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The Carboxyl Terminus of the G-Subunit Is the Latch for Triggered Activation of Heterotrimeric G Proteins

Institute of Pharmacology, Center of Biomolecular Medicine and Pharmacology, Medical University of Vienna (C.N., R.K., Q.Y., E.F., M.F.); and Boehringer-Ingelheim Austria GmbH, Vienna, Austria (H.A.)

Received July 16, 2005; accepted September 30, 2005

	Abstract

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The receptor-mimetic peptide D2N, derived from the cytoplasmic domain of the D₂ dopamine receptor, activates G protein -subunits (G_i and G_o) directly. Using D2N, we tested the current hypotheses on the mechanism of receptor-mediated G protein activation, which differ by the role assigned to the G-subunit: 1) a receptor-prompted movement of G is needed to open up the nucleotide exit pathway ("gear-shift" and "lever-arm" model) or 2) the receptor first engages G and then triggers GDP release by interacting with the carboxyl (C) terminus of G (the "sequential-fit" model). Our results with D2N were compatible with the latter hypothesis. D2N bound to the extreme C terminus of the -subunit and caused a conformational change that was transmitted to the switch regions. Hence, D2N led to a decline in the intrinsic tryptophan fluorescence, increased the guanine nucleotide exchange rate, and modulated the Mg²⁺ control of nucleotide binding. A structural alteration in the outer portion of helix 5 (substitution of an isoleucine by proline) blunted the stimulatory action of D2N. This confirms that helix 5 links the guanine nucleotide binding pocket to the receptor contact site on the G protein. However, neither the -subunit amino terminus (as a lever-arm) nor G was required for D2N-mediated activation; conversely, assembly of the G heterotrimer stabilized the GDP-bound species and required an increased D2N concentration for activation. We propose that the receptor can engage the C terminus of the -subunit to destabilize nucleotide binding from the "back side" of the nucleotide binding pocket.

An alternative hypothesis suggests that R^* may use a discrete "latch" on the surface of the -subunit and thus prompt GDP release from the "back side" of the nucleotide binding pocket (Sprang, 1997). There are several candidate regions, each marked by the receptor "footprint" [the carboxyl (C) terminus, the amino (N)-terminal extension, loops connecting helix 4 to strand 6 and 2 to 4, respectively, as well as side chains linking the N terminus to helix 1; cf. Conklin et al., 1996; Bae et al., 1997; Grishina and Berlot, 2000; Blahos et al., 2001; Herrmann et al., 2004], but which of these regions holds the latch is unclear.

On investigating the mechanism of receptor-catalyzed G protein activation one has to bear in mind that the reaction can be divided in two steps, namely, docking of the receptor to the G protein (coupling) and G protein activation (König et al., 1989; Franke et al., 1990; Onrust et al., 1997). If a structural alteration in any of the -subunit candidate regions reduced receptor-catalyzed activation, this would reflect uncoupling, and, in addition, might indicate that the activating latch had been undone. Hence, reconstitution with a full-length receptor will not allow to distinguish the activating latch from docking sites on the surface of the -subunit, except if they do not overlap (Onrust et al., 1997; Grishina and Berlot, 2000).

Using D2N, a receptor-mimetic peptide derived from the N-terminal portion of the third cytoplasmic loop of the D₂-dopamine receptor, we tested the current hypotheses of receptor-catalyzed G protein activation. D2N directly activates G proteins of the G_i/G_o family and reproduces the G protein selectivity of the D₂ receptor (Voss et al., 1993). There is an additional argument for concluding that the stimulatory activity of D2N is not an accidental property: D2N also represents the receptor segment that binds Ca²⁺-calmodulin (Ca²⁺/CaM), and Ca²⁺/CaM inhibits the regulatory action of the receptor and of D2N with identical affinity (Bofill-Cardona et al., 2000).

Our data suggest that the action of D2N can be integrated into a sequential fit-model where the G C-terminal tail interacts with R^* to catalyze guanine nucleotide exchange, and this occurs consecutively with the initial encounter between R^* and the G protein (Herrmann et al., 2004). Thus, interaction of a single cytoplasmic receptor loop with the -subunit C terminus is sufficient to enhance the guanine nucleotide exchange reaction; the G protein -subunit, although essential for efficient R/G coupling, is not required to open up the nucleotide exit pathway.

	Materials and Methods

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Materials. D2N and peptides derived from the same sequence were synthesized by the solid phase method as described previously (Voss et al., 1993). Peptides derived from the C terminus of G_o3 (CDIIIADNLRGCGLY; in G_i1, TDVIIKNNLKDCGLF) and G_q (CLQLNLKEYNLV) were gifts from Dr. Bernd Nuernberg (University of Duesseldorf, Duesseldorf, Germany).

The polyclonal antibodies sc-262 and sc-392 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). sc-262 is directed against the C-terminal peptide sequence in G_i3 (KNNLKECGLY), which is cross-reactive with G_i1 (KNNLKDCGLF); sc-392 is directed against 19 amino acids within the extreme C terminus of G_q/11. Radiochemicals were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). Restriction enzymes and adenine and guanine nucleotides were from Roche Molecular Biochemicals (Mannheim, Germany).

Plasmids were purified with the use of kits from QIAGEN (Valencia, CA), hexa-histidine-tagged proteins with a cobalt-chelating affinity matrix (Talon; BD Biosciences, San Jose, CA). Oligonucleotides were obtained from GenXpress (Maria Wörth, Austria); the integrity of the DNA constructs was verified by fluorescent sequencing. Pertussis toxin, calmodulin, and 5-(dimethylamino)-1-naphtalenesulfonyl (dansyl) chloride were obtained from Sigma-Aldrich (St. Louis, MO). All other chemicals and reagents were of the highest purity grade available.

Production and Purification of G Protein Subunits. Recombinant G protein -subunits were produced in Escherichia coli and purified from bacterial lysates. Myristoylated G_i1 was generated in the B-MT strain and purified from lysates essentially as described previously (Mumby and Linder, 1994). N-Terminally truncated versions of G_i1 were created by primer-directed amplification of the rat G_i1 cDNA; a hexa-histidine tag was appended to the C terminus by subcloning into a pQE-60 vector, and the E. coli strain BL21(DE3) was transformed with the respective constructs.

To generate N-terminally truncated versions of full-length G_i1, a KpnI restriction site was inserted 5' upstream to the ATG start codon (oligonucleotide sequences available on request); the N-terminal peptide sequence is rather divergent between subtypes up to position 35 in G_i1 from where a conserved sequence links the N-terminal extension to the G1 region. The reverse primer covered a BamHI restriction site present in the sequence of _i1 (codons 211-213).

The truncated constructs _i17 and _i30 could be expressed in E. coli to substantial amounts of protein (expression of proteins corresponding to _i27 and _i34 was not detected). Site-directed mutagenesis to introduce a proline in position 343 was performed on _i17 using the QuikChange kit from Stratagene (La Jolla, CA). Specific activity of G preparations was assessed by binding of [³⁵S]GTPS in the presence of 10 mM MgSO₄. Purification of G from porcine brain extracts was carried out essentially as described previously (Nanoff et al., 1997).

Binding of [³⁵S]GTPS, [-³²P]GDP, and Hydrolysis of [-³²P]GTP. For measuring concentration-dependent effects of D2N and D2N-derived peptides, G protein -subunit (1-10 pmol/assay, alone or mixed with purified brain G) was taken up in HEDL [50 mM HEPES-NaOH, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, and 0.1% Lubrol (detergent, formula not disclosed)], and a Mg²⁺ salt was added as indicated. After a short preincubation of G protein with peptide on ice, the binding reaction was started by the addition of warmed radioligand solution containing [³⁵S]GTPS (30 nM-1 µM final concentration; specific activity 1000 cpm/fmol at low and 8000 cpm/pmol at high GTPS concentrations) and terminated after 2 min (stop solution: 25 mM Tris-HCl, 10 mM MgCl₂, 100 mM NaCl, and 1 µM GTPS). Bound and free radioactivities were separated by filtration over nitrocellulose filters (BA 85; Millipore Corporation, Billerica, MA). Association and dissociation time courses were performed on 0.1 to 0.5 pmol of purified -subunit, and GTPS (1 µM) was used as the trapping ligand.

Release of prebound GDP and steady-state GTPase activity were determined essentially as described by Ross and Higashijima (1994). When measuring the concentration-dependent effect of D2N, dissociation of [-³²P]GDP was initiated by diluting [-³²P]GDP-loaded -subunit in HEDL buffer containing D2N at the indicated concentrations plus MgCl₂ (2 mM) and 100 µM GDP. The reaction was terminated after 3 min at 28°C with ice-cold stop buffer. Steady-state GTPase activity was measured using purified G protein -subunit (5-10 pmol/reaction), the indicated concentrations of D2N, and buffer (HEDL plus 2 mM MgCl₂). The reaction was started by the addition of substrate GTP (0.5 µM [-³²P]GTP, specific activity 100,000 cpm/pmol) and carried out for 2 min at 28°C in a volume of 50 µl. For the determination of concentration-dependent GTP turnover rates, between 0.2 (at a concentration of 30 or 100 nM GTP) and 1 pmol (at 300 or 1000 nM GTP) of -subunit was used. The assay volumes were adjusted to between 40 and 120 µl to allow for substantial excess of substrate over enzyme. The specific activity of [-³²P]GTP was between 20 and 200 cpm/fmol, and initial turnover rates were assessed by five determinations over a time course of 15 min.

Fluorescence Spectroscopy. Fluorescence emission was recorded in a Hitachi F4500 spectrofluorometer at 18°C. The assay volume amounted to 0.4 ml, composed of 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, and 0.01% Lubrol; G_i1 was included at a concentration of 0.3 µM. For recording changes in intrinsic tryptophan fluorescence, the excitation wavelength was 290 nm; five emission wavelength scans were recorded (scanning time 6 s) successively and averaged. Fluorescence changes of dansylated calmodulin were measured as described by Bofill-Cardona et al. (2000).

Miscellaneous Procedures. Native gel electrophoresis of calmodulin in complex with D2N or a D2N analog was carried out as described in Bofill-Cardona et al. (2000). Pertussis toxin-catalyzed ADP-ribosylation of G_i was done as in Nanoff et al. (1995). The final concentration of pertussis toxin was 10 µg/ml (0.13 µM).

	Results

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Activation of G by D2N. The 19-amino acid peptide D2N derived from the third cytoplasmic loop of the D₂-dopamine receptor regulates the activity of the G protein -subunit. Addition of the peptide enhances the release of GDP from the -subunit of G_i (Fig. 1A) and G_o (Fig. 1B). At a concentration of 1 µM D2N, the increment in the GDP dissociation rate was greater with G_i1 (from 0.024 ± 0.004 to 0.167 ± 0.031/min) than with G_o (from 0.10 ± 0.01 to 0.21 ± 0.04/min), but the stimulated release rates were comparable between the two subtypes.

View larger version (16K): [in this window] [in a new window]   Fig. 1. D2N stimulates the release of [-32P]GDP from Gi1 (A) and Go (B). Recombinant myristoylated -subunit (10 pmol) was incubated with [-32P]GTP (1 µM) for 30 min; dissociation of nucleotide was initiated by a 60-fold dilution in buffer with () or without () 3 µM D2N. The reaction was stopped at the indicated time points by the addition of ice-cold stop buffer with a fluoroalumino complex.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Concentration dependence of the effect of D2N. The GDP/GTP exchange reaction was assessed by measuring in parallel the dissociation of [-32P]GDP (; 3 min), the binding of [35S]GTPS (; 2 min), and the release of [32P]phosphate upon hydrolysis of [-32P]GTP (; 2 min). The experiments were carried out in the presence of 1 mM free MgCl2.

View this table: [in this window] [in a new window]   TABLE 1 Sequence and potency of D2N and D2N-derived peptides Sequence of D2N and peptides obtained by D2N sequence permutation. Arrows and asterisks (*) indicate the position of amino acid variation; asterisks mark the flanking residues of the putative CaM-binding motif, the amino acid positions of which are numbered in the top row. Right-hand columns give affinity estimates for the interaction with Gi1, Go, and with Ca2+-activated calmodulin. The EC50 values ± S.E.M.) were estimated from concentration-response curves in GDP release experiments (Gi1 and Go) or by fluorescence changes of dansyl-CaM.

Mg²⁺ exerts multiple effects on G protein activity. Very low (approximately nanomolar) concentrations are required for GTP hydrolysis; at higher concentrations Mg²⁺ supports receptor-catalyzed G protein activation and, in addition, may reduce the spontaneous GDP off rate (in G_o; Higashijima et al., 1987). We examined the way in which Mg²⁺ modulates D2N-mediated G protein activation. The results are compatible with a receptor-type mode of action and similar to those obtained with mastoparan (Higashijima et al., 1990).

If the guanine nucleotide exchange reaction was followed over time by measuring the association of GTPS (Fig. 3A), Mg²⁺ led to decreased association rates. These were observed both in the presence of D2N (k_app = 0.23 ± 0.05/min with MgCl₂ versus 0.48 ± 0.08/min without MgCl₂) and in its absence (0.035 ± 0.003/min versus 0.1 ± 0.03/min; n = 3 experiments carried out in parallel). However, inclusion of 1 mM MgCl₂ did not affect the -fold increase because of D2N. A second finding is that the addition of both D2N and Mg²⁺ enhanced the amount of GTPS binding (Fig. 3A, ). It is most likely that D2N—just as the activated receptor—reduced the Mg²⁺ concentration required for GTPS binding (Senogles et al., 1990).

View larger version (20K): [in this window] [in a new window]   Fig. 3. Interfering effects of Mg2+ and D2N on the binding GTPS. Association of GTPS with (A) and dissociation of GTPS from (B) Gi1 in the absence (open symbols) or presence of MgCl2 (1 mM free concentration, closed symbols). The experiments were performed with the addition of D2N (, fl; 1 µM) or without (, ); in the presence of Mg2+, GTPS was not released (data obtained without D2N not shown). The experiments shown are representative of three performed.

The inhibitory Mg²⁺ effect on guanine nucleotide exchange was further assessed in short-term (2 min) GTPS binding experiments. Figure 4 shows that activation by D2N was gradually reduced by increasing Mg²⁺; in the lower concentration range (10 mM), the inhibition was overcome by increasing D2N (Fig. 4A). If the data were plotted versus the Mg²⁺ concentration, it became evident that the apparent potency of Mg²⁺ was lower at higher D2N concentrations (Fig. 4B). In the absence of D2N, the inhibition occurred at less than 1 mM Mg²⁺, but in the presence of D2N higher concentrations were needed; inhibition was maximal at 10 mM Mg²⁺ (Fig. 4B). Thus, D2N reduced 1) the Mg²⁺-dependent inhibition of GDP release from G (see also Fig. 5B) and 2) the Mg²⁺ requirement for the binding of GTPS. Our data confirm what had been observed with mastoparan on G_o (Higashijima et al., 1990): Mg²⁺ is not necessary for the triggered release of nucleotide, but it is necessary for the conversion to the active GTP-bound form.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Short-term GTPS binding (2 min) to assess the inhibitory effect of Mg2+ on guanine nucleotide exchange. GTPS binding was determined at increasing concentrations of D2N and of Mg2+. The GTPS binding data are plotted versus the concentration of D2N (A) or versus the concentration of Mg2+ (B). The Mg2+ counter ion was , which was held constant at a concentration of 50 mM. The Mg2+ effects could not be reproduced with Ca2+ or Mn2+.

View larger version (23K): [in this window] [in a new window]   Fig. 5. Intrinsic tryptophan fluorescence of Gi1 recorded in the presence of increasing D2N. D2N-peptide was mixed in stepwise, in volumes of 0.1% of the final volume, and recordings were performed in 3-min intervals. A, emission traces from wavelength scans recorded upon the addition of D2N at the indicated concentrations (in micromolar). B, maximum emission (at 340 nm) from wavelength scans recorded with () or without () MgCl2 (10 mM). For the experiment depicted in the bottom inset, GDP was added to a final concentration of 25 µM (). The lower emission in the presence of GDP was due to UV absorbance by the guanine nucleotide.

Mutation of the D2N Sequence. The action of D2N cannot be accounted for by nonspecific stimulation because of the polybasic composition of the peptide. First, D2N did not stimulate G_s at concentrations up to 100 µM (data not shown). Second, the D2N-peptide sequence comprises a CaM-binding motif that conforms to a reversed 1-8-14 pattern; binding of CaM blocked the action of D2N and likewise that of the receptor (Bofill-Cardona et al., 2000). To differentiate binding to CaM from that to G, we mutated the motif-flanking residues in D2N (Table 1, positions 1 and 14 shaded) to alanine. In addition, neighboring basic residues were also replaced by alanine. Reducing the hydrophobicity invariably lowered the affinity for G, both G_i1 and G_o (peptide 2A in Table 1) but not for CaM (data not shown). Likewise, arginine in position 2 was crucial with peptide 3A displaying a further 3-fold reduction in apparent G protein affinity. In contrast, substituting the lysine residue in position 13 (peptide 4A) or lysine in position 3 (peptide 4A') did not result in an additional decrease in affinity (cf. peptide 2A). The affinity of all D2N-derived peptides for Ca²⁺/CaM (assessed by fluorescence change of dansyl-CaM, Table 1; and by native gel electrophoresis of peptide CaM complexes, data not shown) was comparable with—or even higher than—that of the parent peptide (Table 1). The affinity for CaM only dropped by >10-fold when an aspartate residue had been introduced to replace leucine in position 8 (data not shown).

Monitoring the D2N-Induced Conformational Switch by Fluorescence Spectroscopy. G_i1 contains three tryptophan residues. One is located in the switch II region (W²¹¹), the fluorescence of which is sensitive to the conformational state (summarized by Sprang, 1997). Fluorescence emission increases upon binding of Mg-GTP and thus reflects the active state. Conversely, the release of prebound GDP is expected to affect the conformation of the switch II region. As shown in Fig. 5, the action of D2N was detectable by recording the intrinsic tryptophan fluorescence of G_i1. Addition of D2N led to a concentration-dependent decline in fluorescence emission, indicating that the tryptophan residue became more exposed to the aqueous solvent (Fig. 5, A and B). The emission data could be fitted to a monophasic curve where the half-maximal decrement was reached at a concentration of 0.5 µM (Fig. 5B, ). The decrease in tryptophan fluorescence is likely to result from the conformational change that favors GDP release. Therefore, the D2N effect was abolished by the inclusion of a high concentration of GDP (Fig. 5B, bottom inset). As predicted by the experiments shown in Figs. 3 and 4, the addition of Mg²⁺ resulted in a right shift of the concentration-response curve (Fig. 5B, ). When titrating the effect of D2N, we made sure that fluorescence emission was stable before each recording. We can also rule out that longer incubation times in the presence of D2N led to denaturing of the protein because a 1-h preincubation with D2N at 18°C followed by the addition of GTPS gave a robust increase in tryptophan fluorescence (data not shown).

Role of the -Subunit N Terminus and of G in D2N-Mediated G Protein Activation. The N terminus of the -subunit is thought to have an important part in receptor interaction and has been implicated in triggered G protein activation. Both a receptor-mimetic peptide isolated from the third loop of the ₂-adrenergic receptor (₂i3c in Tables 2 and 3; Taylor et al., 1994) and mastoparan (Higashijima and Ross, 1991) were cross-linked to the N terminus of the -subunit; moreover, for activation by mastoparan the N terminus was essential. Because of its flexibility, the N-terminal extension has also been integrated as a lever-arm into the model of receptor-mediated G protein activation (Iiri et al., 1998; Cherfils and Chabre, 2003). We have therefore tested whether the -N terminus was required for the D2N-mediated guanine nucleotide exchange. The experiments depicted in Fig. 6 compared G_i1 to an N-terminally truncated protein (_i30) in which the entire subtype-specific N terminus had been deleted; in addition, _i30 lacked the N-terminal myristate and was engineered to include a C-terminal hexa-histidine tag. The truncated protein demonstrated a basal exchange rate virtually identical (0.041/min) to that of full-length myristoylated G_i1. As shown by two representative experiments using GTP turnover (Fig. 6A) and fluorescence emission (compare Fig. 6B with 5B), _i30 was activated by D2N in a manner indistinguishable from that of the full-length protein. Average maximal activation of GTP turnover was similar in _i30 (8.9 ± 1.3-fold over basal, means ± S.E., three experiments performed in parallel) and full-length G_i1 (6.9 ± 1.3). Thus, we did not find any appreciable defect upon N-terminal truncation of the protein.

View this table: [in this window] [in a new window]   TABLE 2 Alignment of peptide activator sequences Sequences of peptide G protein activators derived from the third (i3) or second (i2) cytoplasmic loop of the 2-adrenergic, D2 dopamine, and M4 muscarinic receptor of mastoparan (MP) and of a peptide fragment of the insulin-like growth factor II receptor (IGF II-R). Segments taken from the C-terminal loop portion are given in the inverse orientation (cn); similarity to D2N (D2i3n) is remote for the amino acids indicated by bold text.

View this table: [in this window] [in a new window]   TABLE 3 Activation characteristics of activator peptides G protein activation characteristics of peptide activator sequences. "G required" indicates whether addition of G was needed for full stimulation by the peptide.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Truncation of the Gi1 N terminus by 30 amino acids. A, D2N-mediated activation of the guanine nucleotide exchange in the truncated -subunit (), compared with full-length myristoylated Gi1 (). Shown are typical activation patterns determined by [32P]phosphate release. B, D2N-induced decrease of the intrinsic tryptophan fluorescence of i30, determined as in Fig. 5 and in the absence of Mg2+. The experiment shown was repeated once with similar results.

According to the lever-arm and gear-shift models, the receptor uses G to open the guanine nucleotide binding pocket and to release GDP. D2N, however, can act on G alone, and, in fact, when G is added to the reaction, it suppresses the stimulatory effect of D2N. Figure 7 shows GTPS binding experiments performed on the heterotrimer. To rule out that an effect by G is caused by scavenging of the peptide by (or contaminants), we examined the D2N effect on the heterotrimer composed of full-length G_i1 (Fig. 7A) and on the heterotrimer composed of an N-terminally truncated version (_i17; Fig. 7B). By gauging the effect of G on pertussis toxin-mediated ADP-ribosylation, we found that the affinity of _i17 for G was lower than that of the full-length protein by approximately an order of magnitude but sufficient to support heterotrimer formation (data not shown; cf. Graf et al., 1992). If purified G was included, activation by D2N at low concentrations was reduced. With full-length G_i1, this inhibition was observed at an excess () and a substoichiometric amount of G (). The concentration-response curve shifted to the right even when the concentration of was low. If instead truncated _i17 was used, G caused a shift only at an excess () but not a substoichiometric amount (Fig. 7B,). Hence, the affinity of the -subunit for G determined the decrease in sensitivity to D2N; stabilization of the inactive state was overcome by increasing D2N.

View larger version (19K): [in this window] [in a new window]   Fig. 7. G inhibits D2N activation of G. Gi (A, Gi1; B, i17) was combined with a buffer control (HEDL plus 2 mM MgCl2; ) and a substoichiometric (0.8;) and an excess (3-fold; ) molar amount of G, and D2N-stimulated [35S]GTPS binding was measured for 2 min. Mean values (± S.E.M.) are representative of four experiments.

Mapping the D2N Binding Site. The C-terminal end—at least the last five amino acids—of G_i are required for recognition by pertussis toxin (Scheuring et al., 1998), which uses the cysteine residue at position -4 as the substrate for ADP-ribosylation. We exploited the ADP-ribosylation reaction to examine whether in the heterotrimer, D2N bound to the G C terminus. If so, D2N should diminish ADP-ribosylation by pertussis toxin; Fig. 8A shows that this was indeed the case. D2N decreased ADP-ribosylation—in the presence of excess G—of both full-length and truncated i17.

View larger version (29K): [in this window] [in a new window]   Fig. 8. D2N binds to the -subunit C terminus. A, D2N inhibits pertussis toxin-mediated ADP-ribosylation. Incubation with pertussis toxin (10 µg/ml) was carried out with Gi1 (0.6 pmol; A) or i17 (2 pmol; B) combined with G, which was in excess over (/i1, 3; /i17, 5). The + indicates inclusion of D2N (3 µM). Band representation was electronically adjusted—according to the staining obtained in the absence of D2N—to approximately comparable levels with i1 and i17. The experiment was repeated once with similar results. B, antibody blockage of the C terminus. GTPS binding was determined in the absence () or presence of a polyclonal antibody (, ) directed against the Gi C terminus (for the epitope, see Materials and Methods). The antibody was added at two concentrations, and the amount of IgG was held constant throughout at a concentration of 20 ng/ml by complementation with irrelevant IgG. The experiment shown is representative of three performed. C, inhibition of D2N by a C-terminal peptide derived from Go3 () and from Gq (). Short-term GTPS binding to Gi1 (0.5 pmol) was assayed in the presence of 1 µM D2N and of the C-terminal peptides at the indicated concentrations. The experiment was repeated once.

Helix 5 connects the G C terminus to the guanine nucleotide binding pocket. When bound to the receptor, the extreme C-terminal residues adopt an -helical conformation (Kisselev et al., 1998). Thus, they can be viewed as a continuous extension of helix 5. A kink engineered in the outer portion of helix 5 (by substituting proline for an isoleucine) disrupted rhodopsin activation of transducin (Marin et al., 2002). This suggests that the C-terminal helix is a key structure in receptor-dependent activation.

To test whether D2N—upon binding to the extreme C terminus—prompts guanine nucleotide exchange by a rhodopsin-related mechanism, we mutated an analogous isoleucine (at position 343) in G_i1. As can be seen in Fig. 9A, the mutant G_i1 was less sensitive to D2N, but the affinity was unchanged compared with an -subunit bearing the wild-type sequence. Maximal activation of the turnover rate (determined by GTPase assays) was 6.2-fold over basal (95% confidence interval = 5.1 to 7.3) with the original sequence and 3.9-fold with the mutant (95% confidence interval = 3.1 to 4.7; n = 3 experiments performed in parallel). Thus, the proline-induced structural alteration in 5 reduced activation by D2N in a manner that could not be overcome by increasing D2N.

View larger version (18K): [in this window] [in a new window]   Fig. 9. D2N engages helix 5 to stimulate the guanine nucleotide exchange. A, effect of a mutation in helix 5 (I343P) on D2N activation. Shown are typical D2N concentration-dependent GTPS binding patterns for i17 () and i17P343 (); the experiment was carried out for 2 min at a final concentration of 1 µM GTPS and 1 mM free Mg2+. B, double reciprocal transformation of the GTP hydrolysis rates obtained with i17 and i17P343 in the absence of D2N. Initial-rate constants were determined at four GTP concentrations in two independent experiments. The Km value for GTP is estimated from the regression line intercept with the x-axis.

	Discussion

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The -Subunit C-Terminal Region Transmits the Activating Impulse. In the present report, we show that D2N, a peptide from the N-terminal portion of the third loop of the D₂ dopamine receptor, directly activated G protein -subunits by engaging the C terminus of G and thereby triggering the conformational switch. D2N promoted the guanine nucleotide exchange reaction and led to a decline in the intrinsic tryptophan fluorescence of G_i. The D2N binding site was delineated by antibody and peptide competition experiments; in addition, a mutation introduced into the C-terminal helix (5) blunted D2N stimulation. The mutation was 12 positions removed from the end of the -polypeptide, and it did not affect the apparent potency of D2N. From this, we infer that D2N only bound to the extreme C terminus, which was consistent with the inhibition of pertussis toxin and the G protein subtype selectivity retained in the peptide (Conklin et al., 1996). Our conclusion is that the C terminus of G_i1—in addition to representing a key to selective G protein recognition—exposes an activation latch; this is supported by the observation that also peptides with divergent sequences (Table 2, mastoparan and IGF-II receptor) and nonpeptide G protein activators (Breitweg-Lehmann et al., 2002) require the -C terminus for activation. However, we refrained from testing a C-terminally truncated version of G_i1 because the effect of C-terminal truncation on nucleotide binding and protein stability is uncertain but may be deleterious (see below).

An involvement of the entire C-terminal helix (i.e., the C terminus and helix 5) in receptor-catalyzed G protein activation was invoked because of the effect of mutations within the G5-box; these are C325S in G_o (Thomas et al., 1993) and A326S in G_i1 (Posner et al., 1998). The G5-box is contained in the loop that precedes helix 5, connecting it to strand 6, and lies adjacent to the guanine ring of the bound nucleotide. The G5 mutations resulted in reduced affinity for GDP but no change in GTPS binding, thus resembling the receptor-activated state of the G protein. Moreover, helix 5 per se is thought to impinge via lattice forces on nucleotide binding (Denker et al., 1995). Substitution of buried (but not solvent-exposed) residues caused enhanced spontaneous activation of Gt (Muradov and Artemyev, 2000; Marin et al., 2001) as did a truncation by the last 14 amino acids in G_o. In G_i2, the same truncation, however, abolished the GTP-induced conformation (Denker et al., 1995). In keeping with this, the proline substitution in the outer portion of helix 5 in G_i1 caused a decreased affinity for GTP, but it did not affect the intrinsic exchange rate. Because the structure of helix 5 is linked to the conformation of the binding pocket, it seems possible that D2N enhanced guanine nucleotide exchange by displacing the C-terminal helix relative to its surrounding. A straightforward explanation is that this motion led to a rearrangement of the G5-box, which mediates the activating effect.

Role of G in D2N-Mediated G Protein Activation. According to the current models of receptor-catalyzed G protein activation, the receptor has to engage the - and -subunit to release GDP (Kisselev et al., 1999; Rondard et al., 2001; Cherfils and Chabre, 2003). However, the requirement for G in receptor/G protein coupling is not absolute; we have previously demonstrated that productive coupling can take place—albeit with low efficiency—between a receptor and its cognate -subunit (Freissmuth et al., 1991; Jockers et al., 1994). Likewise, for activation by D2N G was dispensable. Among the proposed models, only the sequential fit model states that a specific contact to the -subunit (i.e., its C terminus) is sufficient for activation; prior contact to G is thought to make the -C terminus available to the receptor (Herrmann et al., 2004). Thus, D2N mimics the effect of the activated receptor by forming this specific contact and recapitulates the second step in the reaction sequence. The effect of D2N on either the isolated -subunit or the holotrimer, however, failed the predictions of the lever-arm and gear-shift models because these unconditionally rely on G for guanine nucleotide displacement.

A canonical role of G in the unstimulated heterotrimer is to stabilize the inactive conformation. Therefore, combining G with G_i1 reduced the sensitivity to D2N and required higher D2N concentrations for activation. Our data suggest that, in theory, inhibition by G would be overcome if the receptor's binding to the C terminus were governed by very high affinity. It is unlikely, however, that receptors have evolved an excessive affinity toward the -C terminus as this would limit the catalytic efficiency of the receptor (Herrmann et al., 2004). Otherwise, the full-length receptor has to interact with the holotrimer by accessory contacts (on the - or -subunit) in a way that relieves the inhibition.

Alignment of the D2N Sequence. The sequence of D2N compares with some of the receptor-mimetic peptides that directly activate G proteins of the G_i/o subfamily (Table 2). The peptides share a length of 10 to 20 amino acids and an amphiphilic, cationic structure. Structural similarity may be detected upon inspection of the sequences present in the i3 loop of the M₄-muscarinic and the ₂-adrenergic receptor (Table 2). The modeled G binding site of pertussis toxin also exhibits sequence similarity to the N-terminal half of the D2N. This suggests that D2N forms bonds with the last two amino acids of the -subunit (Scheuring et al., 1998; data not shown). By contrast, we were unable to find a similarity to peptides derived from the i2 loops (of the M₄- or ₂-receptor), to mastoparan or to the regulatory sequence derived from the IGF-II receptor. All of these peptides preferentially activated Gi and Go with the exception of the short ₂i3n-peptide; ₂i3n, however, is a nonselective activator with low potency, obviously because of the lack of flanking amino acid residues, which are present in D2N, in ₂i3c and in M₄i3c peptides.

Because the alignment is improved, the sequences of M₄i3c and ₂i3c are rendered in the C- to N-terminal orientation (cn). The similarity is concentrated within eight amino acids in the C-terminal half of D2N where mutations were disruptive to D2N activity (e.g., in 3A, 4A'; Table 1). Conversely, N-terminal truncation of the ₂i3c peptide was systematically more deleterious to its activity than several truncations from the C-terminal end (Wade et al., 1996). This indicates that the congruent portion is oriented away from the plasma membrane, toward the G protein interface when the peptides are inherent to the i3 loop of the receptor.

The properties of D2N could not be predicted in detail from the experimental characteristics of other regulatory sequences (Table 3). The distinctions are as follows. 1) D2N-activated guanine nucleotide exchange independently of G; if this had been assessed, G typically was found to assist activation by receptor-mimetic peptides. 2) The -subunit N terminus was not required for the binding of D2N but was a docking site for the ₂i3c peptide and mastoparan. 3) The activating potency of D2N (D₂i3n) was high, comparable only with that of the M₄-peptides.

In spite of the similarities between D2N and other third cytoplasmic loop peptides in amino acid sequence, subtype-selective action, and orientation, the diversity of activator sequences in general is large (Tables 2 and 3). This is not new if one considers that among G protein-coupled receptors the cytoplasmic domains are unrelated. The question therefore is how different receptor peptides align on the surface of the G protein to operate the C-terminal latch; several configurations must be possible. For example, it has been speculated that those peptides that require G for G protein activation need to bind to G for proper alignment (Taylor et al., 1994). However, D2N possibly represents a sequence optimized to bind to the C terminus of G_i and G_o. If available, the structure of D2N bound to the G protein -subunit could be used to delineate the molecular coordinates of peptide- and receptor-mediated G protein activation.

	Footnotes

 
This study was supported by grants P14273 [GenBank] and P16083 [GenBank] from Fonds zur Förderung der Wissenschaftlichen Forschung, the Austrian Science Fund.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.016725.

ABBREVIATIONS: Ca²⁺/CaM, Ca²⁺-calmodulin; GTPS, guanosine 5'-O-(3-thio)triphosphate; IGF-II, insulin-like growth factor-II; HEDL, HEPES-NaOH/EDTA/dithiothreitol/Lubrol.

Address correspondence to: Dr. C. Nanoff, Medical University of Vienna, Institute of Pharmacology, Waehringer Strae 13, A-1090 Vienna, Austria. E-mail: christian.nanoff{at}meduniwien.ac.at

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