Functional Characterization of a Series of Mutant G Protein alpha q Subunits Displaying Promiscuous Receptor Coupling Properties*

Evi Kostenis, Fu-Yue Zeng, and Jürgen WessDagger

From the Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The N termini of two G protein alpha  subunits, alpha q and alpha 11, differ from those of other alpha  subunits in that they display a unique, highly conserved six-amino acid extension (MTLESI(M)). We recently showed that an alpha q deletion mutant lacking these six amino acids (in contrast to wild type alpha q) was able to couple to several different Gs- and Gi/o-coupled receptors, apparently due to promiscuous receptor/G protein coupling (Kostenis, E., Degtyarev, M. Y., Conklin, B. R., and Wess, J. (1997) J. Biol. Chem. 272, 19107-19110). To study which specific amino acids within the N-terminal segment of alpha q/11 are critical for constraining the receptor coupling selectivity of these subunits, this region of alpha q was subjected to systematic deletion and alanine scanning mutagenesis. All mutant alpha q constructs (or wild type alpha q as a control) were coexpressed (in COS-7 cells) with the m2 muscarinic or the D2 dopamine receptors, two prototypical Gi/o-coupled receptors, and ligand-induced increases in inositol phosphate production were determined as a measure of G protein activation. Surprisingly, all 14 mutant G proteins studied (but not wild type alpha q) gained the ability to productively interact with the two Gi/o-linked receptors. Similar results were obtained when we examined the ability of selected mutant alpha q subunits to couple to the Gs-coupled beta 2-adrenergic receptor. Additional experiments indicated that the functional promiscuity displayed by all investigated mutant alpha q constructs was not due to overexpression (as compared with wild type alpha q), lack of palmitoylation, or initiation of translation at a downstream ATG codon (codon seven). These data are consistent with the notion that the six-amino acid extension characteristic for alpha q/11 subunits forms a tightly folded protein subdomain that is critical for regulating the receptor coupling selectivity of these subunits.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

G protein-coupled receptors (GPCRs)1 regulate the activity of a large variety of effector systems via interaction with specific classes of heterotrimeric G proteins (consisting of alpha , beta , and gamma  subunits) that are attached to the cytoplasmic side of the plasma membrane (1-6). In most cases, an individual GPCR can only activate a distinct subset of the many structurally similar G proteins present in each cell (7, 8). How this selectivity is achieved at a molecular level is currently being explored by a great number of laboratories.

A large body of evidence indicates that multiple regions on the G protein alpha  subunits play key roles in receptor binding and dictating the selectivity of receptor/G protein interactions (2, 5, 6, 8, 9). Specifically, recent studies have shown that residues at the extreme C terminus of the G protein alpha  subunits are of fundamental importance for regulating receptor/G protein coupling selectivity (10-13), probably by directly contacting the receptor protein (14, 15). However, several lines of evidence indicate that other regions of Galpha also contribute to receptor binding and the selectivity of receptor recognition (2, 5, 6, 9). Biochemical studies suggest, for example, that the N-terminal portion of Galpha may also be in contact with the receptor protein (14, 16, 17). An early study demonstrated that a synthetic peptide corresponding approximately to the N-terminal alpha N-helix of alpha -transducin (Fig. 1) was able to prevent rhodopsin/transducin interactions, presumably by directly binding to rhodopsin (14). Moreover, Higashijima and Ross (16) showed that a cysteine-substituted mastoparan, a receptor-mimetic peptide toxin, can be cross-linked to the extreme N terminus of Galpha o subunits. Similar findings were obtained with a photoaffinity derivative of a receptor-mimetic peptide corresponding to the C-terminal portion of the third cytoplasmic loop of the alpha 2A-adrenergic receptor (17).

In addition, functional analysis of an N-terminally truncated alpha q subunit suggests that the N terminus of Galpha subunits is also involved in modulating the fidelity of receptor/G protein recognition (18). Galpha q and Galpha 11 contain a unique six-amino acid extension (MTLESI(M)) that is not found in other Galpha subunits (Fig. 1). This short sequence is highly conserved among all vertebrate species from which these subunits have been cloned so far (19-23). When this sequence was removed by deletion mutagenesis (18), the resulting mutant alpha q subunit (corresponding to q(Delta 2-7) in Fig. 1) gained the ability to be activated by various Gi/o- and Gs-coupled receptors which normally do not couple efficiently to wild type (full-length) alpha q (qWT). Based on these results, we speculated that the N-terminal extension characteristic for alpha q/11 subunits may act as a "filter" by selectively preventing Gi/o- and Gs-coupled receptors from contacting functionally critical alpha q/11 residues. Alternatively, it is conceivable that the N-terminal extension exerts indirect conformational effects on adjacent alpha q/11 domains, such as the functionally critical C terminus, thereby constraining the selectivity of receptor/alpha q/11 interactions.

To examine which specific amino acids within the N-terminal segment of alpha q/11 are critical for maintaining the selectivity of receptor recognition, we generated a large number of mutant alpha q subunits in which the N-terminal residues were progressively deleted or systematically replaced, either individually or in combination, with alanine. The ability of these mutant subunits to be activated by different Gi/o- and Gs-coupled receptors was examined in cotransfected COS-7 cells. Surprisingly, all 14 mutant alpha q subunits studied, but not qWT, displayed pronounced receptor coupling promiscuity. These results indicate that each single amino acid within the N-terminal extension of alpha q/11 subunits is critical for constraining the receptor coupling selectivity of these subunits. The most straightforward explanation for this finding is that the N-terminal portion of alpha q/11 forms a well defined protein subdomain that regulates the fidelity of receptor/alpha q/11 interactions.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Creation of Mutant G Protein Constructs-- A mouse alpha q cDNA (19) cloned into the pcDNAI expression vector (24) was used as a template for polymerase chain reaction mutagenesis. All wild type and mutant Galpha q subunits contained an internal hemagglutinin (HA) epitope tag (DVPDYA; Ref. 24). The presence of the epitope tag, which replaced alpha q residues 125-130, did not affect the receptor and effector coupling properties of qWT (10, 11, 24). The construction of the q(Delta 2-7) deletion mutant (formerly referred to as -6q) has been described previously (18). A silent BssHII site was introduced into q(Delta 2-7) at amino acid codons 18-19 to facilitate the generation of additional mutant G proteins. All mutant alpha q subunits were constructed by replacing a 57-base pair BamHI-BssHII fragment of q(Delta 2-7) with the corresponding DNA fragments (generated via polymerase chain reaction) containing the desired deletions or substitutions (Fig. 1). In the wild type and all mutant alpha q plasmids, the BamHI-site of the pcDNAI polylinker was immediately followed by the initiating ATG codon. The correctness of all polymerase chain reaction-derived DNA sequences was verified by dideoxy sequencing of the mutant plasmids (25).

Coexpression of Receptor and G Protein Constructs-- COS-7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) 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 18-24 h later, cells were cotransfected with the indicated G protein (1 µg of DNA/dish) and receptor constructs (4 µg of DNA/dish) by using a DEAE-dextran procedure (26). The following receptor expression plasmids were used: human m2 muscarinic receptor in pcD (27), human D2 dopamine receptor in pcDNAI (28), and rat beta 2-adrenergic receptor in pSVL (29).

Phosphatidylinositol Hydrolysis Assays-- About 18-24 h after transfections, cells were split into six-well dishes (approximately 0.4 × 106 cells/well) and labeled 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 with the appropriate agonists for 1 h at 37 °C, and increases in intracellular inositol monophosphate (IP1) levels were determined by anion exchange chromatography as described (30).

[3H]Palmitate Labeling and Immunoprecipitation-- Approximately 48 h after transfections, COS-7 cells were washed twice with 10 ml of serum-free DMEM and incubated for 1 h in 5 ml of serum-free DMEM (37 °C, 5% CO2). Following removal of the medium, cells were metabolically labeled for 16 h with 200 µCi/ml [9,10-3H]palmitate (50.0 Ci/mmol; American Radiolabeled Chemicals Inc.) in 3 ml of DMEM containing 10% dialyzed fetal calf serum (37 °C, 5% CO2). After the 16-h labeling period, cells were washed twice with 10 ml of phosphate-buffered saline, resuspended in 50 µl of buffer A (10 mM Tris-HCl, pH 8.0, and 150 mM NaCl) containing 1% SDS, and incubated at 70 °C for 10 min. Subsequently, 200 µl of extraction buffer (composition: 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 2 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride) was added, and samples were tumbled for 30 min at 4 °C. Insoluble material and nuclei were removed by centrifugation for 10 min at 14,000 rpm in a refrigerated Eppendorf 5417R microcentrifuge. Supernatants were mixed with an equal volume of buffer A and 20 µl of Sepharose 4B beads (Sigma) coupled to the 12CA5 monoclonal antibody (Boehringer Mannheim) (the antibody was coupled to CNBr-activated Sepharose 4B according to the manufacturer's (Sigma) instructions). Samples were tumbled for 2 h at 4 °C and then centrifuged for 1 min at 2,000 rpm in an Eppendorf microcentrifuge to gently pellet the Sepharose beads. Pellets were washed twice with 500 µl of a 1:5 dilution of extraction buffer and resuspended in 20 µl of Laemmli sample buffer (Bio-Rad) without reducing agents. After heating the samples for 10 min at 70 °C, the beads were pelleted gently (same conditions as above), and supernatants were used for SDS-PAGE (13%). Gels loaded with 10% of the total sample volumes were subjected to Western blotting (see below). Gels run with the remaining 90% of the samples were soaked in 20-30 ml of Fluoro-Hance (Research Products International Corp.) for 30 min, dried, and subjected to fluorography at -80 °C for 4-6 weeks.

Western Blotting-- All wild type and mutant Galpha q subunits were detected with the 12CA5 monoclonal antibody directed against the HA-epitope tag present in all G protein constructs. Samples containing 20 µg of membrane protein prepared from transfected COS-7 cells were resolved by SDS-PAGE (13%), electroblotted onto nitrocellulose, and probed with the 12CA5 antibody as described (11, 31). Immunoreactive proteins were detected by incubation with horseradish peroxidase-conjugated sheep anti-mouse IgG antibody (Amersham Pharmacia Biotech) and visualized using an enhanced chemiluminescence system (Amersham Pharmacia Biotech).

Drugs-- All ligands used in this study were purchased from Research Biochemicals Inc. The remaining chemicals were obtained through Sigma.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Construction and Expression of Mutant alpha q Subunits-- Initially we prepared two series of N-terminally modified mutant alpha q subunits (Fig. 1). In one series, amino acids 2-7 (TLESIM) of mouse alpha q were progressively removed leading to six alpha q deletion constructs (note that q(Delta 2-7) was previously referred to as -6q; Ref. 18). In the other series, these residues were replaced, either individually (yielding six alpha q single point mutants) or in combination (resulting in q(TLE right-arrow AAA) and q(SI right-arrow AA)) (Fig. 1). All wild type and mutant alpha q subunits contained an internal hemagglutinin (HA) epitope tag replacing alpha q residues 125-130. Previous studies demonstrated that the presence of the epitope tag does not affect the receptor and effector coupling properties of qWT (10, 11, 24). Western analysis using an anti-HA monoclonal antibody (12CA5) showed that all mutant alpha q subunits were properly expressed at levels similar to or somewhat lower than observed with qWT (Fig. 2).


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Fig. 1.   Structure of alpha q deletion and point mutants. Amino acids marked with an arrow were replaced, either individually or in combination, with alanine residues. For comparison, the N-terminal portions of several other G protein alpha  subunits are also shown. Sequences (human) were taken from Refs. 23 and 60 (note that the human alpha q sequence (23) shown here is identical to the corresponding mouse sequence (19)). Gaps were introduced to allow for maximum sequence identity. The position of the N-terminal segment of the alpha N-helix, as revealed by x-ray crystallography (43, 44), is indicated.


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Fig. 2.   Immunoblot analysis of wild type and mutant alpha q subunits expressed in COS-7 cells. COS-7 cells were transfected with vector DNA (pcDNAI), qWT, and different alpha q deletion and alanine point mutants (for mutant structures, see Fig. 1). Equal amounts of membrane protein (20 µg) prepared from transfected COS-7 cells were analyzed by SDS-PAGE (13%) and Western blotting, using the 12CA5 monoclonal antibody as described under "Experimental Procedures." The following mutant constructs were studied: q(M7A) (lane 1), q(Delta 2) (lane 2), q(Delta 2-3) (lane 3), q(Delta 2-4) (lane 4), q(Delta 2-5) (lane 5), q(Delta 2-6) (lane 6), q(Delta 2-7) (lane 7), q(TLE right-arrow AAA) (lane 8), q(SI right-arrow AA) (lane 9), q(T2A) (lane 10), q(L3A) (lane 11), q(E4A) (lane 12), q(S5A) (lane 13), and q(I6A) (lane 14). Protein molecular weight standards (in kDa) are indicated. Two additional experiments gave similar results.

Interaction of Mutant alpha q Subunits with Gi/o-coupled Receptors-- To study the effects of the different mutations on the fidelity of receptor/G protein interactions, all mutant alpha q subunits were initially coexpressed (in COS-7 cells) with the m2 muscarinic receptor, a prototypical Gi/o-coupled receptor (32, 33). Transfected cells were then incubated with the muscarinic agonist, carbachol (0.5 mM), and the ability of the m2 muscarinic receptor to couple to the different G proteins was determined by measuring increases in inositol phosphate production (due to alpha q-mediated activation of PLCbeta ; Refs. 34 and 35). Coexpression of the m2 receptor with either vector DNA (pcDNAI) or qWT, followed by ligand stimulation, resulted only in a relatively small increase in PLCbeta activity (approximately 3-fold; Fig. 3), a response known to be largely due to activation of PLCbeta isozymes by G protein beta gamma subunits released upon m2 receptor-mediated activation of endogenous Gi/o proteins (36, 37). Remarkably, the m2 muscarinic receptor gained the ability to productively couple to all 14 mutant alpha q subunits, resulting in a 6-11-fold stimulation of PLC activity (Fig. 3).


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Fig. 3.   Functional coupling of the m2 muscarinic receptor to mutant alpha q subunits. COS-7 cells were cotransfected with expression plasmids coding for the wild type m2 muscarinic receptor and vector DNA (pcDNAI), qWT, or different alpha q deletion and alanine point mutants (for mutant structures, see Fig. 1). Transfected cells were incubated for 1 h (at 37 °C) with 0.5 mM carbachol. The resulting increases in intracellular IP1 levels were determined as described under "Experimental Procedures." Basal IP1 levels observed with cells transfected with the different mutant alpha q subunits were similar to those found with qWT-expressing cells (305 ± 67 cpm/well). Data are given as means ± S.E. of three independent experiments, each carried out in triplicate.

Essentially similar findings were obtained when we studied the ability of the D2 dopamine receptor, another Gi/o-coupled receptor, to interact with the various mutant G proteins. When coexpressed with qWT, the D2 receptor, upon stimulation with the selective D2 agonist, (-)-quinpirole (10 µM), was unable to induce an appreciable degree of PLC stimulation (Fig. 4). However, in the presence of either of the 14 mutant alpha q subunits studied, a significant increase in inositol phosphate production was observed (ranging from 2- to 6-fold above basal; Fig. 4).


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Fig. 4.   Functional interaction of the D2 dopamine receptor with mutant alpha q subunits. COS-7 cells were cotransfected with expression plasmids coding for the wild type D2 dopamine receptor and vector DNA (pcDNAI), qWT, or different alpha q deletion and alanine point mutants (for mutant structures, see Fig. 1). Transfected cells were incubated for 1 h (at 37 °C) with the D2 selective agonist, (-)-quinpirole (10 µM). The resulting increases in intracellular IP1 levels were determined as described under "Experimental Procedures." Basal IP1 levels observed with cells transfected with the different mutant alpha q subunits were similar to those found with qWT-expressing cells (582 ± 102 cpm/well). Data are given as means ± S.E. of three independent experiments, each carried out in triplicate.

Interaction of Mutant alpha q Subunits with a Gs-coupled Receptor-- We next examined the ability of the Gs-coupled beta 2-adrenergic receptor to interact functionally with selected mutant alpha q subunits (q(Delta 2-7), q(T2A), q(L3A), q(E4A), q(S5A), q(I6A), and q(M7A)). Consistent with its known coupling preference (1), this receptor subtype, when stimulated with (-)-isoproterenol (200 µM), was unable to activate efficiently qWT (Fig. 5), mediating only an approximately 1.5-fold increase in IP production. However, the beta 2-adrenergic receptor gained the ability to interact productively with all examined mutant alpha q subunits, leading to a 3-5-fold stimulation of PLC activity (Fig. 5).


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Fig. 5.   Functional coupling of the beta 2-adrenergic receptor to selected mutant alpha q subunits. COS-7 cells were cotransfected with expression plasmids coding for the wild type beta 2-adrenergic receptor and vector DNA (pcDNAI), qWT, or various mutant alpha q constructs (for mutant structures, see Fig. 1). Transfected cells were incubated for 1 h (at 37 °C) with the adrenergic agonist, (-)-isoproterenol (200 µM). The resulting increases in intracellular IP1 levels were determined as described under "Experimental Procedures." Basal IP1 levels observed with cells transfected with the different mutant alpha q subunits were similar to those found with qWT-expressing cells (473 ± 94 cpm/well). Data are given as means ± S.E. of three independent experiments, each carried out in triplicate.

Translation Start Site in Mutant alpha q Subunits-- As described above, all investigated mutant alpha q subunits showed similar functional properties, displaying coupling promiscuity upon coexpression with different Gi/o- and Gs-coupled receptors. As shown in Figs. 1 and 6A, the N-terminal six-amino acid extension (MTLESI) characteristic for alpha q subunits is immediately followed by a second in-frame methionine codon (codon seven). If this second rather than the first methionine codon is used as a translation start site, all alpha q constructs would direct the synthesis of the same N-terminally truncated protein (corresponding to q(Delta 2-7)). To exclude such a mechanism as a potential cause of the observed functional promiscuity displayed by the different mutant alpha q subunits, an additional experiment was designed. Frameshift mutations were introduced between the two N-terminal ATG codons (codons one and seven), using two mutant alpha q constructs, q(L3A) and q(E4A), as model systems (Fig. 6A). In q(L3A)-FS, the deletion of a single nucleotide (C) within codon three in q(L3A) led to a shift of the amino acid reading frame resulting in a premature stop codon after 10 amino acids of nonsense sequence. In q(E4A)-FS, the insertion of a single nucleotide (C) after codon four in q(E4A) led to a changed amino acid reading frame and a premature stop codon after 59 amino acids of nonsense sequence (Fig. 6A).


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Fig. 6.   Lack of functional coupling of the m2 muscarinic receptor to frameshifted mutant alpha q subunits. A, structure of frameshifted mutant alpha q subunits. For comparison, the amino acid and nucleotide sequences of the N-terminal segment of wild type alpha q (qWT) are shown on top. The two N-terminal in-frame ATG codons (codons one and seven) are underlined. In q(L3A)-FS, the deletion of a single nucleotide (C) within codon three in q(L3A) leads to a shift of the amino acid reading frame resulting in a premature stop codon after 10 amino acids of nonsense sequence. Analogously, in q(E4A)-FS, the insertion of a single nucleotide (C) after codon four in q(E4A) leads to a changed amino acid reading frame and a premature stop codon after 59 amino acids of nonsense sequence. B, COS-7 cells were cotransfected with expression plasmids coding for the wild type m2 muscarinic receptor and vector DNA (pcDNAI), qWT, or the indicated frameshifted alpha q mutant constructs. For comparison, the q(Delta 2-7), (L3A), and q(E4A) mutants were also included in this set of experiments. Transfected cells were incubated for 1 h (at 37 °C) with 0.5 mM carbachol, and the resulting increases in intracellular IP1 levels were determined as described under "Experimental Procedures." Data are given as means ± S.E. of three independent experiments, each carried out in triplicate.

If codon seven is in fact used as a translation start site, q(L3A)-FS and q(E4A)-FS would be expected to exhibit functional properties similar to those of q(L3A) and q(E4A), respectively. However, coexpression studies showed that the two frameshifted mutant alpha q constructs, in contrast to q(L3A) and q(E4A), were unable to interact with the m2 muscarinic receptor (Fig. 6B). Following ligand stimulation (carbachol, 0.5 mM) of the m2 muscarinic receptor, q(L3A) and q(E4A) were capable of mediating a pronounced increase in inositol phosphate production (7-8-fold above basal), whereas the responses observed with q(L3A)-FS and q(E4A)-FS were not significantly different from those found with vector-transfected control cells (approximately 3-fold above basal; Fig. 6B).

In addition to the functional studies, G protein expression was monitored by Western analysis using the 12CA5 monoclonal antibody (Fig. 7). Whereas q(L3A) and q(E4A) were found to be properly expressed in a fashion similar to qWT (detectable at 42-45 kDa), no alpha q protein was detected after transfection with the q(L3A)-FS and q(E4A)-FS mutant constructs (Fig. 7, lanes 3 and 5). In this set of experiments, a weak nonspecific band of >50 kDa was observed in all lanes (including vector control), indicating that it represents a cross-reacting COS-7 cell protein. Taken together, these observations demonstrate that the second methionine codon (codon seven) is not used (at least not to a detectable degree) as a translation start site in the studied mutant alpha q constructs.


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Fig. 7.   Immunoblot analysis of frameshifted mutant alpha q subunits expressed in COS-7 cells. COS-7 cells were transfected with vector DNA (pcDNAI), qWT, and various mutant alpha q constructs (for structures of mutant G proteins, see Figs. 1 and 6A). Equal amounts of membrane protein (20 µg) prepared from transfected COS-7 cells were analyzed by SDS-PAGE (13%) and Western blotting, using the 12CA5 monoclonal antibody as described under "Experimental Procedures." The following mutant constructs were studied: q(Delta 2-7) (lane 1), q(L3A) (lane 2), q(L3A)-FS (lane 3), q(E4A) (lane 4), and q(E4A)-FS (lane 5). Protein molecular mass standards (in kDa) are indicated. Please note that COS-7 cells transfected with q(L3A)-FS (lane 3) and q(E4A)-FS (lane 5) did not express detectable amounts of alpha q protein (observable at approximately 45 kDa in all other lanes, except vector). In this set of experiments, a nonspecific band of >50 kDa was observed in all lanes (including vector control), indicative of a cross-reacting COS-7 cell protein. Three additional experiments gave similar results.

Palmitoylation Pattern of Wild Type and Mutant alpha q Subunits-- Previous studies (24, 38-42) have shown that alpha q and alpha 11, like most other Galpha subunits, are palmitoylated at cysteine residues located near the N termini (corresponding to Cys-9 and Cys-10 in Fig. 1). We therefore wanted to examine whether the promiscuous mutant alpha q subunits differed from qWT in their palmitoylation patterns. Toward this goal, COS-7 cells transfected with qWT or selected mutant alpha q subunits (q(Delta 2-4), q(Delta 2-7), q(TLE->AAA), q(SI->AA), q(T2A), and q(M7A)) were metabolically labeled with [3H]palmitic acid, followed by immunoprecipitation of alpha q subunits by the 12CA5 monoclonal antibody, SDS-PAGE, and fluorography. Western analysis showed that the amounts of immunoprecipitated wild type and mutant alpha q proteins were similar (Fig. 8A). The alpha q subunits were the only immunoprecipitated proteins, since no labeled proteins were detected when cells were transfected with "empty" vector DNA. As shown in Fig. 8B, qWT and all mutant alpha q subunits incorporated significant amounts of [3H]palmitate. However, the strength of the palmitoylation signal was generally weaker in the case of the mutant alpha q subunits (40-75% compared with qWT, as determined by scanning densitometry).


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Fig. 8.   Palmitoylation of wild type and mutant alpha q subunits expressed in COS-7 cells. COS-7 cells were transfected with vector DNA, qWT, and selected alpha q deletion and point mutants. Following labeling with [3H]palmitic acid (16 h; 200 µCi/ml), cells were lysed, and extracts were subjected to immunoprecipitation with the 12CA5 antibody as described under "Experimental Procedures." A, aliquots of the immunoprecipitated proteins (10% of total samples) were analyzed by SDS-PAGE (13%) and Western blotting, using the 12CA5 monoclonal antibody. B, the remaining 90% of the samples were resolved by SDS-PAGE (13%) and analyzed by fluorography, as described under "Experimental Procedures." The following mutant constructs were studied: q(Delta 2-7) (lane 1), q(TLE right-arrow AAA) (lane 2), q(SI right-arrow AA) (lane 3), q(Delta 2-4) (lane 4), q(M7A) (lane 5), and q(T2A) (lane 6). Protein molecular weight standards (in kDa) are indicated. Two additional experiments gave similar results.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The fidelity of receptor/G protein interactions critically depends on the ability of different GPCRs to discriminate between unique structural features of the Galpha subunits (2, 5-9). Characteristically, most Galpha subunits (when bound to beta gamma in a trimeric complex) can be efficiently activated by only certain functional classes of GPCRs (7, 8). In this study, we have identified a series of 14 mutant alpha q subunits that display promiscuous receptor coupling. In contrast to qWT, all 14 mutant subunits investigated here could be activated by both Gi/o- and Gs-coupled receptors. In these mutant G proteins, residues within the six-amino acid N-terminal extension that is characteristic for alpha q/11 subunits were systematically substituted with alanine (either individually or in combination) or progressively deleted.

The most straightforward explanation for the observed functional promiscuity displayed by all mutant alpha q subunits is that the unique N terminus of alpha q/11 subunits adopts a well defined three-dimensional structure that is critical for maintaining the coupling selectivity of these G proteins. Based on this concept, mutations within this structural motif are thought to interfere with its proper folding and its ability to regulate receptor/G protein coupling selectivity.

Although all mutant G proteins showed a qualitatively similar functional profile, quantitative differences in their ability to be activated by different Gi/o- and Gs-coupled receptors (m2 muscarinic, D2 dopamine, and beta 2-adrenergic) were noted. Moreover, the observed patterns of G protein activities also varied among the three receptors used in this study. It is likely that the specific receptor residues involved in G protein binding differ between the three receptors, thus providing an explanation for the observed ability of these receptors to discriminate between different mutant Galpha q subunits which, in turn, are also likely to display subtle structural differences.

Recently, the atomic structures of two different G protein heterotrimers, Galpha i1beta 1gamma 2 (Ref. 43) and Galpha t/i1beta 1gamma 1 (Ref. 44), have been resolved by x-ray crystallography. In these structures (but not in the free alpha  subunits), the N-terminal segment of the alpha  subunit (corresponding to residues 6-26 in alpha t) is alpha -helically arranged and protrudes away from the "bulk" of Galpha . This so-called alpha N-helix (Fig. 1) is engaged in interactions with the beta gamma complex via docking along the first blade of the beta  "propeller," thus determining the location of the acylated N terminus of Galpha (note, however, that the G protein structures were resolved using proteins devoid of lipid modifications). Unfortunately, the extreme N termini of the alpha  subunits (corresponding to amino acids 1-10 in alpha q; Fig. 1) were not observed in the x-ray structures, probably due to their conformational flexibility. The available structural information therefore provides little insight into the potential arrangement of the N-terminal 10 amino acids of alpha q/11 subunits.

Accumulating evidence indicates that several C-terminal regions of Galpha (including the C-terminal tail, portions of the alpha 5-helix, and the alpha 4/beta 6 loop) are directly involved in receptor binding and play a key role in dictating the selectivity of receptor/G protein interactions (10-15, 45-49). X-ray crystallographic studies suggest that the N- and C-terminal segments of Galpha subunits are conformationally linked (43, 50). One possibility therefore is that the N-terminal extension characteristic for alpha q/11 subunits constrains their receptor coupling selectivity by preventing access of Gi/o- and Gs-coupled receptors (either due to steric hindrance or to allosteric effects) to functionally critical C-terminal Galpha regions.

As outlined in the Introduction, biochemical studies suggest that residues within the N-terminal 30-amino acid segment of Galpha may be involved in receptor binding (14). Since this Galpha region is also known to be critical for binding of Galpha to beta gamma complexes (43, 44), binding of the receptor to an N-terminal Galpha site may contribute to triggering dissociation of the G protein heterotrimer into free alpha  and beta gamma subunits. An alternative possibility therefore is that the first six amino acids characteristic for alpha q/11 subunits have a "gate" function by regulating access of different classes of GPCRs to an adjacent N-terminal Galpha region.

It has been noted that a negatively charged cavity that is predicted to face the plasma membrane (and/or the receptor) is located at the alpha /beta interface (43, 44). On the other hand, structure-function analysis of a great number of different GPCRs has shown that several positively charged residues located within the second and third intracellular loops of these receptors play key roles in triggering G protein activation (3, 8). Thus, another possibility is that the N terminus of alpha q/11 subunits controls the access of different functional classes of GPCRs to this anionic surface on alpha q/11. The resolution of the atomic structure of a Gq heterotrimer (that also includes the N-terminal alpha q sequences) is likely to provide clues that would allow us to distinguish between these different possibilities.

Interestingly, the functional properties of the mutant alpha q constructs examined in this study resemble those of two other G protein subunits, alpha 15 and alpha 16 (alpha 15 and alpha 16 appear to be the murine and human versions of the same gene; Ref. 51). Recent studies have shown that alpha 15 and alpha 16, like the mutant alpha q subunits described here, show little receptor selectivity and can be activated by most GPCRs studied to date (52, 53). A sequence comparison indicates that the N-terminal eight amino acids of alpha 15/16 (MARSLTWG(R)) differ considerably from the corresponding alpha q/11 sequence (MTLESI(M)MA) (19, 51, 54). It therefore remains to be elucidated whether the coupling promiscuity displayed by alpha 15/16 and all mutant alpha q constructs examined here is dependent on a similar molecular mechanism.

Like most other Galpha subunits, alpha q and alpha 11 are known to be reversibly palmitoylated at cysteine residues located near their N termini (corresponding to Cys-9 and Cys-10 in Fig. 1; Refs. 24 and 38-42). Studies with mutant alpha q constructs in which both N-terminal cysteine residues were replaced with serine or alanine suggest that palmitoylation facilitates membrane attachment of this alpha subunit (24, 38, 39). However, palmitoylation does not appear to be an absolute requirement for membrane targeting of alpha q (39). In addition, palmitoylation-defective mutant alpha q subunits were found to be severely impaired in their ability to mediate receptor-dependent increases in PLCbeta activity (24, 38). However, in a reconstituted system, enzymatic removal of palmitate from purified alpha q had little effect on the ability of this subunit to interact with receptors and downstream effector enzymes (39), indicating that the N-terminal cysteine residues themselves rather than the palmitate moieties attached to them are of primary functional importance.

In the present study, all examined mutant alpha q subunits, similar to qWT, were found to incorporate significant amounts of [3H]palmitate (Fig. 8B). We noted, however, that the intensity of the palmitoylation signal was reduced in the case of the mutant alpha q subunits (about 40-75% as compared with qWT). On the other hand, Western analysis of immunoprecipitated, [3H]palmitate-labeled G proteins indicated that most mutant alpha q subunits were expressed at levels similar to qWT (Fig. 8A). It is therefore conceivable that palmitoylation plays an inhibitory role in constraining the receptor coupling selectivity of alpha q, perhaps by specifying the spatial orientation and fold of the N terminus of alpha q. Consistent with this notion, biochemical studies have shown that the myristoyl moiety attached to the N terminus of alpha t is essential for the proper folding of the N-terminal alpha t segment (55). In addition, a previous study analyzing the coupling properties of a palmitoylation-defective mutant alpha z subunit suggests that palmitoylation inhibits alpha z signaling by an as yet unknown mechanism (56). Interestingly, Tu et al. (57) recently showed that palmitoylation of alpha z and alpha i1 strongly inhibits the activity of several RGS proteins (regulators of G protein signaling) which are known to stimulate the GTPase activity of these alpha  subunits. This finding indicates that palmitoylation of alpha  subunits may represent a general mechanism for prolonging the lifetime of activated alpha  subunits. However, such a mechanism cannot explain the functional properties of the mutant alpha q subunits investigated in this study, which appear to be palmitoylated less efficiently (as compared with qWT) and would therefore be expected to show reduced rather than increased functional activity.

In eucaryotic organisms, translation initiation is usually restricted to the first in-frame ATG (AUG in the case of mRNA) codon. However, several examples are known where translation is initiated from a downstream ATG codon (a process frequently referred to as "leaky scanning"), particularly when the first in-frame ATG codon is located in a "weak" context that considerably deviates from the consensus sequence (5'-CC(A/G)CCATGG-3') for strong initiation codons (58, 59). The residues that exert the strongest effects on the efficiency of translation initiation are a -3 purine (A or G) and a +4 G (the A of the ATG codon is numbered +1, and nucleotides to the left have negative numbers). Inspection of the nucleotide sequences of the alpha q constructs used in this study indicates that the second N-terminal ATG codon (codon seven) is present in a clearly better context than the first one (codon one) (Fig. 6A). It was therefore important to demonstrate that the mutant alpha q constructs analyzed in this study do not allow translation initiation from codon seven, thus yielding the same, N-terminally truncated protein (q(Delta 2-7)) that is known to be functionally promiscuous (18). Several observations clearly argue against such a mechanism. First, since the first ATG in qWT is located in the same weak context as in all mutant alpha q plasmids (all constructs contain identical 5'-flanking sequences), leaky scanning should also lead to the synthesis of the promiscuous q(Delta 2-7) subunit in the case of qWT. However, no such promiscuous coupling was observed when qWT was coexpressed with different Gi/o- and Gs-coupled receptors. Second, a mutant alpha q construct in which the second ATG codon (codon seven) was replaced with an alanine codon (thus allowing only translation from codon one) showed functional properties similar to all other mutant alpha q constructs examined. Third, we constructed two mutant alpha q subunits, q(L3A)-FS and q(E4A)-FS, that differed from q(L3A) and q(E4A) only by the deletion and insertion of a single nucleotide, respectively (Fig. 6A). To avoid changing the nucleotide sequences immediately adjacent to the two ATG codons, these mutations were introduced 7-11 base pairs upstream of the second ATG codon. Functional studies showed that the two frameshifted mutant alpha q constructs did not allow the synthesis of (mutant) alpha q subunits capable of interacting with the m2 muscarinic receptor (Fig. 6B). Consistent with this finding, no q(Delta 2-7) protein could be detected via Western blotting (Fig. 7). Taken together, these observations clearly exclude the possibility that the functional promiscuity of the N-terminal alpha q mutants studied here is due to leaky scanning and the use of the second methionine codon (codon seven) as a translation start site.

In conclusion, our data suggest a novel mechanism by which receptor/G protein coupling selectivity can be regulated. Elucidating the precise molecular mechanisms involved in this process should eventually lead to new insight into the complex processes governing the selectivity of receptor/G protein interactions.

    ACKNOWLEDGEMENTS

We thank Drs. B. R. Conklin and C. M. Fraser for generously providing us with different receptor and G protein expression plasmids.

    FOOTNOTES

* This work was supported by a grant from the Deutscher Akademischer Austauschdienst (NATO) (to E. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: NIDDK, Laboratory of Bioorganic Chemistry, Bldg. 8A, Rm. B1A-05, Bethesda, MD 20892. Tel.: 301-402-3589; Fax: 301-402-4182; E-mail: jwess{at}helix.nih.gov.

1 The abbreviations used are: GPCR, G protein-coupled receptor; DMEM, Dulbecco's modified Eagle's medium; IP1, inositol monophosphate; PLC, phospholipase C; qWT, wild type Galpha q; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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