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Regions in the G Protein Subunit Important for Interaction with Receptors and Effectors

Laboratory in Pharmacology, College of Pharmacy, Chungnam National University Daejeon, Korea (C.-S.M.); Faculty of Medicine and Health Sciences, Universiti Malaysia Sarawak, Kuching, Sarawak, Malaysia (W.K.L.); Department of Gastroenterology, Showa University, Yokohama, Japan (H.Y.); Departments of Pharmacology and Medicine, University of Michigan, Ann Arbor, Michigan (R.R.N.); and Department of Pharmacology, University of Virginia Health Sciences Center, Charlottesville, Virginia (J.M.D., J.C.G.)

Received September 15, 2005; accepted November 30, 2005

	Abstract

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G dimers containing the ₁₁ or ₁ subunits are often less potent and effective in their ability to regulate effectors compared with dimers containing the subunit. To explore the regions of the ₂ subunit that affect the activity of the dimer, we constructed eight chimeric subunits from the ₁ and ₂ subunits. Two chimeras were made in which the N-terminal regions of ₁ and ₂ were exchanged and two in which the C-terminal regions were transposed. Another set of chimeras was made in which the CAAX motifs of the chimeras were altered to direct modification with different prenyl groups. All eight chimeras were expressed in Sf9 cells with the ₁ subunit, G dimers were purified, and then they were assayed in vitro for their ability to bind to the G_i1 subunit, to couple G_i1 to the A1 adenosine receptor, to stimulate phospholipase C-, and to regulate type I or type II adenyl cyclases. Dimers containing the C-terminal sequence of the ₂ subunit modified with the geranylgeranyl lipid had the highest affinity for G_i1 (range, 0.5-1.2 nM) and were most effective at coupling the G_i1 subunit to receptor. These dimers were most effective at stimulating the phosphatidylinositol-specific phospholipase C- isoform and inhibiting type I adenyl cyclase. In contrast, dimers containing the N-terminal sequence of the ₂ subunit and a geranylgeranyl group are most effective at activating type II adenyl cyclase. The results indicate that both the N- and C-terminal regions of the subunit impart specificity to receptor and effector interactions.

To date, seven and 12 subunits have been identified in mammalian systems (Downes and Gautam, 1999); thus, the dimers, which can be assembled from the known subunits, yield a large number of potentially unique complexes that differ in their interactions with receptors and effectors (Hildebrandt, 1997; Cabrera-Vera et al., 2003). Whereas the ₁-₄ subunits are more than 85% identical in amino acid sequence, the sequence identity of the subunits ranges from 10 to 70% (Downes and Gautam, 1999). The diversity of either the or subunit can impart specificity to the interactions of the dimer with receptors (Richardson and Robishaw, 1999; Hou et al., 2000; McIntire et al., 2001) or effectors (Cabrera-Vera et al., 2003), and the type of prenyl group on the subunit is clearly important in the interactions with both receptors (Kisselev et al., 1995; Yasuda et al., 1996) and effectors (Myung et al., 1999). Multiple experiments suggest that the subunit is divided into three functionally different regions. The central region of all subunits is believed to be involved in the interactions between the and subunits. For example, mutagenesis data indicate that a 14 amino acid sequence (amino acids 36-49 on ₁ or 33-46 on ₂) is critical for the interaction with the ₁ subunit (Lee et al., 1995), and the crystal structure of the dimer confirms that 17 of the 27 residues in the subunit that interact with the subunit are centrally located (Sondek et al., 1996). The N- and C-terminal regions of the subunits are the least conserved (Sondek et al., 1996; Downes and Gautam, 1999), and several lines of evidence suggest that these regions are important for the interaction with receptors (Yasuda et al., 1996; Hou et al., 2000) and effectors (Yasuda et al., 1996, 1998; Hildebrandt, 1997; Akgoz et al., 2002; Cabrera-Vera et al., 2003).

View larger version (36K): [in this window] [in a new window]   Fig. 1. Strategy for constructing the chimeric subunits. A, comparison of the primary sequences and charged amino acids in the N- and C-terminal regions of the 1 and 11 subunits with those of 2. The QLK and DPL sites and the C-terminal CAAX motif are highlighted. The center section of the molecules is omitted. B, schematic view of the chimeric subunits. The sequence of the 1 subunit is indicated in white and the 2 subunit in gray. Both of the 1 and 2 subunits are divided into three regions at the QLK and DPL sites. The molecules were designated as AAA for the 1 subunit and BBB for the 2 subunit. The farnesyl group (C-15) on the 1 subunit is indicated in light gray, and the geranylgeranyl group (C-20) on the 2 subunit is indicated in dark gray. The nomenclature used in the text to identify each chimera is indicated above the schematic representation.

To examine how the N- and C-terminal domains of the subunit and its prenyl group affect the interaction with subunits, receptors, and effectors, we constructed eight chimeric subunits from the ₁ and the ₂ subunits. These two subunits were used for the chimeras because the N and C termini of these proteins are representative of the major differences between the subunits in subfamily I and the other nine subunits. Two chimeras were made in which the N-terminal amino acids of ₁ and ₂ were exchanged. Two other chimeras were made in which the C-terminal 23 amino acids of ₁ and ₂ were exchanged. Finally, another set of chimeras was made in which the CAAX motifs in the four chimeras were altered to direct modification with different prenyl groups. All eight chimeras were expressed in Sf9 cells with the ₁ subunit and purified. Each dimer was tested using five in vitro assays chosen to probe major facets of activity: the ability to bind to the G_i1 subunit, to couple G_i1 to the A1 adenosine receptor, to stimulate PLC-, or to regulate type I and II adenylyl cyclases. The in vitro results indicate that the subunit's C terminus and its prenyl group are important for coupling subunits to the A1 adenosine receptor, for the activation of PLC-, and for the inhibition of type I adenylyl cyclase. In contrast, the subunit's N terminus is very important for the activation of type II adenylyl cyclase.

	Materials and Methods

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Strategy for Construction of the Chimeric Subunits. The ₁ and ₂ subunits have a conserved set of three residues approximately 20 amino acids from the N terminus (QLK) and the C terminus (DPL). The strategy for making the chimeras was based on creating new cDNAs for the ₁ and ₂ subunits, which had restriction sites engineered into the QLK and DPL sequences and are similar to those published previously (Jian et al., 2001). These sites are highlighted in Fig. 1A. In addition, note the similarities in the N- and C-terminal regions of the ₁ and ₁₁ subunits. Thus, whereas the chimeras were made with regions from ₁ because the cDNA for ₁ contained a convenient restriction site in the DPL sequence, the regions were selected to be representative of ₁₁ (Fig. 1). An AflII site was used to create the QLK sequence and a BamHI site to code for the DPL sequence in each molecule. The nomenclature used to describe these chimeric subunits is shown in Fig. 1B. The protein was divided into three regions by the QLK and DPL sites, designated as AAA for ₁ and BBB for ₂. If the C-terminal prenyl group was switched from the native modification, the amino acids in the mutant CAAX motif are indicated (Yasuda et al., 1996). For example, _2-L71S indicates the native ₂ subunit modified to change only the normal C-20 geranylgeranyl to the C-15 farnesyl. A designation of _BBA indicates a chimeric subunit in which the C terminus of the ₁ subunit (molecule A) is added to the N-terminal and middle regions of the ₂ subunit (molecule B) at the conserved sequence. The prenyl group of this chimera would be farnesyl. A designation of _BBA-S74L would indicate the same chimeric subunit mutated to code for the addition of geranylgeranyl at the C terminus.

The ₁ cDNA was excised from a pEV plasmid (HindIII/BamHI) and subcloned into the pGem7zF+ plasmid using the same restriction sites; it was then excised with HindIII/Asp700 for subcloning into the pAlter-1 mutagenesis plasmid at the HindIII and SmaI sites. The ₂ cDNA in pGem4z was excised by digestion with XbaI and SmaI and was subcloned into pAlter-1 using the same restriction sites. To construct the chimeras, identical restriction sites were created in the QLK and DPL sequences in both the ₁ and ₂ cDNAs; an AflII site was engineered to code for the QLK sequence, and a BamHI site was engineered to code for the DPL sequence. Because the BamHI restriction site already existed in the ₁ cDNA in the site corresponding to the DPL residues, a BamHI site was added to the ₂ cDNA through site-directed mutagenesis by altering one base in the ₂ sequence (GAA to GAG, encoding glutamic acid) using the Morph Site-Specific Plasmid DNA Mutagenesis Kit (5 Prime 3 Prime, Boulder, CO), taking care to conserve the protein sequence. The AflII restriction site for the QLK sequence was engineered into both the ₁ and ₂ subunits using the same mutagenesis kit. The resulting ₁ and ₂ cDNAs in the pAlter-1 plasmid containing the new AflII and BamHI sites were used to make the C- or N-terminal chimeric subunits.

Construction of N- and C-Terminal Chimeras and Production of Recombinant Baculoviruses. The ₁ and ₂ C-terminal chimeras (_AAB and _BBA) were constructed by digesting the ₁ and ₂ cDNAs in the pAlter-1 plasmid with BamHI and NheI. The four resulting DNA fragments contain the C termini of ₁ and ₂ and cDNAs encoding for the intact N terminus and middle regions of the ₁ and ₂ proteins. The C-terminal cDNA fragments were gel-purified and ligated back into either the cDNA encoding the N terminus and middle regions of the ₁ or ₂ as desired. This protocol generated the constructs containing the _AAB and _BBA chimeras. To exchange the N termini of the ₁ and ₂ molecules, the respective cDNAs in the pAlter-1 vector were digested with AflII and NheI, and the N-terminal cDNA fragments were gel-purified and ligated back into the cDNA encoding the middle regions and C terminus of either ₁ or ₂ as desired. This protocol generated the constructs containing _BAA and _ABB chimeras. It was desirable to subclone the cDNAs into the XbaI site in the baculovirus transfer vector pVL1393. Thus, the four chimeras in the pAlter-1 plasmid were subcloned into the linear pCNTR plasmid, which added the XbaI restriction sites to the end of each chimera. Each of the four chimeras was subcloned into the pVL1393 baculovirus transfer vector by excising them from pCNTR with XbaI and ligating the fragments into pVL1393 at its XbaI sites. The four chimeras containing altered CAAX sequences—_AAB-L71S (farnesyl), _BBA-S74L (geranylgeranyl), _BAA-S74L (geranylgeranyl), and _ABB-L71S (farnesyl)—were constructed using polymerase chain reaction on the cDNAs in pVL1393 as described previously (Lindorfer et al., 1996). Each of the eight pVL1393 transfer vectors containing the cDNA for a chimeric subunit was sequenced to ensure fidelity. Recombinant baculoviruses encoding for the eight chimeras were produced by cotransfecting each recombinant plasmid DNA with linear wild-type BaculoGold viral DNA into Sf9 cells and purified by one round of plaque purification (Graber et al., 1994). The recombinant baculoviruses encoding the _s, _i1, ₁, ₁, _1-S74L, ₂, and _2-L71S subunits have been described previously (Graber et al., 1994; Lindorfer et al., 1996).

Expression and Purification of G Protein and Subunits. G protein subunits were overexpressed in bacteria-(Sarvazyan et al., 1998) and baculovirus-infected Sf9 insect cells (Graber et al., 1994). Bacterially expressed, myristoylated _i1 was purified from Escherichia coli (BL21/DE3) by following the method of Mumby and Linder (1994). The amount of _i1 protein was determined using the Bradford assay. The G_i1 subunits expressed in Sf9 insect cells were purified to homogeneity as described previously (Graber et al., 1994), and the G_s subunit used in the adenylyl cyclase assays was prepared from a 0.1% (w/v) CHAPS extract of crude cell lysates (McIntire et al., 2001). The specific activity of GTP binding of these preparations was 11 to 15 nmol/mg of subunit, as measured by guanosine 5'-O-(3-[³⁵S]thio)triphosphate binding (Graber et al., 1994; Sarvazyan et al., 1998). Bovine brain dimer was isolated from brain cortex synaptosomal membranes as described previously (Sarvazyan et al., 1998). The membranes were a gift from Dr. T. Ueda (University of Michigan, Ann Arbor, MI). Recombinant subunits were prepared from baculovirus-infected Sf9 insect cells. Sf9 cells were coinfected with the appropriate recombinant baculoviruses encoding the ₁ subunit and native or chimeric subunits in Sf9 cells at a multiplicity of infection of 3 and harvested 48 h after infection. All subunits were extracted from frozen cell pellets with 0.1% Genapol C-100, and the detergent-extracted dimers were purified on a DEAE column followed by affinity chromatography on a G_i1-agarose column (Graber et al., 1996). The combinations used in this study are properly folded because they were purified on subunit affinity column, have a high affinity for the subunit (see Fig. 2), and were released after activation with AlF^-₄. The purified dimers were resolved on 12% polyacrylamide gels and stained with silver, and the concentration of the purified dimers estimated using ovalbumin standards. All preparations of the dimers were highly pure, and examples of their purity have been published previously (McIntire et al., 2001). The experiments shown under Results were performed using at least two different preparations of each dimer with consistent results.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Comparison of the ability of native dimers and those containing chimeric subunits to support the high-affinity agonist binding state of the A1 adenosine receptor. A, Sf9 cell membranes expressing recombinant, bovine A1 adenosine receptors were reconstituted with the Gi1 subunit and the dimer at concentrations of 0, 2, 6, or 40 nM. The ratio of receptor / was approximately 1:25. The amount of agonist binding to the receptor was measured with graded concentrations of 125I-amino-benzyladenosine as described under Materials and Methods. The figure indicates the recovery of high-affinity agonist binding conformation of the receptor as a function of the total amount of G protein heterotrimer reconstituted into the membrane. Each data point is an average of three similar experiments performed in triplicate. B, a plot of the affinity of the Gi1 subunit for eight different dimers measured in the flow cytometer against the EC50 values for the formation of the high-affinity agonist binding state of the receptor measured at 6 nM Gi1 and concentrations of ranging from 0 to 100 nM. Each data point is an average of three similar experiments performed in triplicate. Details of both assays are found under Materials and Methods. , dimers with farnesyl moieties; , dimers with geranylgeranyl moieties.

View this table: [in this window] [in a new window]   TABLE 1 Molecular mass of recombinant, chimeric subunits The molecular weights of the subunits in the dimers used in this study were measured by mass spectrometry as described under Materials and Methods. Molecular masses were determined on two independent preparations of each dimer and were in agreement. The data presented are from one determination.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Comparison of the ability of native dimers and those containing chimeric subunits to stimulate PLC-. A, the indicated concentrations of dimers were reconstituted with recombinant turkey PLC- in phospholipid vesicles containing [3H]phosphatidylinositol bisphosphate, and PLC- activity was measured as described under Materials and Methods. The activity of 1ABB () was compared with 12 (, thick solid line) and 11 (, thick broken line). The effect of the 1ABB dimer was statistically different from 11 (p < 0.0001; Table 2) but not from 12. B, an analogous experiment performed with 11-S74L and 1BAA-S74L and compared with the effects of 11 and 12. See Table 2 for the activity of dimers containing other chimeric subunits. C, an analogous experiment performed with 1BBA and compared with the effect of 11 and 12. The effect of the 1BAA dimer was statistically different from 12 but not from 11 (p < 0.0001; Table 2). Each data point is an average of three independent experiments, each performed in duplicate.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Comparison of the ability of native dimers and those containing chimeric subunits to inhibit type I adenylyl cyclase. A, Sf9 cells were infected with a recombinant baculovirus encoding the type I adenylyl cyclase, membranes were prepared, and the cyclase reaction was performed with the indicated concentrations of dimers. cAMP was measured as described under Materials and Methods. The ability of 1AAB () to inhibit type I cyclase was compared with 12 (, thick solid line) and 1 (, thick broken line). The effect of 1AAB statistically significant was different from 11 but not from 12 (Table 2). Each data point is an average of three independent experiments, each performed in duplicate. B, an analogous experiment performed with 1AAB-L71S () and compared with the effect of 11 and 12. Each data point is an average of three independent experiments, each performed in duplicate. C, an analogous experiment performed with 1BBA (open triangles) and compared with the effect of 11 and 12. The effect of the 1BBA dimer was statistically different from 12 but not from 11 (p < 0.0001; Table 2). Each data point is an average of three independent experiments, each performed in duplicate.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Comparison of the ability of native dimers and those containing chimeric subunits to stimulate type II adenylyl cyclase. A, Sf9 cells were infected with a recombinant baculovirus encoding the type II adenylyl cyclase, membranes were prepared, and the cyclase reaction was performed with the indicated concentrations of dimers as described under Materials and Methods. The ability of 1BAA-S74L () to activate type II adenylyl cyclase was compared with 12 (, thick solid line) and 11 (, thick dotted line). The effect of 1BAA-S74L was statistically significantly different from 11 (Table 3). B, an analogous experiment performed with 1BAA and 1BBA and compared with the effects of 11 and 12. Each data point is an average of three independent experiments, each performed in duplicate. C, an analogous experiment performed with 1ABB-L71S () and compared with the effects of 11 and 12. The difference between the effect of 1ABB-L71S and 12 significant was statistically (Table 3). Each data point is an average experiments, of three independent each performed in duplicate.

Expression of A1 Adenosine Receptors, Phospholipase C-, and Type I and II Adenylyl Cyclase. A recombinant baculovirus encoding the A1 adenosine receptor was used to overexpress the receptor in Sf9 insect cells, and membranes were purified as described previously (Yasuda et al., 1996). Sf9 insect cell membranes overexpressing recombinant type I or II adenylyl cyclase were prepared as described previously (McIntire et al., 2001). Recombinant turkey PLC- was overexpressed in Sf9 cells and purified as described previously (Myung et al., 1999).

Assay of the Activity of Dimers. Different facets of the activity of each dimer were measured in a set of in vitro assays chosen to probe the known protein-protein interaction domains of the dimer. Each of these assays was selected for its ability to highlight a known function of the dimer and because the outcome was directly dependent on activity. The affinity of the dimer for the subunit reflects interactions of the top and sides of the subunit with the switch II and C-terminal regions of the subunit (Cabrera-Vera et al., 2003). The prenyl group on the C-terminal region of the subunit is also important for binding the subunit (Iniguez-Lluhi et al., 1992). The affinity of the dimers for G_i1 was measured in solution with pure proteins by flow cytometry using fluorescein-labeled bacterially expressed myristoylated _i1 and biotinylated bovine brain as described previously (Sarvazyan et al., 1998). In brief, 1 nM biotinylated brain was prebound to streptavidincoated polystyrene beads (SVP-60-5; Spherotech Inc., Libertyville, IL) and incubated with 1 nM fluorescein-labeled, myristoylated _i1 together with varying amounts of recombinant in 20 mM HEPES, pH 8, 1 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl, 1.2 mM MgCl₂, 0.1% Lubrol, and 10 µM GDP. After a 30-min incubation at room temperature, samples were analyzed with a Becton Dickinson FACScan (BD Biosciences, San Jose, CA) capturing events on the forward scatter, side scatter, and fluorescein channels. Histograms of fluorescein fluorescence from singlet bead populations were obtained, and mean channel numbers were calculated using LYSIS II software (BD Biosciences).

The ability of each particular _1x dimer to support coupling of the G_i1 subunit to the A1 adenosine receptor was used to monitor the interactions of the N-terminal, central, and C-terminal regions of the 10 subunit chimeras with the receptor. In this assay, the receptor is expressed in an Sf9 cell membrane in the absence of G proteins, causing it to be in its low-affinity agonist binding conformation (Yasuda et al., 1996). Reconstitution of the G protein heterotrimer into the membrane re-establishes the high-affinity agonist binding conformation of the receptor as measured by a radioligand binding assay using an agonist ligand (Fig. 2A) (McIntire et al., 2002). On the other hand, if an excess of subunit and graded amounts of a dimer are reconstituted into the membrane, the amount of the receptor returned to the high-affinity agonist binding state as measured using a low concentration of the agonist ligand reflects the affinity of the receptor- subunit complex for the dimer (McIntire et al., 2002). Because only the subunit was different in the experiments shown in Fig. 2B, the assay provides a measure of the ability of the dimers containing the different chimeric subunits to interact with the receptor and subunit. This value is expressed as the EC₅₀ value of receptor coupling shown in Fig. 2B. The ability of the three regions of the subunit to interact with PLC- was measured by reconstituting dimers containing the chimeric subunits and pure PLC- into synthetic lipid vesicles (Myung et al., 1999). Sf9 cells infected with recombinant baculoviruses for adenyl cyclases can be used to monitor the direct interaction of the dimer with type I and type II adenyl cyclase (Taussig et al., 1994; McIntire et al., 2001).

To measure type I or II adenylyl cyclase activity, Sf9 membranes overexpressing the desired enzyme (10 µg of membrane protein/assay tube) were reconstituted with the purified guanosine 5'-3-O-(thio)triphosphate-activated G_s subunit and varying concentrations of pure dimers containing either a native or chimeric subunit and held on ice for 30 min (McIntire et al., 2001). Each assay was incubated at 30°C for 7 min, and the reaction was terminated with 1 ml of 0.11 N HCl. The samples were prepared for the assay of cAMP and the assay performed as described previously (McIntire et al., 2001). The activated G_s subunit was used as the coactivator of type I and type II cyclase in these experiments because the dimer does not increase the activity of type II cyclase in the absence of G_s or in the presence of forskolin (Taussig et al., 1994), and the dimer only modestly inhibits type I cyclase if it is preactivated with forskolin or Ca²⁺/calmodulin (Taussig et al., 1994). Pilot experiments with our Sf9 membranes confirmed both of these findings (data not shown).

Calculation and Expression of Results. Experiments presented under Results are the average of three or more similar experiments. Data expressed as concentration-response curves were fit to sigmoid curves using the fitting routines in the Prism software (GraphPad Software Inc., San Diego, CA). The EC₅₀ and V_max values shown in Tables 2 and 3 were taken from these fits. Statistical differences between the fitted curves were determined using all of the individual data points from multiple experiments to calculate the F statistic (Motulsky and Ransnas, 1987).

View this table: [in this window] [in a new window]   TABLE 2 Comparison of the ability of dimers containing native or chimeric subunits to activate PLC- or to inhibit type I adenylyl cyclase. The EC50, IC50, or Vmax values for PLC obtained with the indicated dimers were determined by fitting each data set to sigmoid curves as described under Materials and Methods. Vinhib is the level of cAMP production observed with 10–7 M of the indicated dimer. Control levels of ACI activity were 0.9 nmol cAMP/mg protein/min. Values are the average of three similar experiments expressed as means ± S.E.M.

View this table: [in this window] [in a new window]   TABLE 3 Comparison of the ability of dimers containing native or chimeric subunits to activate type II adenylyl cyclase The EC50 and Vmax values obtained with each dimer were determined by fitting each data set to sigmoid curves as described under Materials and Methods. Values are the average of three similar experiments expressed as means ± S.E.M.

Materials. All reagents used in the culture of Sf9 cells and for the expression and purification of G protein subunits have been described previously in detail (Graber et al., 1996). The baculovirus transfer vector pVL1393 was purchased from Invitrogen (Carlsbad, CA); BaculoGold viral DNA was from PharMingen (San Diego, CA); 10% Genapol C-100 and phosphatidylinositol 4,5-bisphosphate were from Calbiochem (San Diego, CA); phosphatidylethanolamine (bovine heart) was from Avanti Polar Lipids (Alabaster, AL); inositol-2-[³H]phosphatidylinositol 4,5-bisphosphate was from NEN Life Science Products (Boston, MA); CHAPS was from Roche Molecular Biochemicals (Indianapolis, IN); bovine serum albumin (fatty acid-free) was from Sigma (St. Louis, MO); and the pCNTR shuttle vector and the Morph Site-Specific Plasmid DNA Mutagenesis Kit were from 5 Prime 3 Prime, Inc. All other reagents were of the highest purity available.

	Results

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The ₁₁ and ₁₁₁ dimers are measurably less active than the ₁₂ dimer in its ability to activate K⁺ channels (Cabrera-Vera et al., 2003), PLC- (Myung et al., 1999), type II adenylyl cyclase (Myung et al., 1999), and PtdIns 3-kinase (Kerchner et al., 2004), or to inhibit type I adenylyl cyclase (McIntire et al., 2001). Because the dimers tested in these experiments all contained the ₁ subunit, differences in the primary amino acid sequence of the subunits, in their prenyl modification or both must explain the differences in activity. Whereas it was initially attractive to ascribe the unique properties of the ₁ subunit to its specialized role in visual transduction (Cabrera-Vera et al., 2003), the fact that ₁₁ and ₈ have similar amino acid sequences and prenyl modifications (Fig. 1A) and are widely expressed (Downes and Gautam, 1999) makes it important to understand the reasons for the differential activity of this subfamily of subunits. Figure 1A presents the differences in the N and C termini of the _{1, 11}, and ₂ subunits. Note that the N-terminal 20 amino acids of the very similar ₁ and ₁₁ subunits have six negatively charged amino acids, whereas this region of the ₂ subunit has only one negative charge, and that the C-terminal amino acids of the subunits also show marked differences in charge distribution. Thus, differences in the amino acid sequences and charges in the N- and C-terminal regions of these proteins may be important for the signaling specificity of the dimer. To address this issue, we constructed a set of four chimeric subunits in which the N- and C-terminal amino acids of ₁ and ₂ were exchanged. To complete the set, an additional four mutants with altered prenyl modifications was prepared. Figure 1B presents the eight chimeras that were made and the nomenclature used to describe them. The 12 subunits shown in Fig. 1B were expressed with ₁ subunit, and the dimers were purified from baculovirus-infected Sf9 insect cells by _i1-agarose affinity chromatography. The activity of the complete set of dimers was examined in a panel of in vitro assays measuring the affinity of the dimer for the G_i1 subunit, the ability to support coupling of the subunit to the bovine A1 adenosine receptor, and the ability to activate PLC- and regulate type I or type II adenylyl cyclases.

Receptor Coupling. The C-terminal domain of the mammalian subunit and its prenyl group are known to be important for the interaction of the dimer with subunits and receptors (Yasuda et al., 1996; Azpiazu et al., 1999). The affinity of the subunit for the dimer is an important component of the interaction of the heterotrimer with the receptor (Sarvazyan et al., 1998), but this parameter is not commonly measured. Moreover, the differences noted with the different forms of the subunit can be minimized when high concentrations of the and subunits are reconstituted into vesicles or membranes (Fig. 2A). For these reasons, we performed experiments to compare the affinity of dimers containing the chimeric subunits for the G_i1 subunit with the EC₅₀ values determined for the ability of the dimer to support the high-affinity agonist binding state of the A1 adenosine receptor. Figure 2A shows the ability of various concentrations of the G_i1:₁₂ heterotrimer to reconstitute the high-affinity agonist binding conformation of the adenosine A1 receptor overexpressed in Sf9 cell membranes (Yasuda et al., 1996). Four sets of membranes were reconstituted with increasing amounts of heterotrimer, and the amount of total agonist ligand bound was plotted as a function of ligand concentration. Note that concentrations of heterotrimer in the 40 nM range shift the affinity of the receptor for the agonist (amino-benzyladenosine) from approximately 30 (, no G protein) to 0.3 nM (, 40 nM G_i1:₁₂). An intermediate shift in the affinity of the receptor is caused by 2 to 6 nM heterotrimer. When the concentration of the heterotrimer is in the 2 to 6 nM range, it is possible to measure differences in the ability of various combinations to interact with the receptor- subunit complex and support high-affinity binding (McIntire et al., 2002). Therefore, we measured the ability of the six C-terminal chimeras to support the establishment of the high-affinity agonist binding conformation of bovine A1 adenosine receptors at the 6 nM heterotrimer. The results were expressed as the EC₅₀ value of the interaction and compared with the results with the affinity of the dimers for the G_i1 subunit measured in solution. The data in Fig. 2B show that the K_d (x-axis) of the dimer for the G_i subunit varies approximately 20-fold from 0.4 nM (₁₂) to 7.7 nM (_1BAA). Note that the EC₅₀ value for supporting coupling of the G_i1 subunit to the receptor (y-axis) varies 5- to 6-fold from 0.4 nM (₁₂) to approximately 2.2 nM (_1BAA). All dimers containing subunits modified with the farnesyl group () had lower affinities for the G_i1 subunit and lower EC₅₀ values for supporting receptor coupling than did those containing the geranylgeranyl moiety. These results confirm and extend the concept that the C-terminal region of the subunit and its prenyl group are very important for the interaction of the dimer with subunits and receptors in this assay. Moreover, the data show that there is a rough correlation between the EC₅₀ value for receptor coupling and the affinity of the and subunits themselves.

Phospholipase C-. Our previous experiments show that the activity of the ₁₁ and ₁₁₁ dimers on PLC- is significantly less than that of the ₁₂ dimer, and the activity is affected by the type of prenyl group on the subunit (Myung et al., 1999). To determine how the N- and C-terminal regions of the subunit affected the interaction of the dimer with PLC-, dimers containing the 12 subunits shown in Fig. 1B were tested for their ability to stimulate avian PLC- in vitro. The data in Fig. 3A show that ₁₂ markedly activates PLC- with an EC₅₀ value of approximately 5 nM () and that ₁₁ () is approximately 5-fold less potent (EC₅₀ value of 25 nM; Table 2). The activity of the ₁₁ dimer can be made equal to the ₁₂ dimer by replacing the central and C-terminal amino acids of the ₁ subunit with those from the ₂ subunit (, _1ABB). A chimeric subunit containing the first 50 amino acids of the ₁ subunit and the C terminus of the ₂ subunit (_1AAB) is also as equally effective as _1ABB (Table 2). These results suggest that the C-terminal 23 amino acids of the ₂ subunit are important for potent activation PLC- in vitro, a result confirmed by the data in Fig. 3C, which shows that replacing the last 23 amino acids and the geranylgeranyl group of ₂ with the amino acids and farnesyl group of ₁ (_1BBA) yields a dimer with activity very similar to that of ₁₁. The amino acids in the central region do not seem to affect the interaction with PLC-, because _1BAA has an activity equal to that of _1BBA and ₁₁ (Table 2).

Chimeric subunits with switched prenyl groups indicate that the differences in the activity of the dimers shown in Fig. 3, A and C, are due to both the differences in the C-terminal 23 residues and the composition of the prenyl group. Note that switching the prenyl group on the ₁₂ dimer from farnesyl to geranylgeranyl (_1BAA-S74L) provides a dimer with intermediate activity on PLC- (, Fig. 3B). Exchanging the prenyl group on ₁₁ (_11-S74L) produces a similar result (). In fact, all dimers containing subunits with exchanged prenyl groups were intermediate in either their EC₅₀ value or their maximal activity on PLC- (Table 2). These results indicate that the dimers containing the C-terminal sequence of the ₂ subunit modified with a geranylgeranyl group are more potent and effective in activating PLC- in vitro than those with the C-terminal region of the ₁ subunit and/or modified with farnesyl group.

Type I Adenylyl Cyclase. The data in Fig. 4 present the ability of five representative dimers to inhibit type I adenylyl cyclase. Figure 4A shows that the ₁₂ dimer inhibited type I adenylyl cyclase with an IC₅₀ value of 15 nM () and was approximately 2-fold more potent and effective than the ₁₁ dimer (IC₅₀ value of 36 nM; Table 2). However, a chimeric subunit with the C terminal 23 amino acids of the ₁ subunit and its prenyl group exchanged with those of the ₂ subunit (_AAB) produces a subunit with activity equal to that of the ₂ subunit (, Fig. 4A). In contrast, replacing the C terminus of ₂ and the geranylgeranyl group of ₂ with the C terminus of ₁ and the farnesyl group (_BBA) produces a dimer with activity similar to that of ₁₁ (, Fig. 4C). The data in Table 2 indicate that other dimers in which the C terminus of the ₁ subunit is intact and modified with farnesyl were roughly equal to the native ₁₁ dimer in their ability to inhibit type I adenylyl cyclase (e.g., _1BAA or _1BBA). These observations indicate that the C-terminal region of ₂ is most critical for the inhibition of type I adenylyl cyclase in vitro. As was the case with PLC-, both the C terminal amino acids and the prenyl group seem to be important for the interaction of the dimer with type I cyclase. Note that if the prenyl group on the _1AAB dimer is changed to farnesyl (_1AAB-L71S), the activity was intermediate between that of ₁g₂ and ₁₁ (, Fig. 4B). Similar data were obtained with the six other dimers containing subunits with modified prenyl groups (Table 2). Overall, the data in Figs. 3 and 4 demonstrate that the C-terminal amino acid sequence of the ₂ subunit and its native geranylgeranyl group are important determinants for the interaction of the subunit with effectors such as PLC- and type I adenylyl cyclase.

Type II Adenylyl Cyclase. The ₁₂ subunit activates type II adenylyl cyclase more than 10-fold with a nanomolar EC₅₀ value, and surprisingly, neither the ₁₁ nor the ₁₁₁ dimer activates type II adenylyl cyclase well (Myung et al., 1999). In addition, phosphorylation of the ₁₂ subunit in the ₁₁₂ dimer significantly inhibits its ability to stimulate type II adenylyl cyclase (Yasuda et al., 1998). The phosphorylation site has been determined to be at Ser¹ in the N terminus of the molecule (Asano et al., 1998). There are more negative charges in the N-terminal region of ₁ or ₁₁ compared with the ₂ subunit (Fig. 1A), potentially explaining the inability of dimers containing the ₁ or ₁₁ subunits to activate type II adenylyl cyclase. Taken together, these results suggest that the introduction of negative charges in the N-terminal regions of the subunit inhibit the interaction of the dimer with the type II adenylyl cyclase. Thus, we examined the ability of dimers containing N-terminal chimeric subunits to activate type II adenylyl cyclase.

The data in Fig. 5 present the ability of six representative dimers to activate type II adenylyl cyclase performed using concentrations of dimers ranging from 0.1 to 100 nM. As expected (Yasuda et al., 1998), the ₁₂ dimer () activated type II adenylyl cyclase with an estimated EC₅₀ value of 13 nM and was far more potent and effective in activating this effector enzyme than the ₁₁ dimer (Fig. 5A). Adding the N-terminal region of the ₂ subunit to the ₁ subunit and changing its prenyl group to geranylgeranyl (_1BAA-S74L) generated a molecule with activity equal to that of ₁₂ (, Fig. 5A). A similar result was observed with _1BBA-S74L (Table 3). In contrast, a dimer with the poor activity of ₁₁ could be created using a chimeric subunit containing the N-terminal 23 amino acids of ₁ and a farnesyl group (_1AAB-L71S; , Fig. 5C). These results suggest that the N-terminal region and the geranylgeranyl group are very important for the activation of type II adenylyl cyclase in vitro. Indeed, Fig. 5B shows that the _1BAA dimer, which contained the N-terminal amino acids of ₂ and the farnesyl modification, was intermediate in the activation of type II adenylyl cyclase. Indeed, the data in Table 3 show that every chimeric subunit, which is intermediate in activity on type II cyclase, is missing either the N-terminal amino acids of ₂ and/or the geranylgeranyl group. These results indicate that the dimers containing the N-terminal sequence of the ₂ subunit, which has one negatively charged amino acid, are more potent and effective in activating type II adenylyl cyclase than those with the N-terminal region of the ₁ subunit, which has six negatively charged amino acids. A C terminus modified with the geranylgeranyl group is also important. Similar results are observed with the effector PtdIns 3-kinase in that dimers containing ₁ or ₁₁ weakly activate the enzyme (Kerchner et al., 2004).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
There are three major findings of this in vitro study. First, dimers with a subunit containing the C-terminal 23 amino acid sequence of the ₂ subunit and modified with a geranylgeranyl group have the highest affinity for the G_i1 subunit and are the most effective in coupling of the G_i1 subunit to bovine A1 adenosine receptors (Fig. 2). These data provide an important demonstration of the correlation between the affinity of the subunit for the dimer and the ability of the complex to support high-affinity receptor-G protein coupling. Second, dimers with these attributes are most effective at activating PLC- and inhibiting type I adenylyl cyclase in vitro (Figs. 3 and 4). Finally, dimers containing the N-terminal 19 residues of the ₂ subunit are far more effective at activating type II adenylyl cyclase than are those containing the N-terminal 22 residues of the ₁ subunit. The full activity on type II adenylyl cyclase also required modification of the subunit with a geranylgeranyl group, but experiments with dimers such as _11-S74L and _111-S73L (Table 2), which are modified with the geranylgeranyl lipid, indicate that the C-terminal prenyl group is of secondary importance to the N-terminal amino acid residues in the activation of this effector (Myung et al., 1999).

The finding that the C-terminal region of the subunit and its prenyl group is much more important than the N terminus for the interaction with subunits and receptors confirms and extends a large amount of data obtained with rhodopsin in the visual system (Jian et al., 2001), the A1 adenosine receptor (Yasuda et al., 1996), the 5-hydroxytryptamine-1A receptor (Butkerait et al., 1995), the _2A receptor (Richardson and Robishaw, 1999; Lim et al., 2001), and bombesin receptors (Jian et al., 1999). Experiments performed with a similar set of chimeric subunits made between ₁ and ₂ were tested for their ability to support coupling between rhodopsin and the G_t subunit (Jian et al., 2001). These experiments also indicated that the C terminus of the ₂ subunit and its geranylgeranyl group contribute to better coupling between receptor and the heterotrimer (Jian et al., 2001). Moreover, experiments using synthetic peptides to probe the rhodopsin: subunit interaction found that the C-terminal 12 amino acids of the subunit (DKNPFKELKGGC in ₁ or SENPFREKKFFC in ₂) are clearly involved in interacting with subunits and the receptor. When the ₁ peptide was altered to change Phe⁶⁴ to threonine and Leu⁶⁷ to serine, it markedly decreased the interaction with rhodopsin (Azpiazu et al., 1999). Taken together, the results indicate that the C terminus of the mammalian subunit is very important for the interaction between G protein-coupled receptors and the heterotrimer. The receptor also seems to contact multiple domains in the subunit and a region near the C terminus of the subunit (Cabrera-Vera et al., 2003). Thus, each subunit in the heterotrimer is used for efficient activation of the subunit.

Whereas data obtained with mammalian cells point to a role for the C terminus of the subunit in coupling dimers to receptors, studies with the Saccharomyces cerevisiae pheromone mating pathway provide evidence that the C-terminal region of the yeast subunit is not important in receptor coupling. Using yeast strains in which the Ste18 locus (the only yeast subunit) was disrupted and pheromone signaling was inhibited, a variety of mutated subunits was tested for their ability to rescue signaling. Yeast subunits with a variety of C-terminal sequences, mutated by alanine scanning or by deletion of up to eight amino acids immediately preceding the CAAX box, were all able to rescue pheromone signaling (Chinault and Blumer, 2003). These data suggest that this region of the yeast molecule was not important for signaling through yeast G protein coupled receptors in vivo (Chinault and Blumer, 2003). More studies with mammalian cells are needed to reconcile these differences, although the diversity of the G and G subunits expressed in the mammalian genome will complicate these studies.

Whereas the C termini of subunits contain sequences capable of interacting with multiple receptors, subtle differences in the residues and charges must be important in determining specificity. For example, the ₁₁ dimer couples the G_i1 subunit to the ₂-adrenergic receptor poorly (Richardson and Robishaw, 1999), whereas the ₁₁₁ dimer couples well (Lim et al., 2001), yet there are only two differences in the C-terminal 12 amino acids of ₁ and ₁₁ (Fig. 1A). Experiments performed with receptors reconstituted with G protein heterotrimers using various protocols show relatively modest differences in the interaction of the various subunits with receptors (Yasuda et al., 1996; Hou et al., 2001; Lim et al., 2001; Akgoz et al., 2002). However, antisense experiments performed with intact cells suggest an exquisite selectivity between receptors, subunits, and certain combinations (Kleuss et al., 1993). Thus, despite considerable effort, the factors dictating the specificity that must exist in intact cells remain elusive. These data, combined with the studies performed in yeast, suggest that additional work with mammalian cells is needed to reconcile these differences. The diversity of the G and G subunits in the mammalian genome will make these studies complex, but the observation that mice with the ₇ subunit knocked out have an altered phenotype (Schwindinger et al., 2003) offers hope that these studies will provide interesting results.

The data in Fig. 3 indicate that, as with receptors, both the prenyl group and the C-terminal amino acids of the subunit contribute significantly to the interaction of the dimer with PLC- in vesicles. This finding is consistent with experiments showing that native dimers containing the ₁ or ₁₁ subunits have reduced activity on this effector (Myung et al., 1999). In addition, experiments performed with dimers containing the ₅ subunit modified to contain a shortened or extended C terminus are less effective at activating PLC- (Akgoz et al., 2002). A possible explanation for the role of the prenyl group in the activation of effectors may be provided by the hypothesis that the prenyl group on the subunit interacts with the C-terminal region of the subunit and participates in a conformational change that, in part, determines the activity of the dimer at effectors (Myung and Garrison, 2000). On the other hand, the prenyl group may interact directly with PLC-, as suggested by experiments with prenylated peptides (Akgoz et al., 2002). It is also possible that both mechanisms come into play. A more complete understanding of the role of this region of the and subunits in these interactions awaits structural information or biophysical experiments determining the exact sites of contact between these regions of the dimer and receptors or effectors.

A surprising finding is that the C-terminal region of the subunit seems to predominate in the inhibition of type I adenylyl cyclase, whereas the N-terminal region of the molecule is the most important for the activation of type II adenylyl cyclase. However, the regulation of adenyl cyclase is complex, with multiple binding sites identified for the known regulatory molecules (Taussig et al., 1994). The differences identified for regulation by the subunit in the dimer are in keeping with these findings. The observations that the N-terminal domain of is important for the activation of type II cyclase are consistent with earlier experiments showing that phosphorylation of the ₁₂ subunit in the ₁₁₂ dimer with protein kinase C decreases its ability to stimulate type II adenylyl cyclase (Yasuda et al., 1998). The phosphorylation site in ₁₂ is Ser¹ at the N terminus (Asano et al., 1998), suggesting that negative charges in this region decrease the interaction of the dimer with type II cyclase. The ₁, ₈, and ₁₁ subunits contain six negatively charged amino acids in their N terminus (Fig. 1A); thus, dimers containing these subunits might be predicted to interact poorly with type II cyclase. Indeed, dimers containing ₁, ₁₁, or chimeras such as _ABB-L71S or _ABB are not able to activate the enzyme well (Table 3) (Myung et al., 1999). Together, these results strongly suggest that negatively charged amino acids in the N terminus of the subunit lead to an inability to activate type II adenylyl cyclase in vitro. The observation that inhibition of the type I isoform of adenyl cyclase is quite dependent on the sequences in the opposite end of the subunit indicates that the different isoforms of adenyl cyclase must bind to different regions of the and subunits in the dimer. In keeping with this finding, mutations on the top surface of the subunit that dramatically inhibit the activation of type II cyclase do not alter the inhibition of type I cyclase (Li et al., 1998). Thus, the precise determinants of the interaction of the type I and type II cyclases with dimers seem to be different.

The isoform of the subunit is also important in the interaction of dimers with effectors, and certain regions on the top surface of the subunit have been identified as critical for interaction of the dimer with effectors (Ford et al., 1998). A major concept arising from these studies is that whereas different effectors may interact with distinct or overlapping regions of the subunit, certain regions in the subunit interacting with effectors are in the domain covered by the subunit in the heterotrimer (Ford et al., 1998). Thus, formation of the basal state of the :-receptor complex is an efficient mechanism to terminate signaling. The data in Fig. 2B show that the C terminus of the subunit and its prenyl group are important determinants of the affinity of the subunit for the dimer. Thus, this domain of the subunit may be important for the rapid formation of a high-affinity : complex, which speeds reversal of the effects of the dimer on targets such as PLC- or adenylyl cyclase. The present study also demonstrates that the nature of the N and C terminal regions of the subunit imparts significant specificity to signaling. Thus, dimers containing subunits from subfamily I such as ₁ or ₁₁ (and probably ₈, although it has not been studied extensively) seem to have unique signaling properties in that they are unable to activate type II adenylyl cyclase or other important effectors, such as phosphatidylinositol-(4,5)-3-kinase (Kerchner et al., 2004). The distribution of ₁ is restricted to retinal rods (Fung, 1983), and the original report of ₈ (termed _c) suggested that it was restricted to cone cells (Ong et al., 1995). However, the ₁₁ subunit is widely expressed (Morishita et al., 1998), and it now seems that ₈ is also widely expressed (Downes and Gautam, 1999). Thus, it is important to define the signaling properties of dimers containing these farnesylated subunits in multiple cellular systems. The appealing concept that certain dimers may associate selectively with distinct subunits and/or receptors has been used to account for the diversity of these subunits in the G protein family (Hildebrandt, 1997; Richardson and Robishaw, 1999; McIntire et al., 2001; Cabrera-Vera et al., 2003). This hypothesis also predicts that unique dimers will be released upon receptor activation in intact cells, allowing a given receptor to release a dimer with selective signaling properties. If the dimers released were from subfamily I, they might have very selective signaling properties at effectors. The data in this report support this idea; however, future experiments will need to concentrate on the functions of this diverse family of proteins in intact cellular systems.

	Acknowledgements

 
We thank Dr. Ravi Iyengar (Mt. Sinai School of Medicine, New York, NY) for the baculoviruses encoding type I and type II adenylyl cyclases, Dr. T. K. Harden (University of North Carolina, Chapel Hill, NC) for the purified recombinant turkey phospholipase C-, the University of Virginia Diabetes Core Facility for cAMP assays, and the Biomolecular Research Facility for DNA sequencing and mass spectrometric analysis. We also acknowledge Warren J. Clingan III for excellent technical assistance.

	Footnotes

 
This work was supported by a Postdoctoral Fellowship from the Virginia Affiliate of the American Heart Association (to C.-S.M.), a training grant from the Universiti Malaysia Sarawak (to W.K.L.), and the National Institutes of Health grants CA40042, DK19952 (to J.C.G.), and HL46417 (to R.R.N.).

ABBREVIATIONS: PLC-, phosphatidylinositol-specific phospholipase C- isoform; PtdIns, phosphatidylinositol 4,5-bisphosphate; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; Genapol C-100, polyoxyethylene (10) dodecyl ether.

Address correspondence to: Dr. James C. Garrison, Department of Pharmacology, University of Virginia Health Sciences Center, P.O. Box 800735, 1300 Jefferson Park Avenue, Charlottesville, VA 22908-0735. E-mail: jcg8w{at}virginia.edu

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