A previous study has shown that the residues lying on the switch III region and the α3 helix of Gα_q are required for interaction with PLCβ 1. The corresponding two clusters of amino acids on Gα₁₆ are Asn²⁴⁵-Glu²⁴⁷ and Gly²⁵⁹-Thr²⁶⁰ (Figure 1A). Mutations of these two clusters of residues into alanine created 3 mutants (Figure 2A) denoted as 3A (Asn²⁴⁵-Glu²⁴⁷ → Ala), 2A (Gly²⁵⁹-Thr²⁶⁰ → Ala) and 5A (all 5 residues → Ala). The Gln²¹² → Leu (QL) mutation 821 was also introduced to individual alanine mutants to generate a constitutively active phenotype for studying effector interactions in a receptor-independent fashion. Previous studies have already confirmed the capability of Gα₁₆QL to activate various downstream effectors such as ERK, NF-κB, STAT3 and JNK 712.

Since COS-7 cells do not express Gα₁₆ endogenously, they are an excellent platform for studying Gα₁₆-regulated signaling pathways by over-expressing Gα₁₆ or its mutants. A tailor-designed antiserum 22 which recognizes the N-terminus (amino acid resides 13–27) of Gα₁₆ was used for detecting the expression level of Gα₁₆ and its mutants. Upon expression in COS-7 cells, a single band at around 43 kDa was detected by the anti-Gα₁₆ antiserum for each construct (Figure 2B, lower part). Plasma membrane proteins prepared from non-transfected COS-7 cells served as a negative control (Figure 2B, leftmost lane). All of the mutants were expressed in COS-7 cells in a comparable level as Gα₁₆ and Gα₁₆QL, indicating that the mutations introduced did not affect the apparent expression or stability of Gα₁₆.

The same sets of transfected cells examined in expression study were tested for their ability to stimulate PLCβ. Expression of Gα₁₆QL in COS-7 cells significantly increased IP formation by ~7 fold as compared to cells expressing wild-type (WT) Gα₁₆. Expression of constitutively active counterparts of the mutants (2A-QL, 3A-QL and 5A-QL) in COS-7 cells significantly increased IP accumulation as compared to the responses of their corresponding non-QL counterparts. Responses induced by QL forms of the mutants were significantly reduced as compared with the response elicited by Gα₁₆QL (Figure 2B). 2A-QL and 3A-QL retained 42% and 73% of the IP production of Gα₁₆QL, respectively, whereas 5A-QL preserved about 61% of the IP accumulation of Gα₁₆QL. However, the differences between the mutants were not statistically significant. Apparently, the results indicated that the mutations partially impaired the ability of Gα₁₆ to activate PLCβ.

Further experiments were carried out to verify the observed variations of the induced IP formation between different constitutively active mutants. Increasing amounts of individual QL mutant cDNA were transfected into COS-7 cells and the IP accumulation in the transfected cells were measured. For all of the QL mutants, increasing levels of IP accumulation were observed as the cDNA amounts were increased (Figure 2C, upper part). The expression levels of individual QL mutants were correspondingly increased as examined by Western blotting (Figure 2C, lower part). There were obvious differences in the PLCβ responses induced by the different mutants. Gα₁₆QL and 2A-QL induced maximal stimulation of IP production at ~0.1 μg/ml, while maximal responses for 3A-QL and 5A-QL were only obtained at cDNA concentrations of 0.5 μg/ml or higher. Despite similar levels of expression, the maximal response stimulated by 2A-QL was only about half of that elicited by Gα₁₆QL. The estimated EC₅₀ values of IP accumulation were 0.020, 0.039, 0.123 and 0.610 μg/ml for Gα₁₆QL, 2A-QL, 3A-QL and 5A-QL, respectively. These results indicate a progressive decrease of the efficiencies for activating PLCβ when one or more of the putative PLCβ-interacting domains on Gα₁₆ were mutated to alanine. Mutations at the α3 helix (2A) might be particularly detrimental to PLCβ activity, because the maximal stimulatory response of 2A-QL was always lower than those of Gα₁₆QL and the other two alanine mutants. Since 5A-QL could attain a higher maximal PLCβ response than 2A-QL, the incorporation of 3A mutations in the switch III region apparently relieved the functional impairment associated with the 2A mutations in the α3 helix of Gα₁₆.

Diverse downstream effectors have been found to be regulated by Gα₁₆, including ERK, STAT3, NF-κB and JNK 791123, but their dependencies on PLCβ activation have not been well defined. To further investigate the functions of the selected amino acids in regulating these downstream effectors, WT and QL forms of Gα₁₆ and the three mutants were examined for their regulations of these downstream effectors when expressed in HEK293 cells. We employed HEK293 cells in this part of the study because of the regulations of ERK, STAT3, and NF-κB had been fully characterized in this cellular background 711 and all of the mutants could be efficiently expressed in these cells (data not shown). As illustrated in Figure 3A, no observable alteration in the total ERK1/2 levels was found in cells transfected with different cDNA constructs. The induction of ERK phosphorylation by the QL forms of the alanine mutants was significantly higher than their corresponding WT controls, and they were all similar to Gα₁₆QL. It should be noted that 2A-QL and 5A-QL consistently generated a slightly lower level of ERK phosphorylation than 3A-QL and Gα₁₆QL.

As both PLCβ and ERK can serve as upstream regulators of STAT3 7, we examined the ability of the mutants to induce STAT3 Tyr⁷⁰⁵ phosphorylation. All three mutants were capable of stimulating STAT3 phosphorylation at Tyr⁷⁰⁵ albeit weaker than that induced by Gα₁₆QL (Figure 3B). The phosphorylation triggered by 2A-QL, 3A-QL and 5A-QL were not significantly different between each other. Given that Gα₁₆QL-induced STAT3 phosphorylation involves PLCβ and PKC 7, these results were not surprising because the alanine mutants have impaired PLCβ regulation (Figure 2).

Regulation of NF-κB by Gα₁₆ has been demonstrated in recombinant systems as well as in human lymphoblastoma REH cells 11. The phosphorylation of NF-κB induced by constitutively active alanine mutants was examined. As compared to Gα₁₆, the expression levels of total NF-κB were unaffected in cells transfected with the alanine mutants (Figure 3C). The levels of NF-κB phosphorylation caused by the constitutively active alanine mutants were only slightly reduced (but not statistically different) when compared to the Gα₁₆QL responses. There was also no obvious difference between 2A, 3A and 5A mutants.

Since the background phosphorylation of JNK in transfected HEK293 cells are relatively high (Chan AS and Wong YH, unpublished observation), in vitro kinase assay was adopted to determine the activity of JNK activity in COS-7 cells instead. COS-7 cells were co-transfected with Gα₁₆ mutants together with HA-tagged JNK. The expression level of HA-tagged JNK were similar in all transfected cells (Figure 3D). Gα₁₆QL enhanced JNK-mediated [³²P]-labeling of GST-c-Jun significantly by 3.6-fold as compared to that of Gα₁₆. Surprisingly, none of the constitutively active alanine mutants was capable of inducing JNK activation (Figure 3D). It appeared that the sequence integrity on the switch III region and α 3 helix is more critical for the regulation of JNK activation.

As the alanine mutations of the two clusters caused reduction of PLCβ activation, one of the possible explanations would be the impairment of cognate recognition of PLCβ. Co-immunoprecipitation experiments were performed to study the possible alteration of the mutants and PLCβ2, a PLCβ isoform commonly expressed and known to interact with different G_q family members (Figure 4A). Expression levels of different Gα₁₆ constructs were similar when co-expressed with recombinant PLCβ2, as revealed in the total cell lysates. The endogenously expressed PLCβ2 could be detected (lane 1 of the bottom strip in Figure 4A) but the level was too low for co-immunoprecipitation assays. Both Gα₁₆ and Gα₁₆QL could be precipitated with PLCβ2 using either anti-Gα₁₆ or anti-PLCβ2 antibodies, while Gα₁₆QL showed much stronger interaction, indicating that the activation of Gα₁₆ facilitated its interaction with PLCβ2. Similarly, the QL forms of either one of the three mutants showed better interactions with PLCβ2 when compared with their corresponding wild-type forms. However, the fold increases between the three alanine mutants and their corresponding QL forms were much lower than the Gα₁₆ pair (values on top of each lane as indicated in Figure 4A). Such reduction was apparently due to the slight enhancement of the interactions between the alanine mutants and PLCβ2 as compared with Gα₁₆. No significant difference of the fold changes was observed between the three mutant pairs. Nonetheless, the results clearly indicated that the Gα₁₆/PLCβ interaction was impaired by both clusters of alanine mutations.

One of the distinctive features of Gα₁₆-mediated signaling is the association with TPR1, which facilitates the accumulation of active Ras 13 and in turn activate the Raf/MEK/ERK cascade. To study the impact of the alanine mutations on the interaction between Gα₁₆ and TPR1, a Flag-tagged full-length TPR1 was co-expressed with each of the Gα₁₆ constructs as indicated in Figure 4. Co-immunoprecipitation of the proteins of interest was performed using anti-Flag tag or anti-Gα₁₆ antibodies. Both Gα₁₆ and Gα₁₆QL could be co-immunoprecipitated with TPR1 (Figure 4); Gα₁₆QL was bound to TPR1 more efficiently under our experimental condition. A truncated form of TPR1 lacking the C-terminal tail did not bind to Gα₁₆ (Rico K. Lo, Andrew M. Liu and Yung H. Wong, data not shown). The three alanine mutants could also be co-immunoprecipitated with TPR1. Similar to the Gα₁₆-PLCβ2 interactions, the QL versions of the alanine mutants showed stronger interactions with TPR1 than their corresponding non-active counterparts. Fold enhancements between the QL form of the alanine mutants and the non-active forms were weaker than Gα₁₆ pair. The 5A-QL mutant showed the weakest enhancement of TPR1 interaction. Expression levels of 5A and 5A-QL might be less in the experiment as showed in the total cell lysates, but the fold enhancement was still the least among the others. More obvious results were obtained when the TPR1-Gα₁₆ complexes were immunoprecipitated with anti-Gα₁₆ antibody. The results suggested that the interactions between TPR1 and Gα₁₆ were also dependent on the identities of the two amino acid clusters. Alanine mutations at both clusters caused the greatest reduction of the interaction.

G₁₆ is well-known for its receptor coupling promiscuity 4, but it also exhibits different degrees of coupling efficiencies to various GPCRs 2224. Since the receptor- and effector-interacting domains overlap partially on the surface of Gα subunits, it is possible that the alanine mutations can also affect the receptor coupling of Gα₁₆. To test this possibility, a panel of GPCRs was examined for their functional coupling to Gα₁₆ and the alanine mutants using the IP accumulation assay. These GPCRs included G_i-coupled adenosine A₁ receptor (A₁R), complement C5aR receptor, formyl peptide receptor (fMLPR), and the G_s-coupled adenosine A_2A and A_2B receptors (A_2AR, A_2BR) and dopamine D₁ receptor (D₁R). All of these receptors are capable of utilizing Gα₁₆ to elicit intracellular calcium mobilization in FLIPR assays 24.

The selected G_i- or G_s-coupled receptors all induced IP accumulation significantly in the presence of Gα₁₆, but with variable efficiencies (Figure 5). These receptors were also capable of interacting with the alanine mutants. In general, IP accumulation mediated by 2A and 3A were ~50% and 35%, respectively, of that induced by Gα₁₆. In some cases, such as C5aR and A_2AR, the differences between the responses of 2A and 3A mutants were not significant, but it may be due to the weaker overall responses when compared with other receptors. Contrastingly, the differences between the phenotypes of these two mutants in the coupling to A₁R, A_2BR and D₁R were more obvious (≥ 50%). Differences in the absolute responses of various receptors have been demonstrated previously in the measurement of intracellular Ca²⁺ mobilization (see Table 1 of 24). The differential coupling efficiencies of the tested GPCRs with G₁₆ allowed us to detect the differences on GPCR coupling between the alanine mutants. Agonist-induced activation of 5A could only marginally increase IP accumulation in cases of A₁R, fMLPR and D₁R, while the other receptors were totally unable to elicit a response via 5A (Figure 5). The defective phenotype indicated that the two clusters were essential for the effective receptor-mediated PLCβ activation through Gα₁₆. The switch III region (harboring the 3A mutations) appeared to be more influential to the productive receptor coupling events than the α 3 helix.

Upon closer examination of the basal levels of the cells expressing Gα₁₆ or the 3 mutants, it was noticed that the basal levels of IP accumulation in the cells expressing A₁R, A_2AR or A_2BR with Gα₁₆ were higher than with the mutants (Figure 5). This trend of decreasing basal IP accumulation was similar to the receptor-activated responses, with Gα₁₆>2A>3A>5A. The results here showed that the basal activities of all three adenosine receptors were suppressed by the expression of the alanine mutants, particularly for 5A. Such evidence further implied that the alanine mutants might associate with the receptors, but such receptor-G protein complexes were defective and the mutants actually sequestered the spontaneous activities of the receptors (also see Discussion).

Type 2 adenylyl cyclase (AC2) can be synergistically stimulated via the Gβγ complex released from Gα in the presence of activated Gα_s 2526. Measurement of AC2-mediated cAMP production in such circumstance is useful for the detection of receptor-mediated release of Gβγ subunits. Functional interaction of the alanine mutants with G_i-coupled receptors, if any, would be expected to stimulate AC2 upon agonist triggering. HEK293 cells were co-transfected with empty vector pcDNA3 and cDNAs encoding Gα_sQL, fMLPR and AC2. Activation of the fMLPR resulted in the stimulation of endogenous G_i and release Gβγ which led to the activation of AC2 (Figure 6). PTX pretreatment attenuated the corresponding increase of cAMP level. In cells co-expressing Gα₁₆, Gα_sQL, fMLPR and AC2, activation of fMLPR led to an increase in cAMP formation in a PTX-insensitive manner (Figure 6), indicating the functional coupling of fMLPR with Gα₁₆. Both 2A and 3A mutants exhibited comparable significant increases in cAMP production as Gα₁₆. However, 5A was totally incapable of stimulating AC2, which suggested that the receptor-mediated activation of G₁₆ and the subsequent Gβγ release might be severely impaired by the mutations on both switch III and α 3 helix.

Human TPR1 cDNA was kindly provided by Richard D. Ye (Department of Pharmacology, College of Medicine, University of Illinois, Chicago, IL). Human embryonic kidney 293 (HEK293) and monkey kidney fibroblast COS-7 cells were purchased from American Type Culture Collection (ATCC CRL-1573 and 1651). Restriction endonucleases were from Roche Applied Sciences. DNA purification columns were from Qiagen. DNA modification enzymes, custom primers and cell culture and transfection reagents were from Invitrogen. [³H]adenine and chemiluminescence detection kit for Western blotting were from GE Healthcare. [γ-³²P]ATP and [³H]myo-inositol was from PerkinElmer Inc. Antibodies against various molecules are from: Cell Signaling Technology (Danvers, MA, USA) for ERK1/2 (Thr²⁰²/Tyr²⁰⁴), STAT3 (Tyr⁷⁰⁵) and NF-κB (Ser⁵³⁶) and their total forms; Torrey Pines Biolabs (C-terminus; Houston, TX, USA) and Gramsch Laboratories (N-terminus, custom-made; Schwabhausen, Germany) for Gα₁₆; sc-206, Santa Cruz Biotechnology (Santa Cruz, CA, USA) for anti-PLCβ2; anti-FLAG antiserum and anti-FLAG affinity gel were from Sigma (St. Louis, MO, USA). Protein G-agarose and cross-linking reagent dithiobis [succinimidylpropionate] (DSP) were from Pierce Biotechnology (IL, USA). Pertussis toxin was purchased from List Biological Laboratories. All other chemicals were obtained from Sigma.

Complete amino acid sequence alignment of Gα 's was generated by CLUSTAL X 1.83 46. Molecular models were created by SWISS-MODEL web server 47 using the coordinates of the crystal structures of Gα_i1 48 and Gα_t1 49 in their corresponding heterotrimers retrieved from Protein Data Bank maintained by Research Collaboratory for Structural Bioinformatics 50 as the modeling templates. The model was modified and visualized with UCSF Chimera 51.

Polymerase chain reactions (PCR) were performed to construct the mutants. Amplified cDNA fragment was subcloned into pcDNA3 vector, which possessed T7 and SP6 promoter regions for primer attachment in PCR reaction. Human Gα₁₆WT and Gα₁₆QL 21 were used as a template to amplify mutants. A pair of overlapping sense and anti-sense primers was designed encoding the desired mutations to replace particular amino acids into alanines. Primers are listed (from 5' to 3') with the mismatch nucleotides underlined: 16-2A/S: CTC GCA TTG TTT

GCG GCG

CGC CGC

GCA GCT GCA

TGC AGC TGC

HEK293 and COS-7 cells were cultured in Earle's modified essential medium (MEM) and Dulbecco's modified Eagle's medium (DMEM), respectively, with 10% fetal bovine serum (FBS; vol/vol), 50 units/ml penicillin and 50 μg/ml streptomycin. Cells were incubated at 37°C in humidified air with 5% CO₂. At the day before transfection, COS-7 cells were seeded on 12-well plates at a density of 1 × 10⁵ cells/well. For western blot analysis, HEK293 cells were used instead, wherein 2 × 10⁵ cells were seeded on 6-well plates. For adenylyl cyclase assay, 4 × 10⁵ of HEK293 cells were transferred to 12-well plates. For c-Jun N-terminal kinase assay, 3 × 10⁵ of COS-7 cells were seeded on 6-well plates. cDNA transfection was achieved using Lipofectamine™ and PLUS™ reagents following the manufacturer's protocol. 50–75% of the cell population will take up the cDNAs as indicated by co-expressing β-galactosidase as a reporter.

750 μl of inositol-free DMEM containing 5% FBS and 2.5 μCi/ml myo-[³H]-inositol was added to each well of transfected COS-7 cells and incubated for 18–24 hr. The labeling media were subsequently replaced by 1 ml of assay medium (DMEM with 20 mM Hepes, pH 7.5 and 10 mM LiCl) for 10 min and then 1 ml of assay medium containing the appropriate agonist was added to the cells for another 1 h at 37°C. Reaction was stopped by adding 750 μl of ice-cold 20 mM formic acid after aspiration and the plates were stored at 4°C for 30 min. [³H]-IP were separated from labeled inositol by ion-exchange chromatography as described previously 52.

HEK293 cell were labeled with [³H]-adenine (1 μCi/ml) in MEM containing 1% FBS (vol/vol) one day after transfection. After 18–24 h the labeling media were replaced by 1 ml of 20 mM Hepes-buffered MEM containing 1 mM isobutylmethylxanthine (IBMX) and the appropriate drugs and incubated at 37°C for 30 min. The treatment was terminated with 1 ml ice-cold 5% trichloroacetic acid (wt/vol) with 1 mM ATP after aspiration and stored at 4°C for 30 min. [³H]cAMP was extracted from the pool of labeled nucleotides by sequential ion-exchange chromatography as described 53.

For cAMP and IP accumulation assay, absolute values for cAMP or IP accumulations varied between experiments, but variability within a given experiment was in general <10%. The cAMP levels were interpreted as the ratios of the counts per minute of [³H]cAMP fractions to those of the total labeled nucleotide fractions and expressed as cAMP/(cAMP + Total). Similarly, IP levels were expressed as IP/(IP + Total). Data shown in the figures were the mean ± S.D. of triplicates within one single experiment. At least three individual experiments yielded similar results. One-way ANOVA with Tukey-Kramer's test were performed using GraphPad Prism 3.03 to verify the significance between different treatment groups within the experiments.

Transfected HEK293 cells were washed with PBS twice and then treated in the same buffer containing 0.5 mM DSP for 15 min at room temperature to cross-link the membrane proteins. Cells were washed as above and maintained in quenching solution (50 mM glycine in PBS, pH 7.4) for 5 min, and then lysed in RIPA buffer (25 mM HEPES, pH 7.5, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM dithiothreitol, 200 μM Na₃VO₄, 0.7 μg/ml pepstatin, 4 μg/ml aprotinin, 100 μM PMSF, and 2 μg/ml leupeptin). For coimmunoprecipitation, cell lysates were incubated with anti-Gα₁₆ (4 μg/sample) or anti-PLCβ2 antiserum (0.4 μg/sample), followed by the incubation with 30 μl of protein G-agarose (50% slurry) at 4°C for 2 h, or 30 μl anti-FLAG affinity agarose gel (50% slurry) at 4°C overnight. Immunoprecipitates were washed by 400 μl RIPA buffer 4 times, and then resuspended in 50 μl RIPA buffer gel loading buffer and boiled for 5 min. Gα₁₆ and FLAG-TPR1 proteins in the immunoprecipitates were detected by specific primary antisera and horseradish peroxidase-conjugated secondary antisera using Western blotting analysis.

COS-7 cells were grown on 100-mm dishes to 70–80% confluence. Transfection was performed as in 12-well plates with proper adjustments to the surface area of the plate and the amounts of the reagents used. After 48 h in normal growth conditions, cells were washed with Ca²⁺/Mg²⁺-free Dulbecco's PBS (DPBS) and harvested with 3 ml DPBS containing 10 mM EDTA. The following procedures were performed at 4°C. Cells were spun down briefly (200 g, 5 min), resuspended in hypotonic lysis buffer and lysed by one cycle of freeze-thawing followed by 10 passages through a 27-gauge needle. Nuclei were removed by brief spinning and membranes were collected by spinning the supernatants at 15,000 g for 15 min. Membrane pellets were finally resuspended in lysis buffer. Detergent-compatible protein assay system (Bio-Rad) was employed to determine the protein contents in cell lysates or crude membrane preparations. Protein samples were separated by 10% SDS-PAGE. Separated proteins were transferred to nitrocellulose membrane by electroblotting. Antisera against specific proteins were applied and the detection was facilitated by ECL chemiluminescence reagents (GE Healthcare). Band intensities on the autoradiographs were analyzed and quantified using ImageJ 1.40 54.

Details of the assays were basically identical as described in our previous study 55. Briefly, COS-7 cell were transfected with the appropriate constructs and serum-starved overnight before assay. Cells were lysed by chilled detergent-containing lysis buffer with agitation on ice. 50 μl of each supernatant was used for the detection of JNK-HA expression after removing the cell debris by centrifugation, and the remaining was incubated for 1 h at 4°C with anti-HA antibody (2 μg/sample), followed by incubation with 30 μl of protein A-agarose at 4°C for 1 h. Immunoprecipitates were washed twice with lysis buffer and twice with kinase assay buffer, resuspended in kinase assay buffer with 5 μg of GST-c-Jun, and the kinase reactions were initiated by the addition of 10 μl of ATP buffer (50 μM ATP containing 2 μCi of [γ-³²P]-ATP per sample). After 30 min incubation at 30°C with occasional shaking, the reactions were terminated by adding sample buffer, and the samples were resolved by 12% SDS-PAGE. The gel was fixed for 30 min and the radioactivity incorporated to GST-c-Jun was detected and quantified by PhosphorImager (Molecular Dynamics 445 SI).