INTRODUCTION

G protein-coupled receptors (GPCRs),¹ when activated by extracellular ligands, interact with specific classes of heterotrimeric G proteins (consisting of , , and  subunits) which can then, in their activated forms, inhibit or activate various effector enzymes and/or ion channels (1-5). Characteristically, a specific GPCR can interact with only a limited subset of the many structurally similar G proteins that are expressed within a cell. Molecular genetic and biochemical studies have identified distinct intracellular regions (as well as single amino acids contained within these domains) on the GPCR proteins that play key roles in determining the fidelity of receptor-G protein coupling (1-7). In addition, recent studies have shown that residues at the extreme C terminus of the G protein  subunits are also of fundamental importance for the selectivity of receptor-G protein interactions (8-10). However, several lines of evidence suggest that the C terminus of the G subunits is clearly not the only structural determinant on the G proteins that is critical for dictating receptor-G protein coupling selectivity (2, 5).

Interestingly, two G protein  subunits, G_q and G₁₁, contain a unique six-amino acid extension that is not found in other G subunits (Fig. 1). This short sequence is highly conserved among all vertebrate species from which these subunits have been cloned so far (11-15), suggesting that it may be relevant for some aspect of G_q/G₁₁ function.

Comparison of the N-terminal amino acid sequences of selected G protein  subunits.

Previous studies (16, 17) analyzing the biochemical properties of a mutant G_q subunit lacking the N-terminal extension (hereafter referred to as 6q) failed to reveal any major functional differences between wild type G_q (WTq) and 6q. Both studies showed that G_q/11-coupled receptors such as the NK2 neurokinin (16) and the m1 muscarinic receptor (17) were able to activate 6q in a fashion identical to WTq. Similarly, other functional properties, such as their affinity for subunits and their ability to activate downstream effectors (e.g. phospholipase C (PLC)), were also found to be very similar for the two G protein subunits (16, 17).

In this study, we tested the hypothesis that the N-terminal extension in WTq may play a role in maintaining the selectivity of receptor recognition, an issue that had not been addressed yet. Toward this goal, the ability of several different G_i/o- and G_s-coupled receptors to interact with WTq or 6q were examined in cotransfected COS-7 cells. Whereas none of the receptors (upon incubation with the appropriate agonist ligands) was able to activate WTq to a significant extent, most of the receptors gained the ability to couple to 6q with considerable efficiency (measured biochemical response: stimulation of phosphatidylinositol (PI) hydrolysis). These data suggest that the N-terminal extension characteristic for G_q and G₁₁ subunits is critical for constraining the receptor coupling selectivity of these proteins.

EXPERIMENTAL PROCEDURES

To create a construct coding for a mutant G_q subunit lacking the first six amino acids (6q), a pcDNAI-based expression plasmid coding for murine WTq (11, 18) was used. To generate the 6q expression plasmid, a 78-base pair synthetic BamHI-FspI fragment containing the desired deletion was used to replace the corresponding sequence in the wild type plasmid. In both plasmids (WTq and 6q), the BamHI site of the pcDNAI polylinker was immediately followed by the initiating ATG codon. Both plasmids contained a short sequence coding for an internal hemagglutinin (HA) epitope tag (DVPDYA), which replaced WTq residues 125-130 (18). The presence of the epitope tag did not affect the receptor and effector coupling properties of WTq (8, 9, 18). The identity of the two G protein constructs was verified by dideoxy sequencing (19).

COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum at 37 °C in a humidified 5% CO₂ incubator. For transfections, 1 × 10⁶ cells were seeded into 100-mm dishes. About 24 h later, COS-7 cells were cotransfected with expression plasmids coding for WTq or 6q (1 µg DNA/dish) and the indicated receptor cDNAs (4 µg DNA/dish) by using a DEAE-dextran procedure (20). The following receptor expression plasmids were used: m2 muscarinic receptor in pcD (21), D2 dopamine receptor (22) in pcDNAI, -opioid receptor (23) in pcDNA3, somatostatin SSTR1 receptor (24) in pCMV, A1 adenosine receptor (25) in CDM7, D1 dopamine receptor (26) in pcDNAI, V2 vasopressin receptor in pcD-PS (27), and 2-adrenergic receptor (28) in pSVL.

Approximately 24 h after transfections, cells were split into 6-well dishes (~0.4 × 10⁶ cells/well) in culture medium supplemented with 3 µCi/ml [³H]myo-inositol (20 Ci/mmol; American Radiolabeled Chemicals Inc.). After a 24-h labeling period, cells were preincubated for 20 min at room temperature with 2 ml of Hanks' balanced salt solution containing 20 mM HEPES and 10 mM LiCl. Cells were then stimulated in the same buffer with the appropriate agonist ligands (1 h at 37 °C), and increases in intracellular inositol monophosphate (IP₁) levels were determined by anion exchange chromatography as described (10).

In a subset of experiments, transfected cells were incubated with pertussis toxin (PTX; 500 ng/ml) for the last 18-24 h of culture.

Approximately 48 h after transfections, COS-7 cells were metabolically labeled for 1 h with 800 µCi/ml of [9,10-³H]palmitate (60 Ci/mmol; American Radiolabeled Chemicals Inc.) in 5 ml of serum-free medium supplemented with 1% (v/v) dimethyl sulfoxide as described (29). The chemical nature of [³H]palmitate incorporation into G protein  subunits has been characterized earlier (18, 30) in cells radiolabeled under similar conditions.

Cells were fractionated into particulate and soluble fractions as described (29).

The 12CA5 mouse monoclonal antibody (BAbCo) specific for the HA epitope was used for immunoprecipitation and immunoblotting. Immunoprecipitation studies were performed using equivalent amounts of protein (2.5 mg) from total cell suspension in solubilization buffer consisting of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% (w/v) of Nonidet P-40, 0.5% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS in a total volume of 1 ml. Cell suspensions were added to the solubilization buffer and incubated at 4 °C on a rotator for 30 min followed by centrifugation for 10 min at 14,000 rpm in an Eppendorf 5415 microcentrifuge to pellet insoluble material. Supernatants were transferred to fresh Eppendorf tubes, and 5 µg of 12CA5 antibody and 20 µl of a 50% suspension of protein A-agarose (Sigma) were added followed by an overnight incubation at 4 °C on a rotator. Immunoprecipitates were recovered by centrifugation at 1,000 × g in a microcentrifuge, washed twice in 1 ml of solubilization buffer containing of the original detergent concentration, solubilized in 45 µl of gel sample buffer (Novex) in the absence of reducing agents, boiled for 5 min, separated by SDS-PAGE on 10% Tris-Gly gels (Novex), and prepared for fluorography using Amplify (Amersham Corp.) according to the manufacturer's instructions. Fluorograms were exposed for 4-6 weeks at 70 °C.

Immunoblotting was performed by separating equal amounts of protein (100 µg) from subcellular fractions solubilized in gel sample buffer (Novex) with 2.5% (v/v) -mercaptoethanol on 10% Tris-Gly gels (Novex), transfer to nitrocellulose membranes, probing with the 12CA5 antibody conjugated to horseradish peroxidase (Boehringer Mannheim), and development with enhanced chemiluminescence reagents (Amersham Corp.). Protein concentrations were determined using the Bio-Rad protein assay kit with IgG as the standard.

[Arg⁸]vasopressin, ()-isoproterenol, and PTX were purchased from Sigma. All other ligands used in this study were obtained through Research Biochemicals Inc.

RESULTS AND DISCUSSION

Receptor Coupling Properties of WTq and 6q

Initially, a series of receptors that are preferentially coupled to G proteins of the G_i/o family (m2 muscarinic, D2 dopamine, -opioid, SSTR1 somatostatin, and A1 adenosine) were coexpressed in COS-7 cells with either WTq or 6q. Transfected cells were then incubated with the appropriate agonist ligands, and the ability of the different receptors to couple to the two G proteins was determined by measuring increases in inositol phosphate production (due to WTq-mediated activation of PLC; Refs. 31 and 32). Coexpression of the different G_i/o-coupled receptors with either vector DNA (pcDNAI) or WTq, followed by ligand stimulation, resulted only in a rather small increase in PLC activity (Fig. 2A). As shown in Fig. 3 for the m2 muscarinic and D2 dopamine receptors, this small increase in inositol phosphate production could be almost completely blocked by pretreatment of cells with PTX (500 ng/ml). Consistent with previous findings (33, 34), this observation suggests that the m2 muscarinic and D2 dopamine receptors do not couple to WTq to a significant extent and that the small increase in PI hydrolysis seen after stimulation of these receptors is most likely due to activation of PLC by G protein subunits released upon receptor-mediated activation of endogenous G_i/o proteins (35, 36).

Functional interaction of different G protein-coupled receptors with the G subunits, WTq and 6q.

Effect of pertussis toxin on inositol phosphate accumulation in cells cotransfected with WTq or 6q and G_i/o-coupled receptors.

Interestingly, coexpression of the different G_i/o-coupled receptors with 6q resulted in a significantly increased PI response (as compared with WTq) that was most pronounced in the case of the two biogenic amine receptors (m2 muscarinic and D2 dopamine; Fig. 2A). These responses could only be partly blocked by PTX pretreatment (shown for the m2 muscarinic and D2 dopamine receptors in Fig. 3), indicating that they were primarily due to receptor-mediated generation of activated 6q. Complete concentration-response curves for m2 muscarinic and D2 dopamine receptor-mediated activation of 6q are given in Fig. 4. The EC₅₀ values (means ± S.E. of five independent experiments, each carried out in triplicate) for these responses amounted to 7.4 ± 0.3 µM in the case of the muscarinic agonist, carbachol (Fig. 4A), and to 1.4 ± 0.2 µM in the case of the dopaminergic ligand, ()-quinpirole (Fig. 4B), indicating that the interaction of the two biogenic amine receptors with 6q was highly efficient.

Functional interaction of the m2 muscarinic and the D2 dopamine receptor with 6q.

We next examined the ability of three G_s-coupled receptors (D1 dopamine, V2 vasopressin, and 2-adrenergic) to functionally interact with WTq or 6q. Consistent with their known coupling profiles (37), these receptors were unable to activate WTq to an appreciable extent (Fig. 2B). However, two of the investigated G_s-coupled receptors (D1 dopamine and 2-adrenergic) gained the ability to induce a significant increase in inositol production when coexpressed with 6q (as compared with WTq; Fig. 2B).

Two previous studies have shown that G_q/11-coupled receptors such as the NK2 neurokinin (16) or the m1 muscarinic receptor (17) can activate 6q in a fashion identical to WTq. Our data therefore suggest that 6q, in contrast to qWT, can be activated by receptors that are members of all three major functional classes of GPCRs.

The maximum degree of PLC stimulation mediated by the bona fide G_q/11-coupled m3 muscarinic receptor (7) amounted to 6-10-fold (data not shown), indicating that activation of 6q by different G_i/o- and G_s-coupled receptors (most of which mediated a 2-6-fold stimulation of PLC activity; Fig. 2) was not optimal. This observation is consistent with the currently held notion that efficient receptor-G protein coupling involves multiple sites of contact between the receptor protein and the G protein heterotrimer (1-7).

It should also be noted that coexpressed 6q did not improve coupling of the V2 vasopressin receptor to PLC stimulation and that the absolute magnitude of responses mediated by 6q upon coexpression with the -opioid or the SSTR1 somatostatin receptor, respectively, was quite small (Fig. 2), indicating that 6q is not generally promiscuous. One possible reason for the observed differences in the ability of the studied G_i/o- and G_s-coupled receptors to interact with 6q may be that the relative functional importance of individual receptor-G protein contact sites may vary among different GPCRs (e.g. peptide receptors versus receptors for biogenic amines).

Subcellular Distribution of WTq and 6q

To exclude the possibility that the promiscuous nature of 6q was simply due to exceptionally high expression levels (as compared with WTq), the subcellular distribution of the two G protein subunits was studied by cell fractionation and immunoblotting. Both G subunits were detected with the monoclonal antibody, 12CA5, which recognizes the internal HA epitope tag present in both proteins (see "Experimental Procedures"). As shown in Fig. 5A, both G protein constructs were found exclusively in the particulate fraction; however, 6q was expressed at lower levels (approximately 10-20% of WTq, as determined by scanning densitometry; data not shown). The precise reason for this latter observation remains unclear; however, possible factors may include reduced protein stability or translation efficiency. In any case, these data exclude the possibility that the ability of 6q to be activated by multiple classes of GPCRs is due to overexpression of this subunit (as compared with WTq).

Subcellular Localization and Palmitoylation of WTq and 6q.

Palmitoylation Pattern of WTq and 6q

Previous studies (16-18, 38-40) have shown that G proteins of the G_q/11 family, like most other G subunits, are palmitoylated at cysteine residues located near the N terminus of the proteins (corresponding to Cys⁹ and Cys¹⁰ in Fig. 1). To compare the palmitoylation patterns of WTq and 6q, transfected COS-7 cells were metabolically labeled with [³H]palmitic acid followed by immunoprecipitation of WTq and 6q by the 12CA5 monoclonal antibody, SDS-PAGE, and fluorography. As shown in Fig. 5B, WTq and 6q were the only immunoprecipitated proteins, because no labeled proteins could be precipitated when cells were transfected with "empty" vector DNA. Consistent with published results, both WTq (16-18, 38, 39) and 6q (16) incorporated considerable amounts of [³H]palmitate (Fig. 5B). The reduction in signal strength seen with 6q (approximately 10-20% of WTq, as determined by scanning densitometry; data not shown) correlated well with the reduction in 6q levels revealed by immunoblotting (Fig. 5A). This observation suggests that 6q is palmitoylated to an extent similar to that of WTq, as has been observed earlier in transfected COS-7 cells (16). It is therefore unlikely that differences in palmitoylation patterns are responsible for the different functional properties of 6q and WTq.

We have shown that 6q, in contrast to WTq, can productively interact with several different G_i/o- and G_s-coupled receptors, suggesting that the six-amino acid extension that is unique to WTq (as well as G₁₁) is critical for constraining the receptor coupling selectivity of this G protein subunit. 6q and WTq were found to be palmitoylated to a similar extent, suggesting that the functional promiscuity of 6q is not due to a lack of acylation. One possibility is that the N-terminal extension characteristic for G_q/11 subunits constrains the receptor coupling selectivity by preventing access of G_i/o- and G_s-coupled receptors. Alternatively, it is conceivable that this six-amino acid sequence exerts an indirect conformational effect on the structure of G_q/11 that is crucial for maintaining the receptor selectivity of these subunits. Clearly, this issue needs to be addressed in future studies. In summary, our data suggest a novel mechanism by which receptor-G protein coupling selectivity can be achieved and should contribute to a better understanding of the molecular basis of receptor-G protein interactions.