Introduction

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The G proteins are responsible for the efficient transmission of signals from agonist-bound cell surface receptors to different intracellular effectors (Bourne, 1997). Currently, the total number of G protein-coupled receptors (GPCRs) far exceeds the number of known G proteins. Each member of the four different subfamilies of G proteins must therefore be capable of interacting with multiple GPCRs. Depending on their coupling specificity, most GPCRs are often referred as G_q, G_i, or G_s coupled, which reflects their primary signal transduction pathway. Some G proteins are more promiscuous than others by possessing the ability to interact with a large panel of GPCRs. The most notable examples of promiscuous G proteins are the human G₁₆ and its murine homolog, G₁₅. Both G₁₅ and G₁₆ link a variety of G_q-, G_i-, and G_s-coupled receptors to the stimulation of phospholipase C (PLC; Offermanns and Simon, 1995; Lee et al., 1998). Because of their promiscuity, G₁₅ and G₁₆ are ideal candidates for linking orphan receptors (cloned receptors without a known ligand) to PLC and its downstream effectors. Hence, G₁₆ has received considerable attention as a potential tool for drug discovery (Milligan et al., 1996).

Although G₁₅ and G₁₆ are more promiscuous than other G proteins, they cannot be considered as true universal adapters for GPCRs. For example, the CCR2a chemokine receptor (Kuang et al., 1996), the _1A- and _1C-adrenoceptors (Wu et al., 1992) are unable to recognize G₁₆. Indeed, of the 33 different GPCRs examined to date (Wu et al., 1992, 1993; Offermanns and Simon, 1995; Zhu and Birnbaumer, 1996; Kuang et al., 1996; Lee et al., 1998; Parmentier et al., 1998), at least six receptors are incapable of activating G₁₆. Most of the GPCRs that fail to activate G₁₆ belong to the G_i-coupled receptor subfamily. Because approximately 18% of the total number of G_i-coupled receptors examined to date cannot activate G₁₆, the molecular structure of G₁₆ may not be optimal for association to GPCRs with a high preference for G_i proteins. This is perhaps unavoidable if G₁₆ were to possess the ability to recognize G_q- and G_s-coupled receptors as well. The G_i-coupled receptors constitute an exceedingly large GPCR subfamily that encompasses many newly discovered receptors such as those for chemokines. This poses a serious concern for the use of G₁₆ as an adapter of orphan receptors in drug-screening protocols.

The overall receptor contact regions on the -subunit of several G proteins have been mapped (reviewed in Conklin and Bourne, 1993; Onrust et al., 1997), and they are in good agreement with the available crystal structure data (Wall et al., 1995; Lambright et al., 1996). Both the N and C termini are sites for receptor contact. Promiscuity for GPCRs can be increased by altering the amino acid sequence of either the N or C terminus. Modification of the last five residues of G_q (Conklin et al., 1993) or G_s (Liu et al., 1995; Conklin et al., 1996) expanded the number of GPCRs that can use the resultant chimeras to regulate the corresponding effectors beyond those of the parental constructs. Likewise, alterations at the N terminus of G_q resulted in additional coupling to several GPCRs that do not normally use G_q for signal transduction (Kostenis et al., 1998). These studies illustrate the possibility of changing the coupling specificity of G proteins by modifying their putative receptor contact sites on the -subunit. Many G_i-coupled receptors share the ability to inhibit adenylyl cyclase via the pertussis toxin (PTX)-insensitive G_z (Chan et al., 1995, 1998; Lai et al., 1995; Shum et al., 1995; Tsu et al., 1995a,b; Yung et al., 1995). Thus, the incorporation of G_z-specific sequences into a G₁₆ backbone might improve the ability of the resultant chimera to recognize G_i-coupled receptors. The present study describes the construction and characterization of a series of chimeras between the -subunits of G₁₆ and G_z (G₁₆ and G_z). Indeed, some of the resultant chimeras has increased promiscuity toward G_i-coupled receptors and are more efficient than G₁₆ in their interactions with GPCRs.

	Experimental Procedures

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Materials. The G₁₆ cDNA was a kind donation from Dr. M. Simon (California Institute of Technology, Pasadena, CA), and all other cDNAs were constructed or obtained as previously described (Wong et al., 1992; Tsu and Wong, 1996; Lee et al., 1998). Simian kidney fibroblast COS-7 cells were obtained from the American Type Culture Collection (ATCC CRL-1651; Rockville, MD). Various receptor agonists were purchased from Research Biochemicals Inc. (Natick, MA), Sigma Chemical Co. (St. Louis, MO), or Tocris Cookson (Bristol, UK). Sequenase Version 2.0 DNA sequencing kit and ECL enhanced chemiluminescence detection kit were purchased from Amersham Pharmacia Biotech. myo-[³H]Inositol was from DuPont-New England Nuclear (Boston, MA). Plasmid DNA purification columns were obtained from Qiagen (Hilden, Germany). G_z-specific antisera 3A-170 (C-terminal) and 3-18 (N-terminal) were obtained from Gramsch Laboratories (Schwabhausen, Germany) and Calbiochem (La Jolla, CA), respectively. Taq DNA polymerase, restriction endonucleases, custom mutation primers, and cell culture reagents were obtained from Life Technologies Inc. (Grand Island, NY), and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

Construction of Chimeras and Mutants. All of the chimeras were constructed by polymerase chain reactions (PCRs) using human G₁₆ and rat G_z cDNAs (subcloned in the XbaI and EcoRI sites of pcDNAI, respectively) as templates with T7 and SP6 promoter sequences as outer flanking priming regions. A pair of chimeric primers covering both the nucleotide sequences of G₁₆ and G_z were designed as appropriate for each chimeric construct. First, two overlapping fragments that corresponded to the portions of G₁₆ and G_z were generated. The 5' fragment was made with T7 primer and the reversed chimeric primer, whereas the 3' fragment was made with the forward chimeric primer and a primer annealed to the SP6 promoter on the vector sequence (SP6 primer). The two PCR products were annealed together, and the full-length fragments were made using the T7 and SP6 primers. Specific primers used for the construction of the various chimeras were listed below, with the nucleotide sequences of G_z underlined: 16z25/S, CAC TAC ACA TGT GCC ACA GAC ACC AGT AAC ATC; 16z25/AS, GAT GTT ACT GGT GTC TGT GGC ACA TGT GTA GTG; 16z44/S, GGC CCC GAG GGC AGC AAC CGA AAC AAG GAG; 16z44/AS, CTC CTT GTT TCG GTT GCT GCC CTC GGG GCC; 16z66/S, GCT ACC TAT TTC CCC GAG TAC AAG GGT CAG; 16z66/AS, CTG ACC CTT GTA CTC GGG GAA ATA GGT AGC; 30z16/S, GAG AGC CAG CGG CAG CGC GGG GAG CTG AAG; and 30z16/AS, CTT CAG CTC CCC GCG CTG CCG CTG GCT CTC. MgCl₂ (1.5 mM) was included in the PCR mixture, and the PCR products were amplified with thermal cycling at 94°C for 60 s, 50°C for 90 s, and 72°C for 90 s for 30 cycles using Robocycler 40 from Stratagene (La Jolla, CA). The 30z16z44 and 30z16z66 chimeras were constructed in a similar manner except one of the half-products was amplified by PCR using either the 16z44 or 16z66 chimera as the template. Full-length chimeric -subunit cDNAs were subcloned into either pcDNA3 or pcDNA3.1Zeo(+) mammalian expression vectors (InVitrogen, San Diego, CA). DNA sequences of the mutants were checked by dideoxynucleotide sequencing method using Sequenase V2.0 kit and restriction mapping.

Transfection of COS-7 Cells. Simian kidney fibroblast COS-7 cells were cultured with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS (v/v), 50 U/ml penicillin, and 50 µg/ml streptomycin at 37°C in humidified air with 5% CO₂. Then, 1 × 10⁵ cells/well were seeded onto 12-well plates the day before transfection. DEAE-dextran-mediated transfection was performed as described previously (Wong, 1994). Briefly, appropriate amounts of various DNA samples purified by Qiagen column chromatography were mixed with growth medium containing 250 µg/ml DEAE-dextran and 100 µM chloroquine. Cells were incubated with the transfection cocktails for ~3.5 h and then shocked for 1 min at room temperature in PBS containing 10% dimethyl sulfoxide (v/v). After rinsing with PBS, the cells were returned to growth media for 24 h. Approximately 50% of the cell population will take up the cDNAs as indicated by cotransfecting a plasmid DNA encoding -galactosidase as a reporter.

Inositol Phosphates (IP) Accumulation Assay. An aliquot (750 µl) of inositol-free DMEM containing 5% FCS and 2.5 µCi/ml myo-[³H]inositol was added to each well of transfected COS-7 cells and incubated for 18 to 24 h. The labeling media were subsequently replaced by 1 ml of inositol phosphate assay medium (DMEM buffered with 20 mM HEPES, pH 7.5, containing 20 mM LiCl) for 10 min, and then 1 ml of IP assay medium containing the appropriate agonist was added to the cells for an additional 1 h at 37°C. Reactions were stopped by adding 750 µl of ice-cold 20 mM formic acid and stored at 4°C for 30 min. [³H]IP were separated from other labeled inositol species by sequential ion-exchange chromatography as described previously (Tsu et al., 1995a).

Membrane Protein Preparation and Immunodetection of Recombinant Proteins. COS-7 cells were grown on 150-mm dishes to 70 to 80% confluence. Transfection was performed as on 12-well plates with proper adjustments to the volumes and amounts of the reagents used. After 48 h in normal growth conditions, cells were washed with Ca²⁺/Mg²⁺-free PBS and harvested with 5 ml of Ca²⁺/Mg²⁺-free PBS containing 10 mM EDTA. The following procedures were performed at 4°C. Cells were spun down briefly (200g, 5 min); resuspended in hypotonic lysis buffer (50 mM Tris · HCl, 2.5 mM MgCl₂, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine-HCl, and 1 mM dithiothreitol, pH 7.4); 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 15,000g for 15 min. Membrane pellets were finally resuspended in lysis buffer. Protein concentrations were determined using the Bio-Rad Protein Assay Kit. For immunodetection, 50 µg of each membrane protein sample was resolved on a 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes through electroblotting. Protein molecular weight markers were visualized by Coomassie blue staining. Several chimeras were detected by the G_z-specific antiserum from Calbiochem and with enhanced chemiluminescence (ECL kit; Amersham).

Data Analysis. [³H]IP was estimated by determining the ratios of [³H]IP to [³H]inositol plus [³H]IP as previously described (Tsu et al., 1995a). Absolute values for IP accumulation varied between experiments, but variability within a given experiment was less than 10% in general. Unless otherwise stated, data shown in the figures and tables represent the mean ± S.E. of three or more independent experiments performed in triplicate. Bonferroni's t test with 95% confidence limit was adopted to verify the significance between different treatment groups within the experiments.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Design of C-Terminal Chimeras. Although multiple regions in the primary structure of G₁₆ are required for receptor coupling (Lee et al., 1995), the molecular determinants for the promiscuous property of G₁₆ has not been fully delineated. Numerous studies on other -subunits have implicated the C-terminal tail of the -chain as one of the major receptor contact regions (Sullivan et al., 1987; Conklin et al., 1993, 1996; Tsu et al., 1997). As a first step toward enhancing the ability of G₁₆ to recognize G_i-coupled receptors, we constructed a series of G_16/z chimeras by incorporating different lengths of G_z sequences at the C terminus of G₁₆. G_z was selected as a donor for the construction of the chimeras because it recognizes practically all G_i-coupled receptors (Wong et al., 1992; Chan et al., 1995, 1998; Lai et al., 1995; Shum et al., 1995; Tsu et al., 1995a,b; Yung et al., 1995) but is insensitive to PTX. Signals transduced by chimeric G_16/z can be easily discerned from G_i-mediated signals with the use of PTX. Based on the alignment of the C-terminal sequences of G₁₆ and G_z (Fig. 1), we selected three junctional sites for the construction of G_16/z chimeras.

View larger version (49K): [in this window] [in a new window]   Fig. 1.   Alignment of amino acid sequences of G16, Gz, and their chimeras. The amino acid sequences of of the N and C termini G16 and Gz are aligned using the Clustal X program. Gaps introduced for better alignment of the sequences are hyphenated. Residues of Gz that are identical to G16 are shown as dots. Numbers shown represent the relative positions of the amino acids of G16 and Gz. Shaded horizontal columns depict sequences of the chimeras that are identical to G16. Putative secondary structures based on the Gt1 crystal structure are indicated by striped (-helix) and solid (-strand) horizontal columns above the Gz sequence.

Functional Coupling of G_16/z Chimeras to -Opioid Receptor. We used a well established transient expression system to examine the ability of the G_16/z chimeras to interact with G_i-coupled receptors. We have previously shown that coexpression of the -opioid receptor (a typical G_i-coupled receptor) and G₁₆ in COS-7 cells permits the -selective opioid agonist [D-Pen²,D-Pen⁵]enkephalin (DPDPE) to stimulate the formation of IP (Lee et al., 1998). Here, we adopted the same approach to study the G_16/z chimeras. In accordance with our earlier report (Lee et al., 1998), 100 nM DPDPE stimulated IP formation by 3-fold in COS-7 cells coexpressing the G₁₆ and the -opioid receptor (Fig. 2). In contrast, agonist treatment failed to evoke formation of IP in COS-7 cells cotransfected with cDNAs encoding the -opioid receptor and G_z (Fig. 2). In COS-7 cells coexpressing the -opioid receptor with either 16z25 or 16z44, 100 nM DPDPE stimulated the formation of IP by 3- to 4.5-fold (Fig. 2). Interestingly, the DPDPE response was significantly higher in cells transfected with the 16z44 cDNA than those expressing 16z25 or G₁₆ (Fig. 2 and Table 1).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Stimulation of PLC by the -opioid receptor via the 16z25 and 16z44 chimeras. G16/z chimeras were constructed as described in Materials and Methods. Left, schematic representation of the chimeras. Activation of PLC was determined by transfecting COS-7 cells with cDNAs (0.25 µg/ml each) encoding the -opioid receptor and one of the three chimeric G proteins: 16z25, 16z44, or 16z66. Additional transfections with the wild-type G16 and Gz were carried out as controls. Transfected cells were labeled with 2.5 µCi/ml Myo-[3H]inositol 20 to 24 h before the assay. Formation of IP was measured in the absence and presence of 100 nM DPDPE. Results are expressed as percentage stimulation of IP production compared with basal activity. Basal values expressed as the ratio (×103) of IP to total inositols ranged from 10.5 ± 0.8 to 16.2 ± 1.8. *DPDPE-induced IP formation was significantly higher than basal levels. **DPDPE-stimulated IP accumulation was significantly higher than that observed in the G16 transfected cells; Bonferroni's t test, P < .05.

View this table: [in this window] [in a new window]   TABLE 1 Coupling of G16, 16z25, and 16z44 to Various Gi- or Gs-Coupled Receptors COS-7 cells were cotransfected with cDNAs encoding 16z25 or 16z44 and the indicated receptors (0.25 µg/ml per construct). Transfected cells were labeled with 2.5 µCi/ml [3H]inositol 20 to 24 h before assay. IP formations were determined in the absence (basal) or presence of specific agonists for the indicated receptors. fMLP, N-formylmethionyl-leucyl-phenylalanine; hCG, human choriogonadotropin; LHR, luteinizing hormone receptor; PIA, (+)-N6-(2-phenylisopropyl)-adenosine.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Immunodetection of chimeric G16/z subunits. COS-7 cells were transiently transfected with cDNAs encoding the wild-type or constitutively active mutant of 16z25, 16z44, and 16z66. Plasma membranes were prepared 48 h post-transfection. Membrane proteins (50 µg) were separated on an SDS-12.5% polyacrylamide gel and electrophoretically transferred to polyvinylidene difluoride membranes. Protein markers were localized by Ponceau S staining, and the chimeras were immunodetected with the 3A-170 antiserum against the C terminus of Gz. Two independent experiments with different batches of membrane proteins yielded similar results.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Constitutively activated G16/z chimeras stimulate PLC except for 16z66QL. Wild-type G16 and G16/z chimeras were constitutively activated by altering the amino acid at position 212 from Q (Gln) to L (Leu). The cDNAs (0.25 µg/ml) encoding the mutant chimeras were transiently expressed in COS-7 cells. G16 and GzQL were included as negative controls. Transfected cells were labeled with Myo-[3H]inositol and assayed for IP accumulation in the absence of agonist as in the legend to Fig. 2. *Basal IP formation was significantly higher than that obtained with wild-type G16; Bonferroni's t test, P < .05.

Promiscuity of 16z25 and 16z44. The ability of 16z25 and 16z44 to interact productively with the -opioid receptor prompted us to further investigate into their capacity to functionally associate with other G_i- and G_s-coupled receptors. COS-7 cells were cotransfected with G₁₆, 16z25, or 16z44 and a receptor (0.25 µg/ml per construct) chosen from a panel of G_i- or G_s-coupled receptors that were available in our laboratory. The selected receptors include the adenosine A₁, ₂- and ₂-adrenergic, complement C5a, dopamine D₁ and D₂, formyl peptide, luteinizing hormone, opioid receptor-like (ORL₁), vasopressin V₂, somatostatin-1 and -2 (SSTR1 and SSTR2), three subtypes each of melatonin (1a, 1b, and 1c) and opioid (µ, , and ) receptors (Table 1). Transfected cells were assayed for IP formation in the absence or presence of saturating concentrations of the appropriate agonists. All 14 G_i-coupled receptors examined were capable of activating 16z25 and elicit agonist-induced PLC activation (Table 1). The magnitude of PLC stimulation by the various receptors ranged from 1.5- to 4.5-fold. Activation of 16z25 by aminergic receptors (dopamine and melatonin receptors) resulted in up to 3.5-fold stimulation of PLC activity. The receptors for peptide ligands gave slightly higher responses in general. Similar results were obtained with the 16z44 chimera (Table 1). All of the G_i-coupled receptors tested were efficiently coupled to 16z44 and stimulated PLC. The 16z44-mediated PLC responses ranged from 1.7- to 5.5-fold stimulation. It should be noted that none of the G_i-coupled receptors, except SSTR2, was capable of stimulating IP production in the absence of 16z25 or 16z44 (unpublished data; Lee et al., 1998). Collectively, these results indicate that both 16z25 and 16z44 are capable of linking a large variety of G_i-coupled receptors to the stimulation of PLC. In terms of absolute amount of IP formation, 16z25 was generally less efficient than 16z44 in transducing signals from G_i-coupled receptors. When G_i-coupled receptors such as the formyl peptide, melatonin Mel1a, and ORL₁ receptors were tested against the 16z66 chimera, no functional coupling could be detected.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Dose-dependent agonist stimulation of PLC by the Mel1c, SSTR1, and -opioid receptors via G16 or 16z44. COS-7 cells were transiently cotransfected with the cDNAs encoding G16 () or 16z44 () and one of the three selected receptors: Mel1c, SSTR1, or -opioid (at 0.25 µg/ml per construct). Transfected cells were then labeled with Myo-[3H]inositol and assayed for IP formation in the absence or presence of various concentrations of 2-iodomelatonin (0.03-100 nM), somatostatin (1 nM to 1 µM), or DPDPE (1 nM to 1 µM). Data represent the mean ± S.D. of triplicate determinations of a single representative experiment; two additional experiments yielded similar results.

Coupling of 16z25 and 16z44 to G_s- and G_q-Linked Receptors. A distinguishing feature of G₁₆ is its ability to link a large number of GPCRs to the stimulation of PLC, including those receptors that normally utilize G_s for signal propagation. To confirm that the G_16/z chimeras can also recognize G_s-coupled receptors, we assessed the ability of 16z25 and 16z44 to interact productively with four different G_s-linked receptors. COS-7 cells were transiently cotransfected with either 16z25 or 16z44 and a G_s-coupled receptor (₂-adrenergic, dopamine D₁, luteinizing hormone, or vasopressin V₂). Transfected cells were subsequently challenged with the appropriate agonists. In cells coexpressing the ₂-adrenergic, dopamine D₁, or vasopressin V₂ receptors with either 16z25 or 16z44, activation of the receptor led to increased production of IP (Table 1). Among these three receptors, only the vasopressin V₂ receptor has the ability to utilize endogenous G_q to weakly stimulate IP formation (66.5 ± 17.8% over basal, n = 6). The magnitudes of agonist-induced stimulations mediated via 16z25 or 16z44 were all ~3-fold and were generally lower than those observed with G₁₆ in previous reports (Offermanns and Simon, 1995; Lee et al., 1998). In contrast, activation of the luteinizing hormone receptor did not significantly stimulate IP formation in the presence of either 16z25 or 16z44 (Table 1).

Construction and Characterization of N-terminal G_z/16 Chimeras. Recent studies by Wess and coworkers (Kostenis et al., 1998) have focused on the extreme N-terminal region of G_q as a determinant for the selectivity of receptor coupling. Compared with the -subunits of the G_i subfamily, G_q and G₁₁ are longer by six residues at their N termini. Progressive deletion or substitution with alanine of these "extra" residues produced G_q mutants that can effectively interact with G_i-coupled receptors (Kostenis et al., 1998). Alignment of the G₁₆ and G_z sequences revealed that their predicted N-terminal -helices share little homology and that the G₁₆ N terminus is longer than that of G_z by nine residues (Fig. 1). To assess whether the N terminus of G_z is required for efficient coupling to G_i-linked receptors, we replaced the entire N helix with that of the first 30 residues of G_z. The resultant 30z16 chimera is shorter than G₁₆ by nine residues (Fig. 1).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Stimulation of PLC by the -opioid receptor via the 30z16 and 30z16z44 chimeras. Gz/16 chimeras were constructed as described in Materials and Methods. Left, schematic representation of the chimeras. COS-7 cells were cotransfected with cDNAs (0.25 µg/ml each) encoding the -opioid receptor and G16, Gz, or one of three Gz/16 chimeras: 30z16, 30z16z44, or 30z16z66. Transfected cells were assayed as in the legend to Fig. 2. Results are expressed as percentage stimulation of IP production compared with basal activity. Basal values expressed as the ratio (×103) of IP to total inositols ranged from 11.3 ± 1.2 to 14.7 ± 1.1. *DPDPE-induced IP formation was significantly higher than basal levels; Bonferroni's t test, P < .05.

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Coupling of 2-adrenergic (2-AR) and ORL1 receptors to the 30z16 and 30z16z44 chimeras. COS-7 cells were cotransfected with cDNAs (0.25 µg/ml each) encoding the 2-adrenergic or ORL1 receptor with one of three Gz/16 chimeras: 30z16 (A), 30z16z44 (B), or 30z16z66 (C). Transfected cells were assayed for IP formation in the absence or presence of agonist (1 µM isoproterenol or 100 nM nociceptin). *Agonist-induced IP formation was significantly higher than the corresponding basal values; Bonferroni's t test, P < .05.

View larger version (43K): [in this window] [in a new window]   Fig. 8.   Immunodetection of chimeric Gz/16 subunits. COS-7 cells were transiently transfected with cDNAs encoding the wild-type Gz, 30z16, 30z16z44, or 30z16z66 chimeras. Immunodetection of the various chimeras was performed as described in the legend to Fig. 3, except that a Gz N-terminal specific antiserum 3-18 was used.

Effects of Inverse Agonists on Receptors Coupled to 16z44. A notable feature of cells coexpressing a GPCR and 16z25 or 16z44 was their elevated basal IP production. The increased basal IP accumulation can be seen with the vast majority of the receptors tested (Table 1), and it resembles the constitutive activity of GPCRs. This interpretation is supported by the fact that no elevation of basal activities could be observed in COS-7 cells expressing 16z44 alone; coexpression with a G_i- or G_s-coupled receptor is required (Y. H. Wong, unpublished results). If the enhanced linkage of the chimeras to GPCRs promotes the formation of constitutively active receptors, it may offer an opportunity to identify inverse agonists that act at various GPCRs. To test this hypothesis, COS-7 cells were cotransfected with cDNAs encoding 16z44 and either the -opioid or ₂-adrenergic receptor, and the ability of known inverse agonists to suppress the elevated basal IP levels of the transfectants was examined. Compared with cells coexpressing the -opioid receptor and G₁₆, 16z44 transfectants exhibited increased basal IP production, but neither ICI-174,864 (an inverse agonist) nor naloxone (a neutral antagonist) affected the elevated basal levels (Fig. 9). The expression of -opioid receptors in the transfectants was confirmed by the DPDPE-induced stimulation of PLC (Fig. 9). Similar results were obtained with cells coexpressing the ₂-adrenergic receptor and 16z44. Two inverse agonists of ₂-adrenergic receptor, ICI-118,551 and timolol, were incapable of reducing the elevated basal level associated with the coexpression of 16z44 (Fig. 9). These results suggest that although 16z44 may provide enhanced linkage to GPCRs, it does not necessarily promote the formation of constitutively active GPCRs.

View larger version (33K): [in this window] [in a new window]   Fig. 9.   Lack of inverse agonist effect on -opioid and 2-adrenergic receptors coexpressed with 16z44. COS-7 cells were transfected with cDNAs encoding G16 or 16z44 together with either the -opioid or the 2-adrenergic receptor (0.25 µg/ml each). Transfected cells were assayed for IP accumulation in the absence (basal) or presence of the indicated ligands: 100 nM DPDPE, 10 µM ICI-174,864, 10 µM naloxone, 1 µM isoproterenol, 10 µM ICI-118,551, 10 µM timolol, or 10 µM propranolol. *Agonist-induced IP formation was significantly higher than the corresponding basal values; Bonferroni's t test, P < .05.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

As one of the largest protein families found in nature, it is estimated that several thousand different GPCRs may exist in the human genome. The recent discovery of more than 1000 genes encoding known and orphan GPCRs in the Caenorhabditis elegans genome (Bargmann, 1998) lends further credibility to this estimation. Human G₁₆ possesses the rare ability to recognize a wide spectrum of GPCRs, which can facilitate the characterization of orphan GPCRs. Numerous biochemical, structural, and molecular genetic studies have revealed that the docking site for receptors is composed of multiple regions on the G-subunit (reviewed by Bourne, 1997). The five regions of the G-subunit involved in receptor recognition are the 2 helix, the 6-5 loop, the 5 helix, and the two extreme termini. By replacing one or more of these regions in G₁₆ with sequences from G_z, we have successfully created chimeric G_16/z proteins that exhibit enhanced linkage to G_i-coupled receptors. The 16z44 chimera appeared to be particularly effective in this respect.

Compared with G₁₆, both 16z25 and 16z44 can additionally couple to the melatonin Mel1c receptor and produce higher magnitudes of PLC stimulation with some of the G_i-coupled receptors (e.g., µ-opioid and SSTR1). In the 16z25 chimera, the 5 helix of G₁₆ was replaced by that of G_z. Numerous studies have attested to the importance of the 5 helix in receptor coupling. Amino acids within the 5 helix of G_s (Conklin et al., 1996; Sullivan et al., 1987), G_i (Tsu et al., 1997), G_t1 (Martin et al., 1996), and G_q (Conklin et al., 1993, 1996) have been shown to alter the specificity of receptor coupling. The 5 helix of G_z is quite different from that of G₁₆ with less than 35% homology (Fig. 1). Alignment of the 5 helices of G₁₆ and G_z shows that every third or fourth amino acid from position 1 are different between the two G-subunits. Using the crystal structures of G_t1 and G_i1 as a basis, we generated a molecular model to highlight the structural differences in the 5 helices of G₁₆ and G_z (Fig. 10A). When the G_z-specific residues are superimposed on the 5 helix of G₁₆, the receptor-facing plane are predominantly composed of G_z-specific amino acids.

View larger version (55K): [in this window] [in a new window]   Fig. 10.   Molecular models of G16/z chimeras. Coordinates of the hypothetical structural models of various G-subunits were generated by Swiss-Model modeling service available from World Wild Web (http://www.expasy.ch/swissmod/SWISS-MODEL.html) using Gt1 and Gi1 complexed with -subunits as template structures (coordinate codes 1GOT and 1GP2; obtained from the Protein Data Bank of Brookhaven National Laboratory, http://www.pdb.bnl.gov/). Models were visualized by Swiss-PDB Viewer v3.1 (http://www.expasy.ch/spdbv/mainpage.htm) and rendered by POV-Ray for Windows v3.1 (http://www.povray.org/). A, the 5 helix of G16 is shown with the putative receptor-facing side on top, as indicated. The last seven residues have not been mapped to the structure due to the lack of the corresponding residues in the template. Green patches correspond to the residues different from Gz in their nature. B, the structures from G to 5 of 16z25 (blue), 16z44 (yellow),and 16z66 (red) are displayed in an overlapping view. Receptor-facing side of 5 helix is inside the plane. The 4/6 loops of 16z25 and 16z44 are longer than that of the template structures and thus are not in well defined structures. C, the GTPase domain of G16 is in the same orientation as in B. The putative PLC-activating domain is marked in blue color according to the study of Medina et al. (1996), and the five residues important for PLC activation are shown in sticks (Venkatakrishnan and Exton, 1996).

Not only did 16z44 recognize more G_i-coupled receptors than G₁₆, the magnitudes of the stimulations were also higher. Comparison of the EC₅₀ values obtained with 16z44 and G₁₆ transfectants reflected that 16z44 was more efficient in linking the G_i-coupled receptors to the activation of PLC. Another notable feature of 16z44 is its elevated basal activities. Cells coexpressing 16z44 generally exhibited basal PLC activities that were much higher than those coexpressing G₁₆ (Table 1). This elevation in basal IP accumulation resembles the constitutive activity of GPCRs, but we were unable to detect inverse agonist effects on the -opioid and ₂-adrenergic receptors. As a result of elevated basal activities and in terms of percentage stimulation, 16z44-mediated responses were not significantly better than those mediated by G₁₆. However, the absolute levels of IP accumulation transduced by 16z44 were often greater than those of G₁₆. Basal IP accumulation was also higher in cells coexpressing the 16z25 chimera, albeit to a lesser extent.

Structural differences between 16z25 and 16z44 are primarily located in the 4/6 loop (residues 318-335 of G₁₆). The 4/6 loop is one of several secondary structures forming the contact surface for receptors (part of the A1 cluster as described by Lichtarge et al., 1996). The 4 helix and 4/6 loop region of G_i1 are important for specific recognition of receptors (Bae et al., 1997). The 4/6 loop of 16z25 is identical with that of G₁₆ because there is no substitution of G_z-specific residues in this region of 16z25 (Fig. 1). Based on the crystal structure coordinates of trimeric G_t1 (Lambright et al., 1996), the 4/6 loop of 16z44 is predicted to be a more flexible structure with an energy level higher than those of 16z25 and G₁₆ (Fig. 10B). This increased flexibility may partially account for the enhanced potency of 16z44 to transduce signals from G_i-coupled receptors. The model generated for 16z66 (Fig. 10B) fitted well to the known structures of G_t1 and G_i1. Compared with G₁₆, 16z66 has a smaller 4/6 loop and a tighter 4 helix. One would presume that the close resemblance of 16z66 to G_t1 and G_i1 implies enhanced capability of the chimera to interact with G_i-coupled receptors, yet our results were contrary to such a prediction.

Because 16z66 was expressed to the same extent as the other chimeras, its inability to mediate receptor-induced stimulation of PLC might be due to the disruption of effector recognition domains like the 4/6 region. The 4 and the 4/6 loops of G_q are known to be involved in the activation of PLC (Arkinstall et al., 1995; Medina et al., 1996). When the putative PLC regulatory domains of G_q are mapped onto a molecular model of G₁₆ (shown in blue, Fig. 10C), these regions were not substituted by G_z-specific residues in 16z66. Several residues around the switch III region of G_q have also been shown to be critical for the regulation of PLC (Venkatakrishnan and Exton, 1996), and they are conserved in G₁₆: residues 241 to 243 and 254 to 255 (Fig. 10C). We used molecular modeling of G₁₆ to identify amino acids distal to or in the 4 helix that may interact with these PLC-activating residues. Residue Leu²⁵⁴ is predicted to interact with several amino acids in the 4 helix (Ile³¹², Met³¹⁵, Tyr³¹⁶, and Thr³¹⁷) and the 4/6 loop (Asp³²⁵). Four of these five potential sites (except Ile³¹²) were actually replaced by G_z-specific residues in 16z66 (Fig. 1). Like mutating Leu²⁵⁴ itself, the disruption of intramolecular interactions may severely curtail the ability of 16z66 to stimulate PLC, resulting in a lack of constitutive activity of 16z66QL. Another possibility is that 16z66 cannot adopt the GTP-bound active form. Further experiments are needed to distinguish the molecular basis for the lack of function of 16z66.

Although we did not embark on an extensive study to determine the importance of N-terminal sequences of G_z in receptor recognition, the present study shows that the N helix of G_z alone could not confer specificity for G_i-coupled receptors. This is in stark contrast to results obtained in similar studies where the N terminus of G_z alone was sufficient to allow a G_z/t1 chimera to respond to the -opioid receptor (Tsu et al., 1997). Close proximity of the two termini in the crystal structures of G_t1 (Lambright et al., 1996) and G_i1 (Wall et al., 1995) supports the involvement of the N terminus in receptor recognition. Biochemical evidence are also available to substantiate this notion for the coupling of receptors to G_o (Denker et al., 1995) and G_t1 (Dratz et al., 1993), two members of the G_i subfamily. Hence, an intact N terminus may be required for G₁₆ to efficiently associate with GPCRs. In this respect, our results are in accordance with those reported by Lee et al. (1995), where the N-terminal 209 residues of G₁₆ were found to be essential for activation by the G_i-coupled C5a receptor.

Despite their enhanced linkage to G_i-coupled receptors, neither 16z25 nor 16z44 can be considered as a universal adapter for GPCRs. Compared with G₁₆, the ability of 16z25 and 16z44 to recognize the luteinizing hormone receptor was actually diminished. Naturally, one would expect that when the specificity for G_i-coupled receptors increases in a G-subunit, its specificity for G_s-linked receptors will be inversely affected because the two sets of GPCRs are designed to produce opposite effects on adenylyl cyclase. Perhaps it is impractical to engineer a universal G protein adapter for GPCRs even though such a construct is highly desirable for the characterization of orphan receptors. Nevertheless, our study has successfully demonstrated the feasibility of improving the recognition of specific subsets of GPCRs by altering receptor contact regions on G₁₆. With the recent explosion of the number of orphan receptors being cloned, the chimeras described herein may become invaluable tools for their characterization. For example, the 16z44 chimera can be incorporated into a variety of cell-based assays for the rapid detection of receptor activation. Conceivably, the ability of G₁₆ to recognize G_s-coupled receptors can be similarly enhanced by incorporating G_s-specific regions on a G₁₆ backbone. Several chimeric G₁₆ with expanded capability of receptor recognition may collectively serve as a true "universal adapter" for orphan GPCRs.