Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Exchange of GTP for GDP on the  subunit of a heterotrimeric G protein represents the initial activation point for signal transduction mediated via G protein-coupled receptors (GPCRs). It is thus the earliest and most appropriate point to measure the effectiveness of GPCR-G protein interactions (Gierschik et al., 1994; Wieland and Jakobs, 1994). Despite the widespread use of ligand-regulated [³⁵S]GTPS binding assays to monitor these events, it has historically been extremely difficult to observe significant elevations of guanine nucleotide exchange produced by GPCRs that couple to G proteins other than members of the pertussis toxin-sensitive G_i family. At least in part, this reflects a combination of the low-basal guanine nucleotide exchange properties of G_q and the G_s family G proteins and the widespread expression profile of members of the G_i family.

Agonist-independent activation of signaling cascades by GPCRs has attracted great interest in the recent past (Scheer and Cotecchia, 1997; Leurs et al., 1998; Pauwels and Wurch, 1998; de Ligt et al., 2000). It is well established that mutations at a considerable range of positions in GPCRs can uncover or enhance such constitutive activity. The _1b-adrenoceptor has been particularly well studied in this regard (Allen et al., 1991; Kjelsberg et al., 1992; Perez et al., 1996; Scheer et al., 1996, 1997, 2000; Hwa et al., 1997; Lee et al., 1997; Mhaouty-Kodja et al., 1999; Rossier et al., 1999; McWhinney et al., 2000; Stevens et al., 2000). This reflects both that it was the first GPCR in which mutation was observed to generate such a constitutively active phenotype (Allen et al., 1991; Kjelsberg et al., 1992) and because mutations at a range of distinct locations produce such effects. However, although it is well appreciated that different mutations may produce varying levels of constitutive activity (Kjelsberg et al., 1992; Scheer et al., 1997; Stevens et al., 2000), this has been difficult to quantitate effectively. This reflects that this GPCR is coupled predominantly to the elevation of intracellular Ca²⁺ via G_q family G proteins. Measurements of functionality have had to be made, until now, either at the level of elevation of inositol phosphate production or via reporter gene assays. Because both of these are downstream endpoints of the initial G protein activation, they are subject to amplification and regulation that can influence details of pharmacology. Furthermore, mutational alterations can greatly alter the levels of expression of the _1b-adrenoceptor (Lee et al., 1997; Stevens et al., 2000), and changes in both the absolute levels of expression of a receptor and the stoichiometry of receptor to G protein are expected to modulate measured levels of constitutive activity.

By using fusion proteins (Seifert et al., 1999; Milligan, 2000) between forms of the _1b-adrenoceptor and the  subunit of the G protein G₁₁, we now combine the defined GPCR-G protein stoichiometry of such constructs with their effective immunoprecipitation to directly monitor constitutive activity and ligand regulation of [³⁵S]GTPS binding to G₁₁ produced by the wild-type and various constitutively active mutant (CAM) forms of the _1b-adrenoceptor.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials. All materials for tissue culture were supplied by Invitrogen (Paisley, Strathclyde, UK). [³H]Prazosin (80 Ci/mmol) and [³⁵S]GTPS (1250 Ci/mmol) were from PerkinElmer Life Sciences (Boston, MA). Oligonucleotides were purchased from Cruachem (Glasgow, Strathclyde, UK). Receptor ligands were purchased from Sigma/RBI (Gillingham, Kent, UK). Production and characterization of the anti-G_q/G₁₁ antiserum CQ was described by Mitchell et al. (1993). All other chemicals were from Sigma-Aldrich (Poole, Dorset, UK) and were of the highest grade available.

Construction of Fusion Proteins. Production and subcloning of wild-type and mutated _1b-adrenoceptor-G₁₁ fusion proteins was performed in two separate stages. In the first step, the coding sequence of G₁₁ was modified by PCR amplification using the amino terminal primer 5'GAGGACGGTACCACTCTGGAGTCCATG-3'; the initiating Met of G₁₁ was removed and a KpnI restriction site (underlined) and, as a consequence, a two amino acid spacer (Gly-Asn) were introduced. Using the C-terminal primer 5'TTGTGCGGCCGCCGGTCACACCAGGTT-3, a NotI restriction site (underlined) was introduced downstream of the stop codon of G₁₁. The amplified fragments digested with KpnI and NotI were subcloned into similarly digested pcDNA3 expression vector (Invitrogen). To obtain the various _1b-adrenoceptor-G₁₁ fusion proteins, the coding sequence of the wild-type or 3CAM (A²⁹³L, K²⁹⁰H, R²⁸⁸K), D¹⁴²A, and A²⁹³E forms of the hamster _1b-adrenoceptor (all obtained from Susanna Cotecchia, Lausanne, Switzerland) were amplified by PCR. Using the amino-terminal primer 5'-GACGGTACCTCTAAAATGAATCCCGAT-3', a KpnI restriction site (underlined) was introduced upstream of the initiator Met. Using the carboxyl-terminal primer 5'-GTCCCTGGTACCAAAGTGCCCGGGTG-3', a second KpnI restriction site (underlined) was introduced immediately upstream of the stop codon. Finally, the G₁₁ constructs in pcDNA3 were digested with KpnI and ligated together with the PCR product of the _1b-adrenoceptor amplification, also digested with KpnI. The open reading frames thus produced represent the coding sequence of either _1b-adrenoceptor-G₁₁, 3CAM _1b-adrenoceptor-G₁₁, D¹⁴²A _1b-adrenoceptor-G₁₁, or A²⁹³E _1b-adrenoceptor-G₁₁. Each was fully sequenced before its expression and analysis.

Transient Transfection of HEK293 Cells. HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 0.292 g/l L-glutamine and 10% (v/v) newborn calf serum at 37°C in a 5% CO₂ humidified atmosphere. Cells were grown to 60 to 80% confluency before transient transfection in 60-mm dishes. Transfection was performed using LipofectAMINE reagent (Invitrogen) according to the manufacturer's instructions.

[³⁵S]GTPS Binding. [³⁵S]GTPS binding experiments were initiated by the addition of membranes containing 50 fmol of the fusion constructs to an assay buffer (20 mM HEPES, pH 7.4, 3 mM MgCl₂, 100 mM NaCl, 1 µM guanosine 5'-diphosphate, 0.2 mM ascorbic acid, 50 nCi [³⁵S]GTPS) containing the indicated concentrations of receptor ligands. Nonspecific binding was determined in the same conditions but in the presence of 100 µM GTPS. Reactions were incubated for 15 min at 30°C and were terminated by the addition of 0.5 ml of ice cold buffer, containing 20 mM HEPES (pH 7.4), 3 mM MgCl₂, and 100 mM NaCl. The samples were centrifuged at 16,000g for 15 min at 4°C, and the resulting pellets were resuspended in solubilization buffer (100 mM Tris, 200 mM NaCl, 1 mM EDTA, 1.25% Nonidet P-40) plus 0.2% sodium dodecylsulfate. Samples were precleared with Pansorbin (Calbiochem, San Diego, CA), followed by immunoprecipitation with CQ antiserum (Mitchell et al., 1993). Finally, the immunocomplexes were washed twice with solubilization buffer, and bound [³⁵S]GTPS was measured by liquid-scintillation spectrometry.

[³H]Prazosin Binding Studies. Binding assays were initiated by the addition of 3 µg of cell membranes to an assay buffer (50 mM Tris-HCl, 100 mM NaCl, 3 mM MgCl₂, pH 7.4) containing [³H]prazosin (0.05-5 nM in saturation assays and 0.8 nM for competition assays) in the absence or presence of increasing concentrations of phenylephrine. Nonspecific binding was determined in the presence of 100 µM phentolamine. Reactions were incubated for 30 min at 30°C, and bound ligand was separated from free ligand by vacuum filtration through GF/B filters (Semat, St. Albans, Hertsfordshire, UK). The filters were washed twice with assay buffer, and bound ligand was estimated by liquid scintillation spectrometry.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

A fusion protein (_1b/G₁₁) was generated in which the  subunit of the G protein G₁₁ was linked in-frame with the C-terminal tail of the _1b-adrenoceptor from which the stop codon had been removed. The functionality of this protein has previously been established by monitoring the capacity of the agonist phenylephrine to elevate intracellular Ca²⁺ levels in a fibroblast cell line in which the  subunits of both G_q and G₁₁ had been eliminated by targeted gene disruption (Stevens et al., 2001). Immunoblotting membrane fractions of HEK293 cells positively transfected with this construct with an antiserum (CQ; Mitchell et al., 1993), which is directed against the C-terminal decapeptide common to G_q and G₁₁, identified the endogenously expressed forms of these G proteins. It also detected much higher levels of a doublet corresponding to the fusion protein (Fig. 1). Although the predicted molecular mass of the _1b-adrenoceptor and G₁₁ is 56 and 43 kDa, respectively, the predominant form of the fusion protein migrated through SDS-PAGE with apparent molecular mass of 130 kDa. The doublet represents differentially glycosylated forms of the receptor-G protein fusion protein because pretreatment with N-glycosidase F compressed these to a single band, with an apparent molecular mass close to 100kDa (data not shown), as shown previously for a fusion protein between the human -opioid receptor and G_o1 (Moon et al., 2001).

View larger version (45K): [in this window] [in a new window]   Fig. 1.   Expression and immunological detection of an 1b-adrenoceptor-G11 fusion protein. HEK293 cells were mock transfected (1) or transfected to express an 1b/G11 fusion protein (2). Membranes from these cells were resolved by SDS-PAGE and immunoblotted with an antiserum which identifies the C-terminal 10 amino acids common between the  subunits of Gq and G11.

When membranes transiently expressing _1b/G₁₁were subjected to a standard [³⁵S]GTPS binding assay, basal levels of [³⁵S]GTPS binding were high. Although this was stimulated significantly by the addition of a high concentration (10 µM) of the ₁-adrenoceptor agonist phenylephrine (Fig. 2A), the relatively poor signal compared with basal is not suitable for detailed studies. Furthermore, the high basal levels of [³⁵S]GTPS binding were not a reflection of constitutive activity of the fusion protein because these were not different from those observed in membranes of mock transfected cells (Fig. 2A). However, when such samples were subsequently immunoprecipitated with antiserum CQ, the basal levels of [³⁵S]GTPS binding were very low in both the positively and mock-transfected cells. However, samples of the positively transfected cells that had been treated with phenylephrine now contained levels of [³⁵S]GTPS 30-fold higher than those that had not been exposed to the agonist (Fig. 2B).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Phenylephrine stimulates binding of [35S]GTPS to Gq/G11. Membranes of mock-transfected HEK293 cells or those transfected to express the 1b/G11 fusion protein were used in [35S]GTPS binding assays in the absence (basal) or presence (Phe) of 10µM phenylephrine (A). Following such experiments, samples were immunoprecipitated with antiserum CQ before scintillation counting (B).

Because HEK293 cells express both G_q and G₁₁, these results did not demonstrate conclusively that the agonist-induced binding of [³⁵S]GTPS was to the G protein element of the fusion protein. We thus constructed and expressed a version of the fusion protein in which the GPCR was linked to a form of the G protein (G²⁰⁸A G₁₁) anticipated to be unable to release GDP and thus to bind [³⁵S]GTPS (Sprang, 1997). This construct bound the ₁-adrenoceptor antagonist [³H]prazosin with the same high affinity as the fusion protein containing the wild-type G protein sequence (Table 1) and was expressed almost as effectively (Table 1). After a membrane [³⁵S]GTPS binding assay and immunoprecipitation with antiserum CQ, basal levels of [³⁵S]GTPS binding were reduced compared with membranes expressing the fusion containing the wild-type G protein (Fig. 3), and the ability of phenylephrine to stimulate [³⁵S]GTPS binding was almost completely eliminated (Fig. 3). This was not a reflection that the _1b-G²⁰⁸A G₁₁ fusion protein bound phenylephrine much more poorly than the _1b/G₁₁ fusion protein. The capacity of phenylephrine to compete with [³H]prazosin for binding to the two fusion proteins was indistinguishable (Table 1). To monitor whether the receptor of the fusion protein was able to activate G proteins other than that linked to it, the _1b-G²⁰⁸A G₁₁ fusion protein was coexpressed with excess wild-type G₁₁. Now phenylephrine did indeed elevate binding of [³⁵S]GTPS in the immunoprecipitates from the _1b-G²⁰⁸A G₁₁ fusion protein expressing membranes (Fig. 3). This was to a substantially lower level than for the wild-type fusion protein and was dependent upon the presence of the _1b-adrenoceptor because immunoprecipitates from cells transfected to express G₁₁ in the absence of the _1b-G²⁰⁸A G₁₁ fusion protein contained no significant levels of [³⁵S]GTPS with or without treatment with phenylephrine (Fig. 3).

View this table: [in this window] [in a new window]   TABLE 1 Characteristics of 1b-adrenoceptor-G11 fusion proteins The binding affinities of a range of fusion constructs for phenylephrine (Phe) and [3H]prazosin were assessed as was the potency of phenylephrine to increase binding of [35S]GTPS above basal levels. Numbers in parentheses indicate independent experiments performed.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Phenylephrine stimulates binding of [35S]GTPS to the 1b-adrenoceptor- G11 fusion protein. HEK293 cells were transfected to express combinations of the 1b-adrenoceptor-G11 fusion protein (1b/G11), an 1b-adrenoceptor-G11 fusion protein containing a G208A mutation in the G protein segment (1b/G11G208A), and the  subunit of G11 (G11). Membranes from these cells were used in [35S]GTPS binding assays in the absence (basal) or presence (Phe) of 10µM phenylephrine.

The [³⁵S]GTPS binding capacity of the _1b/G₁₁ fusion protein was assessed in studies in which phenylephrine-stimulated [³⁵S]GTPS binding was measured in the presence of increasing concentrations of unlabeled GTPS. When such data were corrected for dilution of specific activity and presented as a pseudo-saturation binding profile, the immunoprecipitate was able to bind 0.46 ± 0.01 mol of GTPS/mol of fusion protein expressed in the membrane fraction (Fig. 4A). However, as parallel assessment of the immunoprecipitation efficiency of antiserum CQ indicated that only 45% of the expressed fusion protein was recovered with the amount of antiserum used (Fig. 4B), this corresponds to a true [³⁵S]GTPS binding capacity of the construct of 1.02 mol/mol. Such data confirmed that the full population of expressed construct was able to exchange and bind guanine nucleotides and was thus properly folded.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   The 1b-adrenoceptor-G11 fusion protein can bind [35S]GTPS effectively. The ability of phenylephrine (10 µM) to stimulate binding of [35S]GTPS to the 1b/G11 fusion protein was measured in the presence of varying concentrations of GTPS. Such data are presented as a Scatchard plot (A). Membranes of HEK293 cells expressing the 1b/G11 fusion protein were either immunoprecipitated (+) or not () with antiserum CQ, resolved by SDS-PAGE, and immunoblotted to detect the fusion protein (B). Densitometric scans of such blots were used to assess the efficiency of immunoprecipitation.

Because there was some evidence for a degree of constitutive activity of the _1b/G₁₁ fusion protein to bind [³⁵S]GTPS (Figs. 2B and 3), we assessed whether this would be blunted by addition of the ₁-adrenoceptor antagonist/inverse agonist phentolamine (Fig. 5). Although this was observed, the low levels of basal [³⁵S]GTPS binding made analysis difficult. The initially described constitutively active mutant of the _1b-adrenoceptor resulted from the replacement of a short segment of the third intracellular loop of this GPCR, with the equivalent section of the ₂-adrenoceptor (Allen et al., 1991). We thus constructed a fusion protein between this form of the receptor, that we designate 3CAM (Stevens et al., 2000), and G₁₁. This protein also bound [³H]prazosin with high affinity (Fig. 5B; Table 1) but was expressed at significantly lower levels than the _1b/G₁₁ fusion protein (Fig. 5B; Table 1). A similar feature has previously been observed for both the isolated 3CAM _1b-adrenoceptor (Lee et al., 1997) and a C-terminal GFP-tagged form of the 3CAM _1b-adrenoceptor (Stevens et al., 2000) compared with equivalent forms of the wild-type receptor. Now, however, addition of an equal amount of the 3CAM _1b-adrenoceptor-G₁₁ fusion protein to a [³⁵S]GTPS binding assay, followed by its immunoprecipitation, resulted in substantially higher levels of bound [³⁵S]GTPS than produced by the _1b/G₁₁ fusion protein (Fig. 5C), demonstrating this form of the receptor to possess greater ability to stimulate its associated G protein in the absence of ligand than the wild-type form of the receptor. The presence of phentolamine (10 µM) during the [³⁵S]GTPS binding assay reduced incorporation of [³⁵S]GTPS into the 3CAM _1b/G₁₁ fusion protein substantially (Fig. 5C), confirming that phentolamine acted as an inverse agonist for the 3CAM _1b-adrenoceptor. Phentolamine, however, was unable to reduce binding of [³⁵S]GTPS to the 3CAM _1b/G₁₁ fusion protein to the very low levels of labeling of the wild-type _1b/G₁₁ fusion protein (Fig. 5C). Such results indicate that phentolamine is a partial inverse agonist at the 3CAM _1b-adrenoceptor. Phentolamine bound the 3CAM _1b/G₁₁ fusion protein with high affinity (K_i = 2.8 ± 0.1 × 10⁸ M) (Fig. 5D), and concentration-response curves to phentolamine showed the inverse agonist effects to have an EC₅₀ of 6.1 ± 0.7 × 10⁸ M (Fig. 5E). Other ligands with affinity at the _1b-adrenoceptor, including WB4101, corynanthine, HV723, and 5-methylurapidil also functioned as inverse agonists at the 3CAM _1b/G₁₁ fusion protein (Fig. 6). WB4101 and phentolamine were similarly effective with the others displaying lower extents of inverse activity.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   A 3CAM mutant of the 1b-adrenoceptor activates G11 in the absence of agonist. HEK293 cell membranes expressing either the 1b/G11 (A) or the 3CAM 1b/G11 (B) fusion proteins were used to measure the specific binding of a range of concentrations of [3H]prazosin. Membranes containing 50 fmol of [3H]prazosin binding sites of each construct were used in [35S]GTPS binding assays followed by immunoprecipitation in the absence (basal) or presence (Phent) of 10µM phentolamine (C). The ability of varying concentrations of phentolamine to compete for the specific binding of [3H]prazosin (D) or to inhibit basal binding of [35S]GTPS (E) to the 3CAM 1b/G11 fusion protein were also assessed. Data of A-C are from representative experiments and for D and E are the means + S.E.M. (n = 3).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   A range of 1b-adrenoceptor blockers are inverse agonists at the 3CAM 1b-adrenoceptor-G11 fusion protein. The ability of phentolamine, WB4101, corynanthine, HV-723, and 5-methylurapidil (all at 10µM) to inhibit the basal binding of [35S]GTPS to the 3CAM 1b/G11 fusion protein was assessed. Data are the means + S.E.M. (n = 4). Significantly different from basal: **, P < 0.01; ***, P < 0.001.

Single point mutations close to the interface of transmembrane helix VI and the third intracellular loop of the _1b-adrenoceptor (Kjelsberg et al., 1992) and also at remote locations, such as the interface of transmembrane helix III and the second intracellular loop, are known to induce constitutive activity (Scheer et al., 1997). The best studied have been D¹⁴²A and A²⁹³E. These mutations were introduced into the _1b/G₁₁ fusion protein to allow direct comparisons of the level of constitutive activity and the effectiveness of phentolamine as an inverse agonist at the different mutants. Following transient expression of the wild-type _1b/G₁₁ and each of 3CAM _1b/G₁₁, D¹⁴²A _1b/G₁₁, and A²⁹³E _1b/G₁₁, fusion protein saturation [³H]prazosin binding studies defined expression levels and allowed the same amounts of each fusion protein to be added to the assays. Each of the mutated fusion proteins bound substantially higher levels of [³⁵S]GTPS in the absence of agonist than did the wild-type (Fig. 7), with the 3CAM mutant binding significantly higher levels than the other mutants. Furthermore, phentolamine acted as a partial inverse agonist at each of the mutated fusion proteins (Fig. 7).

View larger version (23K): [in this window] [in a new window]   Fig. 7.   D142A and A293E mutants of the 1b-adrenoceptor activate G11 in the absence of agonist. HEK293 cells were transfected to express the 1b-adrenoceptor-G11 fusion protein (1b/G11) or fusion proteins incorporating the 3CAM (CAM1b/G11), the A293E (A293E 1b/G11), or the D142A (D142A 1b/G11) mutants of the 1b-adrenoceptor. After saturation [3H]prasozin binding experiments, membrane amounts corresponding to 50 fmol of each construct were used in [35S]GTPS binding assays in the absence (basal) or presence (Phent) of 10 µM phentolamine. Significantly different from basal: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Many constitutively active mutant GPCRs remain responsive to agonist ligands, indicating that the mutations do not result in the adoption of a conformation equivalent to that produced by binding of the agonist. This was true for the 3CAM _1b/G₁₁ fusion protein. Phenylephrine further stimulated the binding of [³⁵S]GTPS to this construct in a concentration-dependent manner (Fig. 8A), and as anticipated from previous studies on the isolated 3CAM _1b-adrenoceptor, the agonist displayed substantially greater potency at 3CAM _1b/G₁₁ than at the _1b/G₁₁ fusion protein (Fig. 8A). As with the 3CAM mutant, phenylephrine was also much more potent in stimulating the binding of [³⁵S]GTPS to the fusion proteins containing either the A²⁹³E or D¹⁴²A single point mutants (Fig. 8A). Although each of the fusion proteins containing the constitutively active receptor mutants bound [³⁵S]GTPS in a ligand-independent manner, this was not to more than 20% of the level that was produced by addition of phenylephrine (Fig. 8B). Such results indicate that these mutants do not represent a very good model of the agonist-activated conformation of the receptor.

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Phenylephrine has a 50-fold greater potency to activate G11 via constitutively active mutants of the 1b-adrenoceptor. Membranes expressing fusion proteins containing G11 linked to the wild-type (circles) and 3CAM (diamonds) A293E (squares) or D142A (triangles) forms of the 1b-adrenoceptor were used to measure the potency of phenylephrine to increase the binding of [35S]GTPS above basal levels (A) (n = 3) or to compete for the specific binding of [3H]prazosin (C) (n = 5). The level of constitutive activity of each construct was assessed by comparison of the level [35S]GTPS binding in the absence of ligand compared with that achieved in the presence of 10 µM phenyleprine (defined as 100%) (B) (n = 4). Significantly different from wild-type fusion protein: *, P < 0.05; **, P < 0.01.

This difference in potency of phenylephrine at the fusions containing the mutant receptors reflected the differences in affinity of the agonist for these forms of the receptor (Fig. 8C). Although [³H]prazosin binding experiments on membranes expressing the various _1b/G₁₁ fusion proteins indicated that antagonist binding affinity was not different from the wild-type (Table 1), phenyleprine displayed substantially higher affinity at the mutant constructs than at the wild-type (Fig. 8C).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Regulation of the binding of [³⁵S]GTPS is by far the most widely used assay to monitor ligand and receptor activation of heterotrimeric G proteins (Wieland and Jakobs, 1994). Despite this, there are many technical limitations that restrict further use of this approach. The most important is that while it can be extremely effective for the study of receptors that couple to the pertussis toxin-sensitive G_i subfamily G proteins, it has not historically provided good signal to noise for receptors that couple to members of the G_s or G_q family G proteins. At least in part, this reflects the relatively high rates of basal guanine nucleotide exchange of the G_i-G proteins compared with other family members. An inability to use this assay effectively is a particular limitation for receptors that couple to the G_q family G proteins. This reflects that assays for second messengers generated via this cascade (Ca²⁺, inositol phosphates, and diacylglycerol) cannot be easily performed on membrane preparations and thus for cells and tissues that have previously been harvested and stored.

Attempts to modify membrane [³⁵S]GTPS binding assays to produce reasonable signals for G_s or G_q-coupled receptors have adopted three strategies. The first of these has been to alter the temperature of the incubation or the concentration of guanine nucleotides and/or other assay reagents (Wieland and Jakobs, 1994). The second has been to express the receptor and G protein in systems, including insect Sf9 cells, that allow high levels of production of foreign proteins but that have low endogenous expression levels of G_i-like G proteins (Barr et al., 1997; Windh et al., 1999). The third, and most promising, incorporates a selective immunoprecipitation step at the end of the assay to eliminate the binding of [³⁵S]GTPS from other G proteins arising from basal exchange processes (DeLapp et al., 1999; Akam et al., 2001; Selkirk et al., 2001; Willets et al., 2001).

Although the immunoprecipitation approach has been used successfully, it retains a number of problems. The greatest of these is that little information is usually provided on the immunoprecipitation efficiency achieved, and thus it is difficult to compare results for receptor activation of different G proteins (DeLapp et al., 1999; Akam et al., 2001). Furthermore, because mutated receptors are regularly expressed at different levels than the wild-type, this poses difficulties in attempts to compare their G protein activation capabilities. In recent years, many groups have taken advantage of receptor-G protein fusions to overcome these limitations because the receptor and G protein are always present with the same 1:1 stoichiometry (Milligan, 2002a,b). By linking the same receptor to two different G proteins, it is then possible to make direct measures of the relative ability of ligands to activate each G protein. Indeed, using this approach, occupancy of the human -opioid receptor by the agonist [D-Ala²,D-Leu⁵]-enkephalin has been shown to result in 3 times greater activation of G_i1 than G_o1 (Moon et al., 2001). By contrast, the maximal capacity of the µ-opioid receptor to activate G_i1 is the same as for the -opioid receptor (Moon et al., 2001). This approach is equally useful in examining the effects of receptor mutations. Although an Asp⁷⁹Asn mutation in transmembrane helix II of the _2A-adrenoceptor reduces the maximal ability of this receptor to activate G_i1 by some 95%, this effect is overcome by addition of a reciprocal Asn⁴²²Asp mutation in transmembrane helix VII (Ward and Milligan, 2002).

Herein, we have combined the receptor-G protein fusion approach with an end of assay immunoprecipitation strategy to provide a highly reproducible means to monitor receptor-mediated regulation of the binding of [³⁵S]GTPS to G₁₁. When we expressed an _1b/G₁₁ fusion protein in HEK293 cells, prepared membranes, and then performed a [³⁵S]GTPS binding assay that was terminated by immunoprecipitation with an antiserum to the C-terminal decapeptide common between G₁₁ and G_q, very few counts were present in the immunoprecipitate. However, when the assay was performed in the presence of phenylephrine a 30- to 40-fold increase in [³⁵S]GTPS binding was achieved. However, as most cells endogenously express combinations of G₁₁and G_q, it was initially unclear if this agonist-induced binding of [³⁵S]GTPS was to the G₁₁ of the fusion protein, endogenous G₁₁/G_q, or some combination thereof. However, when we expressed a fusion protein containing a G²⁰⁸A mutant of G₁₁ that prevents GDP release, and thus [³⁵S]GTPS binding, phenylephrine had virtually no effect on the amount of [³⁵S]GTPS in the immunoprecipitate. It is not that the _1b/G₁₁ is unable to interact with endogenous G protein, however, as was shown in studies in which excess G₁₁ was co-transfected along with the fusion protein (Fig. 3). These observations are consistent with previous work on both an _2A-adrenoceptor-G_i1 fusion protein (Burt et al., 1998) and following fusion of the neurokinin-1 receptor to both G_s and G_q (Holst et al., 2001). Thus, achieving a high ratio of fusion protein to endogenously expressed G protein is required to ensure that virtually all of the bound nucleotide is to the G protein linked to the receptor. This can be ensured by using an anti-receptor antibody rather than the anti-G protein antiserum for the immunoprecipitation. We have recently used this approach for fusion proteins between the _1b-adrenoceptor and forms of G₁₁ mutated at the C-terminus such that they are no longer recognized by antiserum CQ (Liu et al., 2002). The only significant limitation has been that the immunoprecipitation efficiency of the anti-receptor antibody was lower than for the anti-G protein antiserum.

A key requirement for these studies was the ability to add equal amounts of different fusion proteins to the [³⁵S]GTPS binding assays. Prior specific [³H]prazosin binding assays on the membrane preparations allowed this, and the 1:1 stoichiometry of the receptor to G protein thus ensured that the same amount of G protein was present in each assay also. The observation that there was less [³⁵S]GTPS binding to the _1b/G₁₁ fusion protein containing the G²⁰⁸A mutation in the absence of added ligand than to the _1b/G₁₁ fusion protein suggested that the wild-type _1b-adrenoceptor displays a degree of constitutive capacity to activate G₁₁. We thus tested if phentolamine would function as an inverse agonist and reduce basal levels of [³⁵S]GTPS binding to _1b/G₁₁. As it did so, we constructed a series of further _1b/G₁₁ fusion proteins that incorporated mutations in the receptor previously recognized to enhance constitutive activity. We reasoned that the [³⁵S]GTPS binding assay would thus provide a direct monitor of the degree of constitutive activity of these mutants that would neither be limited by potential saturation of signal due to amplification nor be affected by issues of receptor reserve.

The first studied constitutively active _1b-adrenoceptor was produced by substitution of a short segment of the third intracellular loop of this receptor with the equivalent section from the ₂-adrenoceptor (Allen et al., 1991; Lee et al., 1997). Expression of a G₁₁ fusion protein containing this form of the receptor followed by membrane preparation, [³H]prazosin binding, and addition of an equal amount to the [³⁵S]GTPS binding assay indeed demonstrated this construct to bind substantially higher levels of [³⁵S]GTPS in the absence of ligand than the fusion containing the wild-type receptor. Furthermore, addition of phentolamine produced a marked reduction in basal [³⁵S]GTPS binding to the 3CAM form of the fusion protein that corresponded to many more counts than were available for potential inhibition resulting from the weak constitutive activity of the fusion containing the wild-type receptor. Thus, fusion proteins containing constitutively active mutant GPCRs are more suited to the detection and analysis of ligands possessing inverse agonism.

A concern expressed about such fusion proteins is that they may not replicate the basic characteristics of coexpressed, but separate, receptors and G proteins. To address this in relation to the expected characteristics of the 3CAM _1b-adrenoceptor, we monitored both agonist and antagonist binding affinity and concentration-response curves for [³⁵S]GTPS binding. Both the affinity and potency of phenylephrine were some 50-fold higher for the fusion incorporating the 3CAM receptor. However, when assessed by the level of basal [³⁵S]GTPS binding, the degree of constitutive activity of both the 3CAM _1b-adrenoceptor and the other mutants was less than might have been anticipated from previous studies that measured inositol phosphate generation. For example, the capacity of agonist to further stimulate inositol phosphate production in COS-7 cells expressing the A²⁹³E _1b-adrenoceptor has often been noted to be rather small compared with the effect of the unliganded receptor (Scheer et al., 1996; Rossier et al., 1999). The implication that the mutated receptor provides a relatively good model of the agonist-occupied receptor has been important to the generation of computer models of the activated state of the receptor (Scheer et al., 1996). However, as noted earlier, second messenger and other downstream measures of constitutive activity are subject to amplification and possibly other elements of integrative regulation. This study thus represents the first direct analysis of the level of G protein activation produced by constitutively active mutants of a G₁₁/G_q-coupled receptor. This is a much more direct assessment of the conformational alterations related to G protein activation that are imbued by the mutations relative to those induced by agonist-occupancy of the receptor. However, these studies indicate that in the absence of agonist, the various mutants of the _1b-adrenoceptor used are actually rather poor at causing guanine nucleotide exchange on G₁₁ compared with the agonist and are at best able to produce some 20% of the effect (Fig. 8B). This might have been contentious if the G protein element of the fusion protein was not able to bind [³⁵S]GTPS quantitatively in response to agonist. However, by combining saturation [³⁵S]GTPS binding studies with measures of immunoprecipitation efficiency, we were able to demonstrate the capacity of these fusion proteins to bind the theoretical maximum of 1 mol of [³⁵S]GTPS/mol of G protein  subunit.

A number of blockers at the _1b-adrenoceptor were shown to be able to reduce the basal activation of [³⁵S]GTPS binding to G₁₁, indicating them to be inverse agonists (Fig. 6). This characteristic has previously been noted for the isolated 3CAM receptor (Lee et al, 1997) and further validates the premise that such receptor-G protein fusions behave as for the isolated polypeptides. However, in all cases these ligands acted as partial inverse agonists because they were not able to reduce the signal to the basal signal produced by the wild-type receptor. Furthermore, the extent of inverse agonism produced by phentolamine was not substantially different between different CAM mutants of the _1b-adrenoceptor.

By developing a robust [³⁵S]GTPS binding assay appropriate for the G_q family G proteins and using a strategy in which modified forms of the receptor have access to the same number of G proteins, we have produced a range of novel observations on the level of constitutive activity of _1b-adrenoceptor mutants and the effectiveness of ligands as inverse agonists. This approach should be applicable to other receptors that couple selectively to G_q family G proteins and may provide a useful screen for inverse agonists at this class of receptors.