failure of transfected ₅ to activate the MAPK pathway was due to abnormal expression or folding of the recombinant polypeptide, additional analysis was performed using the antibody SGS generated against the C-terminal dodecapeptide of mouse ₅. Like the N-terminal ₅ antibody described by Watson et al. (3), SGS identified a major ~39-kDa band in immunoblots of detergent extracts of membranes prepared from ₅/₂ cotransfected but not control COS cells (Figs. 1C and 2A, left panel). This immunoreactive band comigrated with the major band in both mouse and bovine brain membrane extracts (Fig. 2A, left panel) consistent with previous reports (3). A faint upper SGS-reactive band of M_r ~90,000 was also noted in blots of mouse brain extract. The pattern of C-terminal fragments generated by limited proteolysis of bovine brain ₅ was then compared with that of brain ₁ by immunoblotting with specific antibodies (Fig. 2A, right panel). Such biochemical analysis has proven very useful in assessing the folding of native and recombinant G protein complexes (19, 37, 38, 39). Whereas a stable ~26-kDa C-terminal fragment of ₁ is identified by RA antibody (30) in a tryptic digest of bovine brain proteins, analysis of the same sample with the ₅ C-terminal antibody SGS revealed no stable immunoreactive fragments (Fig. 2A, right panel). Limited digestion of the bovine brain samples with endoproteinase Lys-C produced an SGS-reactive C-terminal fragment visible at the dye front, while digestion with V8 protease yielded a diffuse band centered at ~35 kDa. The RA immunoreactivity at ~36 kDa in the endoproteinase Lys-C and V8 digests remained unchanged from control (Fig. 2A, right panel). Further analysis of the low molecular weight products on Tricine gels (28) revealed a major C-terminal SGS-reactive band of 12 kDa and a faint band of ~22 kDa in the endoproteinase Lys-C digests of both mouse and bovine brains (Fig. 2B). In addition to the major 35-kDa band product, a minor product of ~11 kDa was noted in the Tricine gel blots of V8 protease digests of both brain samples (Fig. 2B). An identical pattern of SGS-reactive C-terminal fragments was seen upon limited proteolysis of detergent extracts of membranes prepared from COS cells cotransfected with ₅ + ₂ (Fig. 2B). Taken together, these data demonstrate that the state of folding of the ₅ polypeptide expressed in transfected COS cells inferred from selective proteolysis is indistinguishable from that of native ₅ found in brain.

C-terminal G₅ antibody immunoblots of control and protease-treated membrane detergent extracts and comparison with G₁.

Ability of ₅ to Assemble with Different G Isoforms and Activate PLC-

Previous studies of -responsive effectors comparing purified recombinant G complexes have found significant differences due to the  component of the heterodimer (40, 41). We therefore wondered if the inactivity of ₅ in the MAPK assay was specific to the ₅/₂ combination and might be overcome by employing other  isoforms. Because certain combinations of  and  subunits fail to assemble as heterodimers (39, 42), we first compared three additional  subunits with ₂ in their ability to assemble with ₅ in cotransfected COS cells. Immunoblots of cholate extracts of COS cell membranes revealed no endogenous ₅ immunoreactivity and no ₅ signal resulting from transfection of any  subunit alone (Fig. 3, upper panel), consistent with the results of Fig. 1C. Transfection of ₅ alone or in combination with ₁ (transducin ) produced only a faint SGS-reactive band at ~39-kDa which may reflect assembly of ₅ with the endogenous pool of  subunits (Figs. 1C and 3, upper panel). In contrast, cotransfection of ₅ with ₂, ₄, or ₇ gave a reproducible increment of ₅ immunoreactivity over that seen with ₅ alone, with a rank order _{2  4} = ₇ (Fig. 3). This increase in the steady state levels of membrane ₅ seen with  cotransfection presumably reflects increased stability and/or membrane targetting of the  subunit engendered by assembly with the exogenous . The reciprocal ability of cotransfected  to stabilize  has been previously documented (43).

Analysis of the expression and function of ₅ cotransfected in COS cells with different  isoforms.

The functional association of ₅ with ₄ and ₇ was also demonstrated by the ability of ₅/₄ and ₅/₇ to stimulate PLC-₂ activity above control levels in parallel functional assays (Fig. 3, lower right). In contrast to results with ₂, ₄, and ₇, cotransfection of ₁ with ₅ produced no increment in PLC activity (data not shown). The ability of the different  isoforms to promote the ₅-dependent activation of PLC-₂ correlated strongly with their ability to increase the steady-state expression of ₅ documented by quantitation of the SGS immunoblots (Fig. 3, lower panels). Simon and co-workers (3) also reported that the ₅/₂ combination stimulated PLC-₂ activity much more strongly in COS cells than combinations employing other  isoforms, although ₅ expression levels were not compared.

Failure of Additional G Isoforms to Support MAPK and JNK Activation by ₅

Despite biochemical and functional evidence of their assembly with ₅ (Fig. 3), neither cotransfected ₄ nor ₇ conferred to ₅ the ability to activate the MAPK pathway (Fig. 4A), resembling ₂ in this regard (Figs. 1B and 4A). On the other hand, ₄ and ₇ both supported MAPK stimulation by cotransfected ₁ (Fig. 4A). The analysis of the effector signaling functions of ₅ was extended to look at potential JNK stimulatory activity. The ability of cotransfected mammalian ₁/₂ to activate JNK through a p21^ras- and p21^rac1-dependent pathway bearing many similarities to the -driven pheromone response pathway in Saccharomyces cerevisiae was described recently (15). We compared the ability of ₁ and ₅ to activate HA-JNK when transfected alone or in combination with ₂, ₄, or ₇. The kinase activity of the cotransfected HA-JNK reporter in ₅ +  transfected cells was consistently the same as or lower than the activity in -only transfected cells, regardless of which  was employed (Fig. 4B). The inactivity of ₅ in this assay was in stark contrast to the activity of ₁ which produced a robust increment in JNK activity when cotransfected with each of the three  isoforms (Fig. 4B). Immunoblotting of cell lysates from such experiments demonstrated that the expression of the HA-JNK reporter enzyme was comparable whether ₁ or ₅ was employed in the cotransfection (data not shown). Additional experiments with the ₅ isoform, shown previously by Watson et al. (3) to promote ₅ stimulation of PLC-₂, revealed that it too supported MAPK and JNK activation by ₁, but not by ₅ (data not shown).

Comparison of the MAPK and JNK stimulatory activities of G protein ₁ and ₅ when cotransfected with different G isoforms.

Implications of the Effector Selectivity of G₅

While signaling through particular G subunits is characteristically confined to a restricted set of effector targets, heterodimers have been found to modulate a wide range of structurally diverse effector molecules, an observation true even when a defined pair of  and  isoforms has been studied. Reconstitution experiments have shown, for example, that purified recombinant ₁₂ can regulate adenylyl cyclase (40), phospholipase C- (40, 44), inwardly rectifying potassium channels (41), and -adrenergic receptor kinase activities (45). Differences in effector regulation ascribed to the G component of defined heterodimers noted previously in cotransfection and reconstitution experiments have been only modest (45, 46). The present results offer the strongest evidence to date that G protein  subunits are sufficient to determine the effector selectivity of and suggest that the structural features of the complex mediating effector regulation may differ depending on the effector.

The G protein ₅ fails to activate critical intermediates in the MAPK and JNK pathways in COS cells when complexed with ₂, ₄, ₅, or ₇ in heterodimers. In vertebrates, the G protein complex triggers the MAPK cascade by a p21^ras-dependent mechanism (12, 14) involving an unknown number of upstream intermediaries which may include nonreceptor tyrosine kinases (47), phosphatidylinositol 3-kinase (48), and Shc (49) acting through Ras guanine nucleotide exchange factors including Sos (50) and Ras-guanine nucleotide releasing factor (51). The upstream mediators of the Ras- and Rac1-dependent activation of JNK by subunits (15) remain unknown, but activation of a homologous pathway in S. cerevisiae appears to involve the direct interaction of with the scaffolding protein Ste5p (52) and the guanine nucleotide exchange factor Cdc24p (53). It is tempting to speculate that the structure of ₅ precludes its interaction with the mammalian counterparts of these or perhaps other critical intermediates in the kinase pathways. Such a loss of function might represent an evolutionary goal of ₅. It is possible to speculate, for example, that expression of ₅ might promote the survival of certain neurons by failing to activate JNK, a pathway linked to apoptosis in neuronal cell culture (54). An alternative speculation is that the inability of G₅ to activate the kinase cascades is an incidental consequence of evolution to gain a function such as an ability to regulate novel effector molecules in brain.