Two Amino Acids within the alpha 4 Helix of Galpha i1 Mediate Coupling with 5-Hydroxytryptamine1B Receptors*

Hyunsu BaeDagger §, Theresa M. Cabrera-VeraDagger , Karyn M. Depree, Stephen G. Graber, and Heidi E. HammDagger parallel

From the Dagger  Institute for Neuroscience, Northwestern University, Chicago, Illinois 60611 and the  Department of Pharmacology & Toxicology, West Virginia University, Morgantown, West Virginia 26506

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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We previously reported that residues 299-318 in Galpha i1 participate in the selective interaction between Galpha i1 and the 5-hydroxytryptamine1B (5-HT1B) receptor (Bae, H., Anderson, K., Flood, L. A., Skiba, N. P., Hamm, H. E., and Graber, S. G. (1997) J. Biol. Chem. 272, 32071-32077). The present study more precisely defines which residues within this domain are critical for 5-HT1B receptor-mediated G protein activation. A series of Galpha i1/Galpha t chimeras and point mutations were reconstituted with Gbeta gamma and Sf9 cell membranes containing the 5-HT1B receptor. Functional coupling to 5-HT1B receptors was assessed by 1) [35S]GTPgamma S binding and 2) agonist affinity shift assays. Replacement of the alpha 4 helix of Galpha i1 (residues 299-308) with the corresponding sequence from Galpha t produced a chimera (Chi22) that only weakly coupled to the 5-HT1B receptor. In contrast, substitution of residues within the alpha 4-beta 6 loop region of Galpha i1 (residues 309-318) with the corresponding sequence in Galpha t either permitted full 5-HT1B receptor coupling to the chimera (Chi24) or only minimally reduced coupling to the chimeric protein (Chi25). Two mutations within the alpha 4 helix of Galpha i1 (Q304K and E308L) reduced agonist-stimulated [35S]GTPgamma S binding, and the effects of these mutations were additive. The opposite substitutions within Chi22 (K300Q and L304E) restored 5-HT1B receptor coupling, and again the effects of the two mutations were additive. Mutations of other residues within the alpha 4 helix of Galpha i1 had minimal to no effect on 5-HT1B coupling behavior. These data provide evidence that alpha 4 helix residues in Galpha i participate in directing specific receptor interactions and suggest that Gln304 and Glu308 of Galpha i1 act in concert to mediate the ability of the 5-HT1B receptor to couple specifically to inhibitory G proteins.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The interaction of heptahelical receptors with their cognate heterotrimeric guanine nucleotide-binding proteins (G proteins) represents an initial step in the transmission of extracellular signals across the plasma membrane (2-4). The receptor-G protein interaction modulates specific second messenger systems that result in a unique physiologic response to the extracellular signal. The particular downstream effect of G protein activation is not the result of an explicit interaction between each heptahelical receptor and a unique heterotrimeric G protein. On the contrary, G protein-coupled receptors have repeatedly been demonstrated to couple to several related members within the same family of G protein alpha  subunits, albeit with differing levels of efficiency (5-11). Clawges et al. (12) demonstrated that the serotonin (5-HT)1 1B receptor couples to heterotrimers containing either Galpha i1, Galpha i2, Galpha i3, or Galpha o. Nevertheless, this receptor does not couple to heterotrimers containing another member of this same family, the Galpha t subunit (12). Therefore, the 5-HT1B receptor represents one receptor system that can be exploited to investigate the precise molecular determinants governing selective receptor-G protein interactions.

Numerous biochemical studies have suggested that several subregions of Galpha (13-21) in addition to regions on Gbeta (20, 21) and Ggamma (22-25) may act in concert to determine selective receptor-G protein interactions. The carboxyl-terminal domain of Galpha subunits, in particular, has been demonstrated to play a key role in eliciting several specific receptor-G protein interactions. However, the carboxyl-terminal regions of Galpha i1, Galpha i2, Galpha i3, and Galpha t are highly homologous, and therefore, the carboxyl-terminal domain is not likely to be the primary determinant of 5-HT1B receptor-G protein selectivity between Galpha i/o and Galpha t. The selectivity profile of the 5-HT1B receptor has facilitated the use of Galpha i/Galpha t chimeras to map the residues that play a role in determining the specific interaction of the 5-HT1B receptor to inhibitory G proteins.

By using this approach, we previously demonstrated that substitution of the alpha 4 helix and alpha 4-beta 6 loop (amino acids 299-318) regions of Galpha i with the respective sequence from Galpha t markedly reduced the ability of this chimera to couple to the 5-HT1B receptor (1). These studies determined that the region corresponding to amino acids 299-318 in Galpha i1 plays a key role in determining the selective interaction between Galpha i1 and the 5-HT1B receptor (1). The intent of the present study was to define more precisely which amino acids within this domain are critical for selective 5-HT1B receptor coupling to inhibitory G proteins.

In addition to providing a useful model for receptor-G protein selectivity, the 5-HT1B receptor plays an important modulatory role in the central nervous system. 5-HT1B receptors are the primary terminal autoreceptors within the brain serotonin system (26). Activation of these receptors inhibits the release of 5-HT into the synaptic cleft (27, 28). In addition, drugs that selectively interact with 5-HT1B receptors have proven to be clinically useful for the treatment of migraine headache (29). Thus, deducing the molecular events that are essential to 5-HT1B receptor-catalyzed G protein activation may aid our understanding of both normal and pathologic processes in brain serotonin systems.

By utilizing a series of Galpha i/Galpha t chimeras coupled with site-directed mutagenesis, the present study reveals that alpha 4 helical residues Gln304 and Glu308 of Galpha i1 are critical determinants of 5-HT1B receptor coupling to inhibitory G proteins. This conclusion is supported by the observed marked reduction in receptor-catalyzed GDP/GTP exchange on Galpha i1 Q304K, E308L, and Q304K-E308L mutants. Moreover, whereas Gln304 and Glu308 are absolutely conserved among all Galpha i/o isoforms, they are divergent between Galpha i/o and Galpha t subunits. The crystal structure of Galpha i reveals that Gln304 and Glu308 are surface-exposed (30, 31), and mutation of these residues (Q304K and E308L) may alter the surface potential of the alpha  subunit. Hence, these residues may interact directly with the 5-HT1B receptor to mediate receptor coupling. Mutation of these residues may also indirectly influence the secondary structure of neighboring domains resulting in an inability of the 5-HT1B receptor to couple to the mutant inhibitory alpha  subunits.

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ABSTRACT
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Materials-- Nucleotides and enzymes were purchased either from Boehringer Mannheim or from Amersham Pharmacia Biotech. Serotonin was obtained from Sigma. [35S]GTPgamma S (1250 Ci/mmol) and [3H]5-HT (22-30 Ci/mmol) were purchased from NEN Life Science Products.

Construction of Galpha Mutant Genes-- The present study used the Escherichia coli expression vectors pHis6Galpha i1 or pHis6Chi3 that contain a nucleotide sequence encoding a hexa-histidine tag under the control of a T7 promoter (32). BamHI and HindIII digestion of pHis6Chi3 released a 450-base pair DNA fragment corresponding to amino acid residues 214-354 of Chi3. This fragment was subcloned into pUC19 (pUC19-Chi3). To construct Chi22, Chi24, and Chi25, duplex oligonucleotides containing the desired mutations were ligated into the NaeI- and BglII-digested pUC19-Chi3. Sequences were confirmed, and the correct pUC19-based chimeras were cloned into the pHis6Galpha i1 as a BamHI and HindIII fragment. Generation of the BamHI site in pHis6Galpha i1 did not mutate any residues. Site-directed mutagenesis of either Chi22 or Galpha i1 was carried out using the QuikChangeTM Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Either pHis6Galpha i1 or pHis6Chi22 served as the template for polymerase chain reaction-based mutagenesis.

Expression and Purification of Galpha i1 and Galpha Mutants in E. coli-- Hexa-histidine-tagged Galpha i1 or chimeric Galpha subunits were expressed in E. coli BL21(DE3) cells and purified as described (32) with minor modification. Briefly, cell pellets were resuspended in buffer A (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM MgCl2) supplemented with 50 µM GDP, 0.1 mM phenylmethylsulfonyl fluoride, and 5 mM beta -mercaptoethanol, sonicated, and then centrifuged at 100,000 × g for 60 min. The supernatant was loaded onto a Ni2+-nitrilotriacetic acid-agarose resin column (His-Bond, Novagen). Eluted samples were dialyzed overnight against buffer A in the presence of 20% glycerol, 0.1 mM phenylmethylsulfonyl fluoride, and 2 mM beta -mercaptoethanol and then further purified by high performance liquid chromatography (Waters Protein-Pak QHR-15, Waters Chromatography). Protein concentrations were determined using the Coomassie Blue method (33) with bovine serum albumin (Pierce) as the standard.

Expression and Purification of G Protein Subunits-- For affinity shift assays, the expression and purification of the G protein alpha  subunits in Sf9 cells was performed as described (34, 35) except that the final chromatography step was performed on 15-micron Waters Protein-Pak QHR (Waters Chromatography). Recombinant beta 1gamma 2 subunits were purified from Sf9 cells using a His6-gamma 2 as described by Kozasa and Gilman (36). The native retinal beta gamma subunits used for the GTPgamma S binding experiment were purified as described (37).

Preparation of Sf9 Membranes Containing 5-HT1B-expressed Receptors-- Sf9 cells were infected with recombinant baculovirus containing cDNA for the 5-HT1B receptor, cultured, and harvested as described (34). Membranes were prepared according to a previously published protocol (1). Briefly, harvested cells were thawed and resuspended in ice-cold homogenization buffer (10 mM Tris-HCl, pH 8.0, 25 mM NaCl, 10 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 20 µg/ml benzamidine and 2 µg/ml each of aprotinin, leupeptin, and pepstatin A) and burst by N2 cavitation. Cavitated cells were centrifuged at low speed; the supernatant was removed and then centrifuged at 28,000 × g for 30 min at 4 °C. The resulting pellets were resuspended in 5 mM NaHEPES, pH 7.5, containing 1 mM EDTA and supplemented with the aforementioned protease inhibitors. The membranes were washed twice and resuspended in the same buffer (1-3 mg of protein/ml).

Reconstitution of Receptors with Exogenous G Proteins-- Frozen Sf9 membranes were reconstituted as described (1). Briefly, membranes were pelleted and resuspended in reconstitution buffer (5 mM NaHEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 500 nM GDP, 0.04% CHAPS). G protein subunits were diluted in the same buffer, and the mixture was incubated at 25 °C for 15 min and then held on ice. The reconstitution mixture was diluted with binding assay buffer as described (1).

[3H]5-Hydroxytryptamine Binding Assay-- [3H]5-HT binding to 5-HT1B receptors reconstituted with Galpha and Gbeta 1gamma 2 was determined as described previously (1). To estimate receptor number in individual membrane preparations, the membranes were reconstituted with a large excess (5 µM) of G protein heterotrimers containing Galpha i1 and the 5-HT1B receptors labeled with 1, 10, and 40 nM [3H]5-HT. It has been shown that such reconstitution effectively converts all of the expressed receptors to a coupled, high affinity for agonist state (12). Bmax values were estimated by nonlinear regression analysis with GraphPad PRISM of the specific binding data using a fixed value (0.62 nM) for the high affinity KD (12). For affinity shift assays, high affinity binding to 5-HT1B receptors was determined using a single concentration of [3H]5-HT near the KD value for the radioligand (0.8-1.3 nM).

5-HT1B Receptor-stimulated GTPgamma S Binding Assay-- A GTPgamma S binding assay was used to quantitate receptor-catalyzed GDP/GTP exchange on Galpha subunits as described (1). Briefly, membranes were incubated with 1 mM AMP-PNP at 37 °C for 1 h, and receptor coupling was reconstituted with Galpha and Gbeta gamma subunits on ice in 70 µl of reaction buffer A (25 mM Hepes, pH 7.4, 5 mM MgCl2, 1 mM EDTA, 100 mM NaCl, 1 mM dithiothreitol) for 30 min and then diluted with 200 µl of reaction buffer A containing 150 nM GDP and 60 nM GTPgamma S. The addition of 30 µl of [35S]GTPgamma S (~7 × 106 cpm) initiated the reaction which was incubated at 25 °C. The following final concentrations were used for the GTPgamma S assay: 1.28 nM 5-HT1B, 40 nM Galpha , and 40 nM retinal beta gamma . For agonist activation, 1 µM of 5-HT was included. Aliquots (20 µl) were withdrawn at various times, and the reaction was terminated by filtration. Radioactivity retained on the filters was quantitated with a liquid scintillation counter. As the actual receptor densities and protein concentrations varied in different experiments, some degree of variation in the absolute level of GTPgamma S binding was observed. As rhodopsin can couple to either Gt or Gi heterotrimers with equal efficiency (32), GTPgamma S binding data were normalized to the percent of maximal binding that occurred after incubating samples for 30 min in the presence of an excess amount (500 nM) of light-activated rhodopsin.

Fluorescence Assay-- To ensure that recombinant Galpha proteins are properly folded, instrinsic fluorescence of the subunits was measured as described (1, 32). Briefly, the AlF4--dependent conformational changes of activated Galpha subunits were monitored by intrinsic tryptophan fluorescence changes with excitation at 280 nm and emission at 340 nm. The relative increase in fluorescence of 200 nM Galpha subunits was determined from absorbance readings before and after the addition of 10 mM NaF and 20 µM AlCl3.

Statistics-- Affinity shift activity data and the initial rates of GDP/GTP exchange (Table I) were analyzed separately using a one-way analysis of variance (ANOVA). Differences between groups were determined by a Newman Keuls' post hoc test only after the ANOVA yielded a significant main effect.

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ABSTRACT
INTRODUCTION
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The ability of 5-HT1B receptors to couple selectively to heterotrimers containing members of the Galpha i/o family of G proteins (12) facilitated the use of a series of Galpha i/Galpha t chimeras to determine which amino acids within Galpha i mediate this specific interaction. By using this approach, we previously demonstrated that Galpha i1 amino acid residues 299-318 (corresponding to the alpha 4 helix and alpha 4-beta 6 loop regions of Galpha i1) play a major role in determining the selective interaction with the 5-HT1B receptor (1). Additional residues in the amino-terminal domain of Galpha i1 were also determined to play a secondary role in 5-HT1B receptor coupling (1).

The alpha 4 Helical Domain of Galpha i1 Mediates the Ability of the 5-HT1B Receptor to Couple to Inhibitory G Proteins-- The present study initially used the same approach described above to identify which subdomain within this stretch of amino acids (Galpha i1 299-318) is critical for functional 5-HT1B receptor coupling to inhibitory G proteins. As shown schematically in Fig. 1, three chimeric Galpha i/Galpha t proteins were generated in which either the alpha 4 helix (Chi22) or portions of the alpha 4-beta 6 loop region (Chi24 and Chi25) of Galpha i1 were replaced with the corresponding sequence from Galpha t. Prior to the assessment of coupling activity, the ability of each chimeric Galpha subunit to bind GDP and undergo conformational change upon binding to GTP was tested by determining if the proteins undergo an AlF4--dependent increase in tryptophan fluorescence (32). This assay is based on the ability of AlF4-, which mimics the gamma -phosphate of GTP, to induce the active conformation, which then results in an increase in intrinsic fluorescence of Trp211 in Galpha i1. Tryptophan fluorescence of all mutants and Galpha i1 used in this study increased 40-45% upon the addition of AlF4- (Fig. 1), consistent with our previous results (1, 32).


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Fig. 1.   Secondary structure and AlF4--dependent tryptophan fluorescence change of Galpha subunits. Numbers above the wild type forms of Galpha i1 and Galpha t represent the corresponding residues in each respective alpha  subunit. Numbers above chimeric structures indicate the junction points of Galpha t and Galpha i1 sequences and refer to amino acid positions in Galpha t. *, diagram of secondary structural domains common to Galpha subunits. SI, SII, and SIII refer to the switch regions of Galpha . a Percent increase in tryptophan fluorescence in the presence of 10 mM NaF and 20 µM AlCl3 compared with basal (see "Experimental Procedures" for detail). The increase in tryptophan fluorescence indicates that all constructs were capable of undergoing conformational change in the presence of AlF4-. b As full-length Galpha t is not easily expressed or purified, this Galpha i1/Galpha t chimera was generated previously, crystallized, and shown to exhibit alpha t-like coupling activity (32, 51).

Functional coupling between the 5-HT1B receptor and the chimeric proteins was assessed by examining both the ability of the 5-HT receptor agonist, serotonin, to stimulate receptor-catalyzed GDP/[35S]GTPgamma S exchange on the alpha  subunit, and the ability of the chimeric protein to induce the high affinity agonist binding state of the receptor (affinity shift activity). The underlying principle of the affinity shift assay is that activated receptors can be converted to a high affinity agonist binding state by the appropriate G protein heterotrimers (12). By using a low concentration of agonist near the KD for the high affinity state and well below the KD for the low affinity state, the formation of a functional agonist-receptor-G protein complex is readily detected as an enhanced level of ligand binding. This assay can detect changes in coupling with either native or recombinant receptor and G protein preparations (12, 38-40).

As shown in Fig. 2A, 5-HT stimulation of the 5-HT1B receptor results in a time-dependent increase in [35S]GTPgamma S binding to recombinant Galpha i1. Substitution of the alpha 4 helix of Galpha i1 with the corresponding sequence from Galpha t (Chi22) dramatically reduces (-90%) the ability of the 5-HT1B receptor to couple to the chimeric G protein, as indicated by a marked reduction in the ability of 5-HT to stimulate [35S]GTPgamma S binding to the Chi22 alpha  subunit (Fig. 2B and Table I). In contrast, substitution of the alpha 4-beta 6 loop Galpha i1 residues 308-314 with the corresponding region from Galpha t (Chi24) failed to alter serotonin-stimulated receptor-catalyzed incorporation of [35S]GTPgamma S into the chimeric alpha  subunits (Fig. 2C and Table I). Interestingly, an overlapping chimera, Chi25, with Galpha i residues 305-314 substituted with the corresponding Galpha t residues shows a modest (28%) but significant (p < 0.05) reduction in the initial rate of GDP/GTP exchange (Table I).


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Fig. 2.   Time-dependent 5-HT1B receptor-catalyzed GDP/GTP exchange on Galpha i1 and Galpha i1/Galpha t chimeras. Membranes expressing the 5-HT1B receptor were reconstituted with either 40 nM Galpha i1 (A), Chi22 (B), Chi24 (C), or Chi25 (D) in the presence of 40 nM beta 1gamma 1. Data are expressed as the percentage of maximal GTPgamma S binding obtained in the presence of excess (500 nM) light-activated rhodopsin. Squares indicate GTPgamma S binding in the presence of 1 µM 5-HT (added at the 8.5-min time point), and triangles indicate the binding in the absence of agonist. Data represent the mean ± S.E. from three independent experiments.

                              
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Table I
Affinity shift activity and initial rates of GDP/GTP exchange
Affinity shift data represent the means ± S.E. from 3 to 18 independent experiments. Initial rate data represent the means ± S.E. from 3 independent experiments. Affinity shift and initial rate data were analyzed separately using a one-way analysis of variance followed by a Newman Keuls' post hoc test.

The coupling ability of these same chimeras (Chi22, Chi24, and Chi25) was also examined by agonist affinity shift activity. Consistent with the [35S]GTPgamma S experiments, the affinity shift activity of Chi22 was significantly reduced (-53%) in comparison to recombinant Galpha i1 (Fig. 3 and Table I). Likewise, as expected from the GTPgamma S data, the affinity shift activity of Chi24 did not significantly differ from Galpha i1. In contrast, although the affinity shift activity of Chi25 was reduced by 22%, this reduction did not reach statistical significance (Table I, Fig. 3). Taken together, these results localized the major molecular elements underlying the selective interaction of the 5-HT1B receptor with Galpha i1 to the alpha 4 helical domain.


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Fig. 3.   Affinity shift activity of Galpha i1/Galpha t chimeras with 5-HT1B receptors. Affinity shift activities refer to the -fold enhancement above buffer controls of high affinity [3H]5-HT binding to 5-HT1B receptors reconstituted with G protein heterotrimers containing the indicated alpha  subunits. Data represent the mean ± S.E. from 5 to 9 independent determinations using three separate membrane preparations where 5-HT1B receptors were expressed between 5.2 and 11.7 pmol/mg membrane protein. Exogenous G proteins were 1.3-3.3 µM during reconstitution and 45-112 nM during the binding assays which was a 35-50-fold molar excess over receptors. The concentration of [3H]5-HT was 0.8-1.3 nM in all experiments. Data were analyzed using a one-way ANOVA followed by a Newman Keuls' post hoc test as described under "Experimental Procedures." *, significantly lower than Galpha i1 (p < 0.05).

Amino Acid Residues Gln304 and Glu308 of Galpha i Are Critical Determinants of Coupling to 5-HT1B Receptors-- In order to map more precisely the specific amino acids that are critical for 5-HT1B receptor coupling to Galpha i1, systematic site-directed mutagenesis studies were conducted based on either Chi22 or Galpha i1 (Fig. 4). Within Chi22 there are 5 residues that are divergent between Galpha i1 and Galpha t: Galpha i1-A300G, A301N, Q304K, C305V, and E308L (Fig. 4). Previous studies demonstrated that mutation of Galpha i1 residue Ala300 to glycine did not impair coupling to the 5-HT1B receptor (1). Therefore, the present studies focused only on divergent residues between Galpha i1301 and Galpha i1308. The aim of the substitutions within Chi22 was to determine whether coupling to the 5-HT1B receptor could be restored by replacing Galpha t residues individually or in combination with the corresponding residues from Galpha i1. Mutants based on Chi22 (Chi22-N297A, Chi22-K300Q, Chi22-V301C, Chi22-L304E, and Chi22-K300Q-L304E) demonstrated varying degrees of receptor-catalyzed GDP/GTP exchange (Fig. 5A and Table I) and affinity shift activity (Fig. 5B and Table I). Similar to Chi22, Chi22-N297A exhibited only very weak coupling to the 5-HT1B receptor as exemplified both by the low levels of agonist-stimulated GTPgamma S binding (Fig. 5A, Table I) and by the low affinity shift activity of the mutant chimeras (Fig. 5B). Mutation of valine 301 to cysteine in Chi22 clearly failed to elevate the initial rate of GTPgamma S binding above Chi22 (Table I and Fig. 5A). Likewise, the affinity shift activity of this mutant did not significantly differ from Chi22. Mutants Chi22-K300Q and Chi22-L304E exhibited significant (p < 0.05) 2-4-fold increases in agonist-stimulated GDP/GTP exchange in comparison to the parent Chi22 (Table I). Consistent with these data, enhanced 5-HT1B receptor coupling was also observed with Chi22-L304E as measured by a significant 72% increase in the affinity shift activity over Chi22 (Fig. 5B). Although Chi22-K300Q caused a 42% increase in affinity shift activity over that observed for Chi22, this increase did not reach statistical significance. The critical nature of residues Gln304 and Glu308 is supported by the observation that the double mutant Chi22-K300Q-L304E completely restored GDP/GTP exchange and affinity shift activity to wild type Galpha i1 levels (Fig. 5 and Table I).


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Fig. 4.   Primary sequence alignment of the alpha 4-alpha 4/beta 6 loop region of bovine Galpha i1 (residues 296-318) and Galpha t (residues 292-314). Numbers immediately above the primary sequence of Galpha i1 correspond to residues in Galpha i1. Numbers immediately below the primary sequence of Galpha t correspond to residues in Galpha t. The box indicates the region of Galpha i1 that was substituted with the corresponding sequence from Galpha t to generate Chi22. Boldface letters indicate residues within Galpha t which diverge from Galpha i1 residues. Single or double mutations of Galpha i1 or Chi22 are listed below the sequence alignment.


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Fig. 5.   Functional coupling of the 5-HT1B receptor to Chi22 mutants. Membranes expressing the 5-HT1B receptor were reconstituted with the indicated Chi22 mutants and beta gamma subunits. A illustrates 5-HT1B receptor-catalyzed GDP/GTP exchange on Chi22 point mutants. Data are expressed as the percentage of maximal GTPgamma S binding obtained in the presence of excess (500 nM) light-activated rhodopsin. Curves depict the difference between the rates of GTPgamma S binding in the presence and absence of 1 µM 5-HT. Data shown represent the mean ± S.E. of three independent experiments. The initial rates of nucleotide exchange calculated from these data are shown in Table I. B depicts the affinity shift activity of Chi22 mutants with 5-HT1B receptors. Affinity shift activities refer to the -fold enhancement above buffer controls of high affinity [3H]5-HT binding to 5-HT1B receptors reconstituted with G protein heterotrimers containing the indicated alpha  subunits. Data represent the mean ± S.E. from 3 to 9 independent determinations using three separate membrane preparations where 5-HT1B receptors were expressed between 5.2 and 11.7 pmol/mg membrane protein. Exogenous G proteins were 1.3-3.3 µM during reconstitution and 45-112 nM during the binding assays which was a 35-50-fold molar excess over receptors. The concentration of [3H]5-HT was between 0.8 and 1.3 nM in all experiments. Data were analyzed using a one-way ANOVA followed by a Newman Keuls' post hoc test as described under "Experimental Procedures." *, significantly greater than Chi22 (p < 0.05); #, significantly greater than Chi22-K300Q.

To confirm and further support the contention that Galpha i residues Gln304 and Glu308 are particularly important determinants of 5-HT1B receptor coupling, single or double mutations were constructed in Galpha i1 (A301N, Q304K, C305V, and E308L). The aim of these experiments was to determine whether coupling to the 5-HT1B receptor could be reduced or eliminated by replacing these specific Galpha i1 residues with the corresponding residues from Galpha t. As illustrated in Fig. 6A and Table I, the single amino acid substitution Galpha i1-E308L markedly (-70%) and significantly (p < 0.05) reduced agonist-mediated stimulation of GTPgamma S binding to the mutant alpha  subunit. Likewise, mutation Galpha i1-Q304K resulted in a moderate (>40%; p < 0.05) reduction in agonist-mediated GTPgamma S binding in comparison to recombinant Galpha i1. Consistent with these data, the double mutant Galpha i1-Q304K-E308L exhibited an even greater reduction in agonist-mediated GTPgamma S binding than either single mutation alone (Fig. 6A and Table I). Galpha i1-C305V did not significantly alter coupling to the 5-HT1B receptor as evidenced by the lack of effect on agonist-stimulated GDP/GTP exchange with this mutant (Table I). Quite unexpectedly, Galpha i1-A301N resulted in a small (+12%) but statistically significant (p < 0.05) increase in the initial rate of GDP/GTP exchange in comparison to Galpha i1 (Table I). In contrast to the marked reductions in [35S]GTPgamma S binding observed with Galpha i1 mutants E308L, Q304K, and Q304K-E308L, no significant differences in affinity shift activities were observed between these mutants and Galpha i1 (Table I and Fig. 6B). Affinity shift activity also did not significantly vary between Galpha i1 and Galpha i1-A301N, C305V, or C305V/E308L (Table I).


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Fig. 6.   Functional coupling of the 5-HT1B receptor to Galpha i1 mutants. Membranes expressing the 5-HT1B receptor were reconstituted with the indicated Galpha i1 mutants and beta gamma subunits. A illustrates 5-HT1B receptor-catalyzed GDP/GTP exchange on Galpha i1 point mutants. Data are expressed as the percentage of maximal GTPgamma S binding obtained in the presence of excess (500 nM) light-activated rhodopsin. Curves depict the difference between the rates of GTPgamma S binding in the presence and absence of 1 µM 5-HT. Data shown represent the mean ± S.E. of three independent experiments. The initial rates of nucleotide exchange calculated from these data are shown in Table I. B depicts the affinity shift activity of Galpha i1 mutants with 5-HT1B receptors. Affinity shift activities refer to the -fold enhancement above buffer controls of high affinity [3H]5-HT binding to 5-HT1B receptors reconstituted with G protein heterotrimers containing the indicated alpha  subunits. Data represent the mean ± S.E. from 3 to 6 independent determinations using 2 separate membrane preparations where 5-HT1B receptors were expressed between 5.2 and 11.7 pmol/mg membrane protein. Exogenous G proteins were 1.3-3.3 µM during reconstitution and 45-112 nM during the binding assays which was a 35-50-fold molar excess over receptors. The concentration of [3H]5-HT was between 0.8 and 1.3 nM in all experiments. Data were analyzed using a one-way ANOVA followed by a Newman Keuls' post hoc test as described under "Experimental Procedures." There were no significant differences between the affinity shift activity of Galpha i1 and those of the mutant Galpha subunits.

This lack of correspondence between affinity shift activity and [35S]GTPgamma S binding data for the Galpha i1 mutants is likely to result from differences in the sensitivity of these assays stemming from technical aspects involved in these measures. For example, the initial exchange rates (Table I) are determined from linear regression analysis of the [35S]GTPgamma S binding data generated over the course of 10 min following the introduction of agonist to the assay (see Figs. 5A and 6A) with saturation of [35S]GTPgamma S binding occurring by 30 min (Fig. 2A). In contrast, as is required for radioligand binding assays, the affinity of [3H]5-HT for the 5-HT1B receptor is determined only after equilibrium is reached (i.e. following a 1.5-h incubation with the agonist) and in the absence of GTP. Therefore, if GDP release is impaired in the Galpha i1 mutants (as would be suggested by alterations in [35S]GTPgamma S binding) but not prevented, sufficient GDP could be released over the course of the experiment (1.5 h) such that at equilibrium the amount of high affinity receptors present in the preparation is similar for both Galpha i1 and the Galpha i1 mutants. Alternatively, the divergence between affinity shift activity and [35S]GTPgamma S binding for the Galpha i1 mutants may be indicative of a change in GTPgamma S binding in the absence of a change in GDP release. This situation could only arise if GTPgamma S binding (rather than GDP release) has become the rate-limiting step as a result of the Galpha i1-Q304K and Galpha i1-E308L mutations. If this were the case, we might expect to observe this same phenomenon with the Chi22-K300Q, Chi22-L304E, and Chi22-K300Q-L304E mutants, which we do not. Therefore, although the studies herein do not preclude the possibility of changes in GTPgamma S binding in the absence of a change in GDP release, further studies will be required to assess the likelihood of this potential outcome.

Nonetheless, despite the variations between the affinity shift data and the GDP/GTP exchange rates for the Galpha i1 mutants, the affinity shift activity appeared to show overall trends in the same direction as the [35S]GTPgamma S binding data. We, therefore, determined whether there was a correlation between affinity shift activity and the initial rates of GDP/GTP exchange as measured by [35S]GTPgamma S binding. Fig. 7 illustrates the correlative comparison between these two data sets. Correlation analysis yielded a Pearson correlation coefficient of 0.80, indicating a significant correlation between the GDP/GTP exchange rate and the affinity shift activity.


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Fig. 7.   Correlation between GDP/GTP exchange and affinity shift activity. The data plotted represent the means of each data set as reported in Table I. Filled circle represents the Galpha i1 control value; open circle represents Chi22; open squares represent Chi22 mutants; filled squares represent Galpha i1 mutants. Chi24 is represented by ×, and Chi25 is represented by an asterisk. The Pearson correlation coefficient was 0.80, representing a significant correlation between both data sets.

Computer Simulation of Double Point Mutations Reveal Potential for Direct Interaction of Gln304 and Glu308 with the 5-HT1B Receptor-- Whereas the present study demonstrates the critical role of two specific amino acids within the alpha 4 helical domain of Galpha i1, this mutagenesis approach could not assess whether the 5-HT1B receptor directly interacts with Gln304 and Glu308 or whether these residues have an indirect effect on receptor coupling as a result of structural changes that occur secondary to amino acid substitution. As shown in Fig. 8, both of these residues are potentially available for direct interaction with the 5-HT1B receptor. In fact, the residues might represent one contact point within a receptor-binding surface which also includes the carboxyl-terminal alpha -helix of Galpha i1 and perhaps the beta 6 strand (Fig. 8). To determine whether the Q304K-E308L mutation could alter the surface electrostatic potential of Galpha i1, we utilized the GRASP program (developed by A. Nicholls and B. Honig, Columbia University) to compare the mutant alpha  subunit (Galpha i1-Q304K-E308L) to native Galpha i1. The net molecular charge of Galpha i1 is -3, whereas the net charge of the mutant is -1. As shown in Fig. 9A, Galpha i1 residues Gln304, Glu308, and Thr321 produce a pocket of negative charge at the surface. The Q304K-E308L mutation of Galpha i1 changes the surface potential near these residues to a net positive charge. In addition, the surfaces immediately surrounding these residues are now more neutral than in native Galpha i1. The marked change in the surface potential as a result of these mutations may alter the strength of receptor contact with the alpha  subunit.


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Fig. 8.   Space filling model of the GTP bound form of Galpha i1 highlighting both known and hypothetical receptor contact sites on the Galpha subunit. Residues within the alpha 4 helical domain (purple), which were determined to be critical for 5-HT1B receptor coupling (Gln304 and Glu308), are colored orange to illustrate that these residues are surface-exposed and could potentially directly interact with the 5-HT1B receptor. A hypothetical receptor-binding site may also include portions of the beta 6 strand (green) as well as the alpha 5 helical-carboxyl-terminal domain (fuchsia). The guanine nucleotide (orange) is shown buried within the core of the alpha  subunit. The model was generated using INSIGHT II (Biosym Technologies, San Diego, CA) with the crystal structure coordinates from (30).


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Fig. 9.   Solvent-accessible surface of alpha  subunits colored according to the electrostatic potential. The solvent-accessible surface of GTPgamma S-bound Galpha i1 (30) and GDP-bound Chi6 (51) are shown in A and C, respectively. Using INSIGHT II (version 2.3.0 Viewer Module), computer-simulated models of the crystal structures of Galpha i1-Q304K-E308L and Chi6-K300Q-L304E were generated. The solvent-accessible surfaces of these mutants were then calculated using the GRASP program (developed by A. Nicholls and B. Honig, Columbia University). These models simulate the impact of the double mutation on the surface electrostatic potential of the alpha  subunit. The electrostatic potential is contoured in the range from -10kBT (red) to +10 kBT (blue) where kB is Boltzmann's constant and T is the absolute temperature (K). Amino acids are labeled based on their relative sequence positions (30, 31, 51, 52). C, carboxyl terminus.

Alternatively, rather than affecting direct contact sites with the receptor, the amino acid substitutions within Galpha i1 may have resulted in secondary structural changes in neighboring domains. The alpha 4-beta 6 loop region of Galpha i1 and the beta 6 strand are possible candidate domains for secondary disruptions consequent to amino acid mutation. The three-dimensional crystal structures of both Gt and Galpha i1 show that the conformations of the alpha 4 helix and alpha 4-beta 6 loop are almost identical between these subunits (Fig. 10A). Therefore, as the amino acid substitutions that were generated in the present study were substitutions of Galpha t residues for Galpha i1 residues, it is unlikely that these substitutions would have a marked effect on the secondary structure in this domain. This contention is supported by the fact that all mutant Galpha subunits examined in the current study remain capable of full activation by light-activated rhodopsin (data not shown). However, as illustrated in Fig. 10B, Gln304 of Galpha i1 forms a hydrogen bond with both the side chain carboxyl groups of Glu308 and with the gamma  hydroxyl group of Thr321. In contrast, Galpha t residue Lys300 forms a van der Waals interaction with the delta  carbon on Leu304. Substitution of Galpha i1 residues Gln304 and Glu308 with the corresponding amino acids from Galpha t (Lys300 and Leu304) results in the loss of strong side chain interactions (compare Galpha i and Galpha t contacts in Fig. 10B). This suggests that these mutations might weaken the interaction of the alpha 4 helical domain with the beta 6 strand resulting in an alpha  subunit structure that is less responsive to 5-HT1B receptor-mediated conformational change.


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Fig. 10.   Secondary structure and residue contact sites in Galpha i1 and Galpha t. Superimposed Calpha traces of alpha 4 helix through carboxyl-terminal residues (A) of free Galpha i-GTP (green) and Galpha t-GTP (blue). This overlay demonstrates that there is little difference between the secondary structure of Galpha i and Galpha t within this domain. In addition, the TCAT motif is "linked" to the carboxyl-terminal domain via the alpha 5 helix and to the alpha 4 helix via the beta 6 strand followed by the alpha 4-beta 6 loop. Ribbon representation of portions of the alpha 4 helix and beta 6 strand of free Galpha i1-GTP (B) and Galpha t-GTP (C). The side chains from Galpha i1 residues Gln304, Glu308, and Thr321 in addition to Galpha t residues Lys300, Lys304, and Ser317 are drawn as stick models to illustrate physical contacts of critical residues. Fuchsia residues represent oxygen atoms, and green residues represent nitrogen atoms. Dashed lines indicate intramolecular contacts between residues. Amino acids are labeled based on their respective sequence positions (30, 52). The GTP-bound alpha  subunits are illustrated because the GDP-bound form of Galpha i contains a microdomain that changes the conformation of the carboxyl terminus.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

By using Galpha i/Galpha t chimeras and site-directed mutagenesis, the present study determined that two residues (Gln304 and Glu308) within the alpha 4 helical domain of Galpha i1 are required for 5-HT1B receptor coupling to Galpha i1. These results are consistent with our previous work implicating the alpha 4 helix and alpha 4---beta 6 loop region of Galpha i1 in 5-HT1B receptor coupling to inhibitory G proteins. Taken together, these studies provide evidence for a previously unappreciated role for the alpha 4 helix of alpha  subunits in directing G protein-coupled receptor interactions.

Previously published work has shown that there are several receptor-binding regions present in heterotrimeric G proteins. The primary receptor recognition region is believed to be localized to the carboxyl-terminal domain of Galpha subunits (13-18), although at least three other regions in Galpha are involved in receptor interaction: the amino-terminal domain (18, 20, 41); the alpha 2 helix and alpha 2-beta 4 loop regions (16, 19); and the alpha 4 helix and alpha 4-beta 6 loop domain (1, 16, 42). In addition, segments of the beta  and gamma  subunits may contribute to the receptor interacting surface of heterotrimers (20-25). Whether individual G protein-coupled receptors interact simultaneously with several regions on heterotrimers and/or whether the profile of physical contacts for a particular receptor may direct more subtle features of specificity such as the efficiency of receptor coupling remains to be determined.

Other studies implicating the alpha 4 helix and/or alpha 4-beta 6 loop regions of Galpha in receptor interactions include studies from alanine scanning mutagenesis on Galpha t (16), patterns of evolutionary conservation (43), and tryptic digestion of Galpha t bound to rhodopsin (42). Interestingly, the recently resolved crystal structure of Galpha s by Sunahara et al. (44) indicates that although the overall structures of Galpha s and Galpha i are quite similar, the alpha 4 helix and alpha 4-beta 6 loop region varies between these subunits both in the length of the helical domain and the positioning of this region within the molecule itself (44). Thus the sequence divergence and structural differences between these Galpha 's is consistent with an important role in specific receptor recognition.

According to the crystal structure of Galpha i (30), in three-dimensional space Gln304 and Glu308 are situated on the same molecular surface as the carboxyl-terminal tail of the Galpha subunit which has been well established as a receptor-binding site (Fig. 8). The alpha 4 helix is connected to the carboxyl-terminal domain via the alpha 4-beta 6 loop, followed by the beta 6 strand and the beta 6-alpha 5 loop which contains a conserved guanine nucleotide binding motif TCAT (Fig. 10A). Several biochemical studies have reported that mutations within this TCAT motif dramatically decrease the affinity of the alpha  subunit for GDP (16, 45, 46). Therefore, one could hypothesize that the 5-HT1B receptor interaction with both the alpha 4 helical domain and the carboxyl-terminal region might trigger changes in the beta 6-alpha 5 loop. Upon agonist activation of the receptor, both domains may translate a conformational change to the TCAT motif resulting in a lowered affinity of GDP for the nucleotide binding pocket. Key mutations within either one of these domains (i.e. the alpha 4 helix or carboxyl terminus) may alter the transmission of the receptor-induced conformational signal to the TCAT domain and result in an inability to release GDP upon agonist binding.

Consistent with this idea, it has been suggested that the agonist-activated receptor interacts with the GDP-bound form of the heterotrimeric G protein, and the release of GDP from the Galpha subunit is the rate-limiting step in G protein activation (47-49). This guanine nucleotide free form of the G protein exists in a highly stable complex with the agonist-bound receptor in the absence of guanine nucleotides in the medium (50). Therefore, mutations of Galpha i that generate defects in agonist-activated receptor-catalyzed GDP release from the Galpha subunit should also result in a failure to establish the high affinity ternary complex of agonist, receptor, and G protein. In fact, as shown in Fig. 7, a good overall correlation does exist between the two measurements of GTPgamma S binding and affinity shift activity.

Sequence alignment and comparison of the primary structure between Galpha i/o family members reveals that alpha 4 helical residues Gln304 and Glu308 are absolutely conserved among the members of the Gi/o family of alpha  subunits shown in Fig. 11. In contrast, residues in the homologous position on Galpha t are different. The conservation of these critical residues across Galpha i1, Galpha i2, Galpha i3, and Galpha o members of the Gi/o family is consistent with the ability of the 5-HT1B receptor to couple selectively to heterotrimers containing any one of these members within this family of alpha  subunits (12). These data are also consistent with published studies indicating that the 5-HT1B receptor is incapable of coupling to heterotrimers containing Galpha t (1, 12). Clawges et al. (12) demonstrated that whereas Gi1, Gi2, Gi3, or Go can couple to the 5-HT1B receptor, these subunits exhibited a rank order profile of coupling efficiency (Gi3 approx  Gi1 > Go > Gi2) to the receptor. Together, the current data and previously published work suggest that residues other than those identified within the alpha 4 helical domain may mediate more subtle differences in coupling efficiency between G proteins within the same subfamily.


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Fig. 11.   Primary sequence alignment of alpha 4 helical region of Galpha subunits. Numbers above the sequences refer to amino acid positions in the context of Galpha i1. Boldface letters represent identical amino acid residues among Galpha i/o subunits. The circled amino acid residues in Galpha i1 are those that were determined to be critical for 5-HT1B receptor coupling. Notice that these amino acids are conserved among several members of the Galpha i/o family of alpha  subunits but diverge from the corresponding residues in Galpha t. bov., bovine; hum., human; cavpo., guinea pig.

In summary, 2 amino acids within the alpha 4 helix of the Galpha subunit play a key role in directing the specificity of 5-HT1B receptor coupling. These residues are essential for ensuring both the formation of the high affinity state of the receptor in the presence of agonist and receptor-catalyzed GDP/GTP exchange. Conservation of these residues across several members of the Gi/o family of alpha  subunits strengthens the importance of these residues in agonist G protein activation and suggests that other receptors that distinguish between Galpha i and Galpha t may utilize these residues as well. It remains to be determined whether these residues interact directly with the receptor or act indirectly by affecting the secondary structure of Galpha or the transmission of conformational changes to the GDP-binding pocket. Future work on the generalizability of these results to other Gi-coupled receptors will contribute to the understanding of the mechanism of receptor-catalyzed G protein activation and the nature of selectivity governing various cellular responses elicited by different biological stimuli.

    ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. Maria Mazzoni and Tarita Thomas for critically reading the manuscript and for providing editorial assistance. We also thank Drs. Tohru Kozasa and Alfred G. Gilman for kindly providing the baculovirus expressing His6-gamma 2.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant EY10291 (to H. E. H), American Heart Association Ohio-West Virginia Affiliate grant-in-aid (to S. G. G) and Research Fellowship Award 5T32CA70085-02 (to T. M. C.-V.).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.

The atomic coordinates and structure factors (code 1TND) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

parallel To whom correspondence should be addressed: Institute for Neuroscience, Northwestern University, 320 E. Superior, Searle Bldg., Rm. 5-555, Chicago, IL 60611. Tel.: 312-503-1109; Fax: 312-503-7345; E-mail: h-hamm{at}nwu.edu.

§ Current address: Dept. of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115.

    ABBREVIATIONS

The abbreviations used are: 5-HT, 5-hydroxytryptamine; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; AMP-PNP, adenosine 5'-(beta ,gamma imino)triphosphate; ANOVA, analysis of variance; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

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