©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Mapping of Effector Binding Sites of Transducin -Subunit Using G/G Chimeras (*)

(Received for publication, September 18, 1995; and in revised form, October 23, 1995)

Nikolai P. Skiba Hyunsu Bae Heidi E. Hamm (§)

From the Department of Physiology and Biophysics, College of Medicine, University of Illinois, Chicago, Illinois 60612-7342

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The G protein transducin has been an often-used model for biochemical, structural, and mechanistic studies of G protein function. Experimental studies have been limited, however, by the inability to express quantities of mutants in heterologous systems with ease. In this study we have made a series of Galpha(t)/Galpha chimeras differing at as few as 11 positions from native Galpha(t). Ten chimeras are properly folded, contain GDP, can assume an AlF(4)-induced activated conformation, and interact with beta(t) and light-activated rhodopsin. They differ dramatically in their affinity for GDP, from G(i)-like (initial rates 225 µmol/mol s) to G(t)-like (initial rates 4.9 µmol/mol s).

We have used these chimeras to define contact sites on Galpha(t) with the effector enzyme cGMP phosphodiesterase. Galpha(t)GTP but not Galpha(t)GDP activates it by removing the phosphodiesterase (PDE) inhibitory subunit. In solution, Galpha(t)GTP interacts with PDE (K 12 nM), while Galpha(t)GDP binds PDE more weakly (K 0.88 µM). The interaction of Galpha(i)GDP with PDE is undetectable, but Galpha(i)GDP-AlF(4) interacts weakly with PDE (K 2.4 µM). Using defined Galpha(t)/Galpha(i) chimeras, we have individuated the regions on Galpha(t) most important for interaction with PDE in the basal and activated states. The Galpha(t) sequence encompassing alpha helix 3 and the alpha3/beta5 loop contributes most binding energy to interaction with PDE. Another composite P interaction site is the conserved switch, through which the GTP-bound Galpha(t) as well as Galpha interact with P. Competition studies between PDE and truncated regions of PDE provide evidence for the point-to-point interactions between the two proteins. The amino-terminal 1-45 segment containing the central polycationic region binds to Galpha(t)'s alpha3 helix and alpha3/beta5 loop, while the COOH-terminal region of P, 63-87, binds in concert to the conserved switch regions. The first interaction provides specific interaction with both the GDP- and GTP-liganded Galpha(t), while the second one is conserved between Galpha(t) and Galpha and dependent on the activated conformation.


INTRODUCTION

G proteins are key intermediates in signal transduction by a large class of G protein-coupled receptors that respond to sensory, hormonal, and neurotransmitter signals in the environment. The important roles of G proteins in shaping the specificity and temporal features of cellular responses to a variety of signals are under intense investigation (1, 2, 3, 4) . Receptor-mediated GTP/GDP exchange on G protein alpha subunits regulates the production of both activated GalphaGTP and free Gbeta subunits, which in turn regulate the activity of a variety of effector enzymes and channels; GTP hydrolysis by Galpha then determines the turn-off of the response. Four classes of mammalian Galpha subunits with sequence homology from 45 to 80% along with an unknown number of betas (five beta and at least eight genes) (5, 6) make up heterotrimeric G proteins that interact with a large number of different receptors and a smaller number of effectors(7, 8, 9) .

One of the best studied G protein-mediated transmembrane signaling cascades from the physiological, biochemical, and structural points of view is the visual transduction system of rod outer segments. The abundance of the signaling proteins including the receptor, rhodopsin, the G protein, transducin, and the effector, cGMP phosphodiesterase, has led to a range of studies that makes them prototypical members of these classes of signaling proteins(10, 11, 12, 13) . Another reason for the intense scrutiny of this signaling system is that a single species of receptor, G protein, and effector is present, so the confounding influence of closely related isoforms in other cell types is eliminated. A third reason for the fascination with the rod visual signal transduction system is its highly specialized function as a single photon detector and consequent high sensitivity(14) .

To understand the molecular details of interaction between Galpha(t) and its partners Gbeta(t), rhodopsin, and cGMP phosphodiesterase, relatively large amounts of recombinant functionally active Galpha(t) are required for mutational analysis of the molecule and functional testing in biochemical assays. Numerous unsuccessful attempts to express functional Galpha(t) in various expression systems have been made. The only expression of functional Galpha(t) has been reported by Faurobert et al.(15) in the Sf9/baculovirus system, but protein yields were relatively low (50 µg/liter). The Escherichia coli expression system to express functional Galpha(t) was tried extensively in several labs without success. A high yield of insoluble protein was obtained, mainly in inclusion bodies. Refolding of recombinant Galpha(t) from urea-solubilized material did not result in proper protein folding.

In this work we have obtained functionally expressed Galpha(t) variants using a chimera approach. Galpha, with 68% homology of amino acid sequence, has a very similar overall three-dimensional structure (19) to Galpha(t)(16, 17, 18) , although functionally, the two proteins couple different receptors to different effectors. The fact that recombinant Galpha is soluble and functional (20) , but recombinant Galpha(t) is not, provides a strategy to ``rescue'' recombinant Galpha(t) by systematic replacement of Galpha(t) regions, which inhibit proper folding, with Galpha regions, which are permissive for proper folding. We have constructed a set of Galpha(t)/Galpha chimeras, which are soluble and functional and express to high levels in E. coli. The most Galpha(t)-like chimera contains only 11 amino acids different from native Galpha(t) and is functionally almost indistinguishable from Galpha(t).

We have used these chimeric Galpha(t)/Galpha proteins to investigate the structural basis of high affinity interaction of Galpha(t) with its effector, cGMP PDE. (^1)The inactive retinal rod cGMP PDE is composed of two membrane-anchored catalytic subunits, alpha (99.2 kDa) and beta (98.3 kDa), and two identical inhibitory subunits (9.7 kDa)(21, 22, 23, 24, 25) . Normally, PDE becomes activated when light-activated rhodopsin activates GTP/GDP exchange on G(t). Galpha(t)bulletGTP interacts with phosphodiesterase and activates it by displacing P from inhibitory site on catalytic subunits Palphabeta(26, 27) . The activated PDE rapidly hydrolyzes cytoplasmic cGMP, resulting in closure of plasma membrane cationic channels and hyperpolarization of the rod cell (10) .

Sites on Galpha subunits of effector interaction have been investigated for both Galpha(t) and Galpha(s). We have demonstrated that the synthetic Galpha(t) peptide corresponding to residues 293-314 activates PDE (28) through interaction with a COOH-terminal region of P: residues 46-87(29) . Another important site for P interaction is Trp-207 of Galpha(t)(15) . Mutational analysis of Galpha(s) has revealed regions involved in interaction with adenylyl cyclase(30, 31) .

The chimeric Galpha(t)/Galpha molecules described in this study defined three regions of Galpha(t) involved in interaction with the PDE effector molecule. The presence of single or combinations of these regions in a systematic series of chimeras led to increasing affinity for PDE. Competition studies with fragments of PDE have allowed us to define the point-to-point interactions that are important for productive binding of activated Galpha(t) to PDE leading to enzyme activation.


EXPERIMENTAL PROCEDURES

Materials

GTP, GTPS, GDP, deoxyribonucleotides, and imidazole were purchased from Boehringer Mannheim. All restriction and DNA modification enzymes were obtained from Boehringer Mannheim and Pharmacia Biotech Inc. [alpha-P]ATP (600 Ci/mmol) was from ICN. [S]GTPS was a product of DuPont NEN. Polyclonal antipeptide (344-354) anti-Galpha(i) antibodies (116) were generously supplied by Dr. D. Manning (University of Pennsylvania, School of Medicine). All other reagents were from sources described previously(29, 32) .

Construction of Chimeric Galpha(t)/Galpha Genes and P-1-45

An expression vector pHis(6)Galpha, which contains Galpha cDNA preceded by a nucleotide sequence encoding a His(6) amino acid stretch as an affinity tag under the control of a T7 promoter, was generously provided by Dr. M. Linder, Washington University, St. Louis, MO. This vector was the basic construct for preparing Galpha(t)/Galpha chimeras. A bovine Galpha(t) cDNA was generously supplied by Dr. M. Simon, Caltech. To insert Galpha(t) cDNA into pHis(6)Galpha expression vector instead of Galpha cDNA one of two NcoI sites flanking the sequence encoding the His(6) tag that is located upstream of this DNA fragment was eliminated. This plasmid was digested with NcoI and HindIII to remove Galpha cDNA, and the large fragment was then ligated with NcoI and HindIII treated DNA fragment containing Galpha(t) cDNA. The resulted vector expressing Galpha(t) was defined as pHis(6)Galpha(t).

Chimeric genes were constructed by introduction of unique restriction enzyme sites, flanking target fragments, into Galpha and Galpha(t) cDNAs using PCR amplification with corresponding oligonucleotide primers-mutagenes and then replacement of Galpha cDNA fragments with corresponding Galpha(t) cDNA fragments and vice versa. In no case did insertion of novel restriction sites change protein sequences. Table 2shows the location of restriction enzyme sites along the Galpha(t) sequence which are the joining points for a combinatorial arrangement of the Galpha and Galpha(t) sequences in Galpha/Galpha(t) chimeras. The quadruple Galpha mutant, L232M/A235V/E238D/M240V (Chi1), changed Galpha's switch III region to that of Galpha(t). Two regions of Galpha cDNA encoding residues 1-240 and 241-354 were PCR-amplified in separate reactions. The downstream primer for synthesis of 5`-terminal fragment was an oligonucleotide mutagene directing not only substitutions of indicated 4 amino acids but also introduction of SphI site. The upstream oligonucleotide primer for PCR amplification of the 3`-terminal fragment directed introduction of the SphI site as well. The resulting fragments were cut with EcoRI and SphI (5`-terminal fragment) and SphI and HindIII (3`-terminal fragment), and simultaneously ligated with the large fragment of pHis(6)Galpha digested with EcoRI and HindIII. Chi1 plasmid DNA was used as an intermediate construct for making Chi8, Chi5, Chi4, Chi7, and Chi10, where Galpha switch III region was replaced with the corresponding Galpha(t) region. The DNA sequence of all chimeric genes around joining points (usually 150-200 base pairs) was confirmed by DNA sequencing. Chi6 and Chi9 genes were sequenced nearly completely. No misincorporations of deoxynucleotides in PCR-amplified fragments were detected.



P-1-45 expression vector was constructed by PCR amplification of the corresponding P gene fragment using the P expression vector DNA (32) as a template. The downstream oligonucleotide primer directed an insertion of a TAG terminator codon after Lys-45 codon of P gene, following by BamHI site. The resulting PCR product, cut with NdeI and BamHI, was ligated with the large fragment of pET11alpha cut with the same restrictases. The DNA sequence of this construct was confirmed by DNA sequencing over the PCR-amplified region.

Expression and Purification of Highly Soluble Chimeras

E. coli BL21(DE) cells harboring plasmids encoding all chimeras except Chi9 and Chi10 were grown in 1-3 liters of 2 times YT medium in presence of 100 µg/ml ampicillin at room temperature up to OD of 0.5 and then induced with 30 µM IPTG for 16-20 h. The cell pellet was resuspended in 1:20 of a cell culture volume of buffer containing 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM MgCl(2), 50 µM GDP, 0.1 mM phenylmethylsulfonyl fluoride, and 5 mM beta-mercaptoethanol (buffer A) and then disrupted by ultrasonication. The crude cell lysate was cleared by centrifugation at 100,000 times g for 60 min. The supernatant was collected and adjusted to 500 mM NaCl and 20 mM imidazole concentrations by adding 8 times binding buffer: 160 mM Tris-HCl, pH 8.0, 4 M NaCl, and 160 mM imidazole. The resulting mixture was loaded onto 10 ml of the Ni-nitrilotriacetic acid-agarose resin column (His-Bond, Novagen) prepared according to the manufacturer's protocol. The column was washed with 10 volumes of 1 times binding buffer, and bound material was eluted with 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 100 mM imidazole (buffer I-100). NaCl and imidazole were removed from the protein sample by overnight dialysis against buffer A in the presence of 20% glycerol. The protein samples were directly applied to the 20-ml Waters HPLC AP-1 column (Waters Chromatography Division, Millipore Corp.) packed with the Protein-Pak Q 15 HR anion-exchange resin (Millipore Corp.) equilibrated with buffer A free of GDP and beta-mercaptoethanol. This was followed by protein elution using a NaCl gradient in buffer A. The concentration of chimeras in the eluates, which did not contain GDP, was immediately determined spectrophotometrically. GDP, beta-mercaptoethanol, and phenylmethylsulfonyl fluoride were added to the eluates to a concentration of 25 µM, 2 mM, and 0.1 mM, respectively, and samples were aliquoted and stored at -80 °C for several months with no loss of functional activity. The final yield of highly soluble chimeras and Galpha ranged from 3 to 5 mg of more than 95% pure protein/liter of bacterial culture.

Expression and Purification of the Low Soluble Chimeras Chi9 and Chi10

4-8 liters of cells containing the plasmids encoding Chi9 and Chi10 were grown and induced at conditions described above. Cell pellets were resuspended in 200 ml of buffer A, and insoluble material was spun out at 100,000 times g for 60 min. Supernatants were collected and adjusted to 500 mM NaCl and 20 mM imidazole concentrations by adding 8 times binding buffer. 1 ml of Ni-nitrilotriacetic acid-agarose resin equilibrated with 1 times binding buffer was resuspended in 2 ml of 1 times binding buffer and added to the supernatant. The mix was gently shaken for 10 min and then centrifuged at 1000 times g for 5 min. The supernatant was discarded and the resin was resuspended in 5 ml of 1 times binding buffer and then packed into the 3-ml column. The rest of the nickel-chelate affinity chromatography procedure was done at standard conditions as described above. Proteins eluted in I-100 buffer fraction were dialyzed against buffer A containing 20% glycerol and subjected to a second purification step using anion-exchange chromatography on the 2-ml Waters AP Minicolumn, packed with Protein-Pak Q HR15 resin, as described earlier. Typical yield of Chi9 and Chi10 was 0.1-0.2 mg of 80-90% pure protein/liter of bacterial culture.

Peptide Synthesis

Peptides corresponding to residues 63-87 of P and residues 232-259 of Galpha(t) were synthesized by the solid-phase Merrifield method on an Applied Biosystems automated peptide synthesizer. Peptides were purified by reversed-phase HPLC on a preparative Aquapore octyl column (25 times 1 cm) (Applied Biosystems). The purity and chemical formula of peptides were confirmed by fast atom bombardment mass spectrometry and analytical reversed-phase HPLC.

GTPS Binding Assay

To determine the rate of intrinsic nucleotide exchange, Galpha(t), Galpha, and chimeras were diluted in 100 mM HEPES, pH 8.0, 1 mM EDTA, 10 mM MgSO(4) and 10 mM dithiothreitol (buffer G) to make final concentration of protein 4 µM and incubated on ice for 15 min. The reaction was started by adding 5 µl of the diluted sample to 15 µl of radioactive buffer G containing 5 µM [S]GTPS (5000 cpm/pmol) and proceeded at room temperature. To determine the rate of rhodopsin-dependent GTPS binding, sample proteins (4 µM) were preincubated with 4 µM of Gbeta(t) at room temperature. The reaction was initiated by adding 100 nM dark rhodopsin and exposing the samples to the light at room temperature. Aliquots (20 µl) were withdrawn at the indicated times, passed through the Millipore Multiscreen-HA 96-well filtration plate and washed 10 times with ice cold wash buffer (200 µl/well) containing 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 25 mM MgCl(2). Before loading, sample filters were presoaked by passing ice-cold wash buffer twice. Filters were dried, punched by Millipore Multiscreen Puncher and counted.

Fluorescent Assay

Binding of PLY to Galpha(t)GTPS, Galpha(t)GDP, Galpha, synthetic peptide Galpha(t)(232-259), or chimeric polypeptides and competition between PLY and its fragments (P-1-45 and P-63-87) for binding to indicated G proteins were studied by methods given in (29) and (32) . Fluorescent measurements were performed on a Perkin Elmer LS5B spectrofluorimeter at room temperature in 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, and 2 mM MgCl(2) (buffer B) using excitation at 430 nm and emission at 520 nm. The fluorescence at each point has been corrected for dilution, and the initial value of the fluorescence change in the absence of any Galpha has been set to 1.0 for direct binding and 0% for competition binding. The fluorescence of PLY-Galpha complex in the competition binding measurements was set as maximal fluorescent change of 100%. K(d) values for the binding of chimeric proteins Galpha(t) and Galpha to PLY, and its fragments were determined from their binding or competition curves based on calculated EC values, the concentration of PLY in the assay, and K(d) values for PLY-Galpha(t)GDP complex in the presence or absence of AlF(4). To monitor AlF(4)-dependent conformational change of Galpha(t)bulletGDP, Galpha, and chimeras, tryptophan fluorescence was determined with excitation at 280 nm and emission at 340 nm. Fluorescence of 200 nM of Galpha in buffer B was measured before and after addition of 10 mM NaF and 20 µM AlCl(3).

Protein Concentrations

The concentrations of Galpha subunits were determined spectrophotometrically using calculated extinction coefficients. Measured concentrations were corrected for amount of functional protein based on fluorescent assay detecting AlF(4)-dependent increase in Trp fluorescence, which reflects conformational change upon G protein activation. Maximal fluorescent change detected in this assay for best preparations of homogeneous Galpha(t)bulletGDP, containing two Trp, was repeatedly 70%. We thus estimated that 70% increase in Trp fluorescence corresponds to 100% of functionally active protein. Maximal fluorescent change for the best preparations of homogeneous Galpha, containing 3 Trp residues, was 55%. The concentrations of functional Galpha(t)GDP and chimeras Chi9 and Chi10 (2 Trp residues) were calculated using the formula C = n%/70% times C, where n% is AlF(4)-dependent fluorescent change and C is protein concentration determined spectrophotometrically. For calculation of concentrations of functional Galpha and the rest of other chimeras (3 Trp residues) formula was C = n%/55% times C. ROS concentration is expressed in terms of rhodopsin content as determined by absorbance of rhodopsin at 500 nm before bleaching.

Miscellaneous Methods

The inhibitory subunit of PDE and its NH(2)-terminal fragment P-1-45 were expressed in E. coli and purified to homogeneity as described by Skiba et al.(32) . P was specifically labeled at its single Cys-68 using the sulfhydryl-specific reagent lucifer yellow vinyl sulfone as described by Artemyev et al.(29) . The calculated stoichiometry of P labeling with lucifer yellow was in the range of 0.6-0.7.

G(t), Galpha(t), Galpha(t)GDP, Galpha(t)GTPS, Gbeta(t), and rhodopsin containing ROS membranes treated with urea were prepared as described in(33) .

SDS-polyacrylamide gel electrophoresis of proteins was performed according to the method of Laemmli(34) . For immunoblotting analysis, proteins were transferred from SDS-polyacrylamide gel to nitrocellulose using standard semi-dry transfer method. Peroxidase-labeled secondary antibodies immobilized on membranes were detected using a luminol-based chemiluminescent detection system (LumiGLO substrate kit, Kirkegaard & Perry Laboratories, Gaithersburg, MD). Curve-fitting of the experimental data was performed with nonlinear least squares criteria using GraphPad Prism software.


RESULTS

Our experimental strategy took advantage of the significant structural similarity of Galpha(t) and Galpha (see Fig. 1A for structure-based alignment) and functional divergence to use them as parent proteins for construction of chimeric Galpha proteins that fold properly. We systematically replaced regions of Galpha(t) that inhibit proper folding with corresponding Galpha regions that are permissive for proper folding.


Figure 1: A, alignment of amino acid sequences of Galpha(t) and Galpha. Sequences of Galpha(t) and Galpha were taken from Yatsunami and Khorana (64) and Nukada et al.(65) , respectively. Yellow shows alpha helixes, blue shows beta sheets, and boxes indicate locations of the conformational switch regions. B, G(i)G(t) chimera. Panel is a ribbon drawing highlighting regions that inhibit folding in Galpha(t) (red). C, Galpha(t) (red) and Galpha(i) (blue). Panel shows superposition of Galpha(t)GTPS (Noel et al., 1993) and GalphaGTPS (Coleman et al., 1994).



Insoluble Galpha(t)/Galpha Chimeras

His(6)-Galpha(t) and chimeras listed in Table 1were exclusively located in the insoluble fraction. Western blotting analysis of soluble fractions revealed only very low amounts of these proteins (less than 1-2% of total expressed protein), which were nonfunctional and aggregated. The analysis of the structures of insoluble chimeras indicated that they contain either region 216-228 of Galpha(t) (beta4-beta4/alpha3 element, insert II) or Galpha(t) region 271-294 (alphaG/alpha4 loop, insert III) or both regions. Numerous attempts to reconstitute functional chimeras and Galpha(t) from the insoluble fraction after solubilization in urea or guanidine HCl were unsuccessful (data not shown).



Soluble Galpha(t)/Galpha Chimeras

Avoiding the two ``problem'' regions, a series of chimeric proteins was expressed. Table 2shows schematically the structures of soluble Galpha(t)/Galpha chimeras. The distribution of recombinant protein between soluble and insoluble fractions of the bacterial lysate after centrifugation (supernatant and pellet) was analyzed by gel electrophoresis and Western blotting with an anti-peptide antibody against the carboxyl-terminal 10 amino acids of Galpha. This antibody recognizes both Galpha(t) and Galpha, which have only one amino acid difference in this sequence, and all Galpha(t)/Galpha chimeras. Another criterion of protein solubility was the amount of purified protein from 1 liter of cell culture. Since the purification procedure for all soluble chimeras and losses of protein during purification was probably similar, the final yield of pure chimera closely reflects the initial amount of soluble polypeptide in the bacterial cell. Chimeras 1-8 were fully soluble and were expressed at 3-5 mg/liter.

Chimeras 9 and 10, which contained region 237-270 of Galpha(t), had significantly reduced solubility. Only approximately 5-10% of the expressed polypeptide was located in the soluble fraction when cells were induced at room temperature with lowered concentration of IPTG (30 µM). The final yield of purified chimeras from this group was 0.1-0.25 mg/liter of cell culture. The region 237-270 is composed of the alpha3 helix, the alpha3/beta5 loop, and the beta5 sheet; the alpha3 helix and the alpha3/beta5 loop contain the bulk of differences in amino acid sequence (Fig. 1A). Thus, the decreased solubility of chimeras containing this Galpha(t) region may be explained by substantial inhibition of their assembly caused by either misfolding of the alpha3 helix or improper interaction of the alpha3-alpha3/beta5 motif with other parts of polypeptide upon synthesis in bacterial cells.

Analysis of Functional Activity of Soluble Chimeras

The soluble chimeras listed in Table 2were tested for their ability to undergo an increase in intrinsic fluorescence in the presence of aluminum fluoride. This assay tests the presence of GDP in the guanine nucleotide binding pocket and also the ability to undergo an AlF(4)-dependent activating conformational change(17, 35, 36) . Table 2shows the percent increase in tryptophan fluorescence for soluble chimeras in the presence of 10 mM NaF and 20 µM AlCl(3) compared to ground state fluorescence in the inactive GDP-bound form. The fluorescence of all chimeras increased in the presence of AlF(4), implying that all recombinant polypeptides were properly folded, had GDP bound, and were able to undergo the activating conformational change. However, the maximal increase in fluorescence differed according to how many tryptophans were present in the sequence. Chimeras 1-8 containing 3 Trp had a maximal fluorescence increase of 55% similar to Galpha, while chimeras 9 and 10 with 2 Trp had maximal response approaching 70% similar to wild type Galpha(t). It is known from mutagenesis (15) and crystallographic data (17) that only the environment of Trp207 changes upon activation. Thus, Trp located in the switch II region contributes more fluorescent change for chimeras containing 2 Trp residues (70%) than for chimeras with 3 Trp in their sequence (55%). The good correlation between the experimentally measured fluorescence change and the predicted increase in fluorescence in the presence of AlF(4) is strong evidence for proper folding of all the soluble chimeras, as well as the stoichiometric presence of GDP in the nucleotide binding pocket(37) .

The rate-limiting step of G protein activation is the release of GDP. The intrinsic GDP release rate of the chimeras was measured by determining the rate of [S]GTPS binding and compared to literature values(38, 39) . Fig. 2A shows that at 25 °C native Galpha(t) does not appreciably bind GTPS and thus the dissociation of GDP that precedes GTPS binding is very slow (initial rate of 4.9 µmol/mol s, Table 3). Recombinant His(6)-Galpha showed significantly faster nucleotide exchange rate (initial rate of 225 µmol/mol s, Table 3; K 0.05 min, Fig. 2A), in good agreement with data for authentic NH(2)-terminally acylated Galpha and recombinant non-myristoylated protein (39) . Thus either lack of NH(2)-terminal acylation or presence of an unrelated extra sequence at its NH(2) terminus (His(6) tag) does not significantly alter guanine nucleotide exchange properties of the protein. The rate of intrinsic guanine nucleotide exchange for chimeras ranged from very low, similar to Galpha(t), to high, similar to Galpha. Chimera 2, containing the 56 COOH-terminal amino acids of Galpha(t) in a Galpha context had a high GTPS binding rate, similar to Galpha (Fig. 2A, Table 3, K of 0.056 and initial rate of 227 µmol/mol s). The replacement of the NH(2)-terminal Galpha fragment 1-215 with that of Galpha(t) (Chi6) resulted in a significant decrease in the rate of GTPS binding (Fig. 2A, Table 3, initial rate of 15.9 µmol/mol s). Chimera Chi9 containing additional residues 237-270 of Galpha(t) had a lower GTPS binding rate approaching Galpha(t) (Fig. 2A, Table 3, initial rate of 7.9 µmol/mol s).


Figure 2: Time course of GTPS binding to Galpha(t) (squares), Galpha (circles), chi2 (diamonds), chi6 (triangles), and chi9 (reversed triangles). Proteins (1 µM) alone (A), in presence of 100 nM rhodopsin and 1 µM Gbeta(t) (B), or in presence of 100 nM rhodopsin alone (C) were incubated at room temperature in buffer G containing 5 µM [S]GTPS. Duplicate aliquots were withdrawn at the indicated times, filtered, and counted. Binding of GTPS to proteins is expressed as percent of maximal, calculated based on protein concentration and fit using nonlinear least squares criteria to the equation B = B(max) (1 - e). Data shown are the mean ± S.E. of four experiments.





The rates of rhodopsin-catalyzed exchange of GDP for [S]GTPS were measured in the chimeras to determine their ability to interact with Gbeta(t) and activated rhodopsin. The addition of equimolar concentrations of Gbeta(t) (1 µM) and 100 nM light-activated rhodopsin to Galpha(t), Galpha, and chimeras caused a stimulation of GTPS binding to all proteins (Fig. 2B). For Galpha and Chi2, the rhodopsin-catalyzed increase in GTPS binding was just 2-5-fold (initial rates: 1304 and 1352 µmol/mol s; K of 0.095 and 0.097 min , respectively) and less than for Galpha(t) (780-fold) (initial rate of 3798 µmol/mol s, Table 3; K = 0.3/min, Fig. 2B). Chi6 and Chi9 had significantly increased but similar rates of nucleotide exchange in the presence of Rho and Gbeta(t) (K 0.047 and 0.032 min; initial rates 738 and 554 µmol/mol s, respectively; Fig. 2B and Table 3) but 6-8-fold lower than for ROS Galpha(t). Rhodopsin alone in the absence of Gbeta(t) stimulated the nucleotide exchange of only Galpha(t), due to low levels on contamination with Gbeta(t).

Mapping of Effector Binding Sites of Galpha(t) Using Galpha(t)/Galpha Chimeras

Table 2shows that Galpha(t), Galpha and all chimeras are also capable of binding to PLY. Binding to P of Galpha(t) activated in two different ways, either by GTPS binding, or by binding of AlF(4) to Galpha(t)bulletGDP yielded essentially the same affinity constants for PLY (K(d) = 12 nM, Table 2). Thus, in further testing of chimeras binding to PLY, activation was produced by addition of AlF(4). Galpha(t)GDP was also capable of binding to PLY; however, affinity was 70-fold lower than in its activated form (K(d) = 0.88 µM, Table 2). The binding of GalphaGDP to PLY was undetectable at concentrations up to 20 µM; however, upon activation by AlF(4), it formed a low affinity complex with P (K(d) = 2.4 µM, Table 2, Fig. 3), confirming the studies of Otto-Bruc et al.(40) and indicating that this activation-dependent interaction engages the switch regions of Galpha to contact P. The binding of PLY to these proteins was dose-dependent and completely reversible by addition of unlabeled P, showing the specificity of the interaction. The maximal fluorescent change for the complex of Galpha(t)GDP-AlF(4) with PLY (F/F(o) = 2.8) was slightly higher than for complexes with Galpha(t)GDP and Galpha (2.2-2.5), presumably because the environmentally sensitive lucifer yellow moiety was in a slightly more hydrophobic environment.


Figure 3: Binding of Galpha(t)GDP, Galpha, and chimeras to PLY in the presence of AlF(4). The relative increase in fluorescence (F/F) of PLY (50 nM) was measured after addition of increasing concentrations of Galpha(t) (filled circles), Galpha (open triangles), Chi6 (diamonds), Chi9 (squares), Chi10 (filled triangles), and Chi8 (open circles). The solid lines represent the best fit to a four parameter logistic equation (sigmoidal curve) where the fluorescence at each point has been corrected for dilution. K values for the binding of indicated proteins to PLY were determined based on calculated EC parameters.



Binding of Chimeric Proteins to PLY

To obtain more detailed information about binding surfaces of Galpha(t) and effector molecules and to understand how the GTP-dependent conformational switches are involved in the formation of a high affinity interaction between Galpha(t) and P, we constructed a set of Galpha(t)/Galpha chimeras. Several potential P binding regions (Switch III, alpha3 and the alpha3/beta5 loop, alpha4 and the alpha4/beta6 loop, and the amino-terminal half of the molecule, which contains Switch I and II and the extra domain) within folding-permissive regions of Galpha(t) were put into the context of Galpha singly or in combinations. Based on their interaction with P, the chimeras can be divided into three groups. Chimeras from the first group contain only single effector interaction regions of Galpha(t) (Chi1, Chi2, and Chi3) and had similar P affinities as Galpha in the presence of AlF(4) (Table 2). Thus Galpha(t) region 295-314 by itself (Chi3, K(d) = 1.8 µM) or as a part of the COOH-terminal Galpha(t) region 295-350 (Chi2, K(d) = 1.9 µM) makes only a minor contact with PLY, resulting in a fluorescent change. The quadruple mutant of Galpha, L232M/A235V/E238D/M240V, which changes the few residues of switch III of Galpha to the conservative substitutions on Galpha(t), also had a very small effect on affinity to PLY (Chi1, K(d) = 2.2 µM).

Chimeras from the second group (Chi4, Chi5, Chi6, Chi7, and Chi8) contain various combinations of two effector interaction regions, and have a 2-4-fold increased affinity to PLY compared to Galpha (Fig. 3, Table 2). The presence of any two of the regions (295-314, switch III, and the amino-terminal 1-215 region) thus appear to collaborate to form a composite P binding site. Chi8 contains all three of these Galpha(t) regions, and had a 4-fold increase in affinity to PLY (K(d) 550 nM, Fig. 3, Table 2).

Chimeras from the third group are represented by Chi9 and Chi10. Their sequence contains only 15 and 11 amino acid residues derived from Galpha, respectively (Table 2, Fig. 1A), and the rest of the sequence originates from Galpha(t). These two chimeras both formed a high affinity complex with PLY in the presence of AlF(4) (K(d) 70 and 38 nM, respectively; Fig. 3, Table 2), just 3-6-fold lower than the affinity of wild type Galpha(t)GDP-AlF(4) for P. Comparison of the primary structures of these two chimeras to the structure of Chi6 indicates that the additional Galpha(t) region 237-270 increases the affinity of the chimera for P by 13-fold. This region consists of the alpha3 helix and alpha3/beta5 loop as well as the well conserved beta5 sheet and the NKXD guanine ring binding element. Comparison of the sequences in this region shows that the main differences between the two proteins occur in the alpha3 helix and the alpha3/beta5 loop (Fig. 1A). By ruling out the conserved regions, we can narrow down the most important determinant for high affinity P binding to residues 237-257 (blue residues in Fig. 6).


Figure 6: High affinity effector binding surface of Galpha(t). Highlighted residues indicate: pink, COOH-terminal P site interface (alpha4-alpha4/beta6 and switch III); blue, site of interaction with central region of P (alpha3-alpha3/beta5); tan, switch II; maroon, switch I.



Chi9 and Chi10, similar to Galpha(t)GDP, were able to form a low affinity complex with PLY in the absence of AlF(4) (K(d) 3.7 and 3.6 µM, respectively; Table 2). Neither Galpha or any of the other chimeras had detectable affinity for P in their GDP-bound inactive state (Table 2). This indicates that the main determinant for binding of Galpha(t)GDP to P is contained in this area. To test this idea, we synthesized a peptide encompassing this region (residues 232-259) and tested its binding to P. The peptide increased the fluorescence of PLY in a dose-dependent manner (Fig. 4). The binding was completely reversible by adding an excess of unlabeled P to the Galpha(t)(232-259)-PLY complex. The K(d) for this complex calculated from the binding curve was 4.3 µM.


Figure 4: Binding of the synthetic peptide Galpha(t)(232-259) to PLY. Peptide binding to PLY (100 nM) was estimated by the relative increase in fluorescence (F/F) after recording the fluorescence of PLY plotted as a function of peptide concentration. The kinetic parameters calculated from sigmoidal binding curve are EC = 4.35 mM, F/F max = 1. 94.



The chimeras Chi9 and Chi10 that contain this sequence both had 4-fold lower affinity than Galpha(t)GDP for PLY, suggesting that another P interaction site is found within one of the problem regions.

Assignment of Point-to-Point Interaction Sites between Galpha(t) and PDE

Using this family of chimeric Galpha proteins, we can use competition studies to define the regions involved in binding the two major binding sites on P, the central and COOH-terminal regions, which were defined in previous studies(29, 32, 41, 42, 43) . Competition studies between an amino-terminal construct, 1-45, and full-length P (Fig. 5A, Table 2) showed that only chimeras containing residues 237-270 bind to this region, and their affinity is similar to Galpha(t)GDP (5.3 µM). This is evidence that alpha3 and the alpha3/beta5 loop are the major contact sites of Galpha(t)GDP with the central region of P. Binding of Galpha(t)GDP to full-length P was 6-fold higher affinity (0.88 µM), demonstrating that it also interacts with other regions. The activated conformation of Galpha(t) binds to this region with 12-fold higher affinity (0.44 µM), showing that this region also makes contact with one or more of the switch regions of Galpha(t).


Figure 5: Binding of P fragments to parent proteins and chimeras. A, competition between PLY and P-1-45 for binding with Galpha(t) and Chi9. The fluorescence of the complexes of PLY (50 nM) with Galpha(t)GDP (800 nM) (triangles), Galpha(t)GDP-AlF(4) (50 nM) (circles), and Chi9-AlF(4) (75 nM) (reversed triangles) was measured before and after addition of increased concentrations of P-1-45. B, competition between PLY and P-63-87 for binding to chimeras in the presence of AlF(4). The fluorescence of PLY (50 nM) in the presence of Chi6 (1 µM) (diamonds), Chi7 (750 nM) (squares), and Galpha (2.5 µM) (triangles) as well as all other chimeras and Galpha(t) (see Table 2for details) was measured before and after addition of increased concentrations of P-63-87. The fluorescent change is expressed as a percent of maximal change (100% is fluorescence of PLY-Galpha complex before adding P fragment; 0% is fluorescence PLY alone) and plotted as a function of P fragment concentration. The K values were calculated from the competition curves as described under ``Experimental Procedures.''



On the other hand, Galpha and all the chimeras interacted, to different degrees, with the COOH-terminal region of P, but only in their activated state (Fig. 5B, Table 2). The relative affinities of interaction shown in Table 2match quite closely with affinities for full length P, except for chimeras 9 and 10. This suggests that the several effector regions cooperate for binding to P's carboxyl-terminal region. The presence of region 295-314 adds about 3-fold strength of interaction (compare chimeras 7 and 1); likewise, residues within the first half of the molecule add about 3-fold (compare chimeras 1 and 5). Interestingly, the switch III region of Galpha(t) plays an important role in binding the COOH terminus; those chimeras lacking the transducin sequence in switch III (chimeras 2, 3, 6) have lower affinity for this region of P. Two of the three regions are sufficient for binding the carboxyl terminus, and adding the third region does not help (for example, compare chimeras 8 and 5 or chimeras 5 and 4). The most surprising result is that the affinity of chimera 7 for the COOH-terminal region of P is indistinguishable from Galpha(t)GDP-AlF(4) (0.7 µM). This chimera differs from Galpha at only about 10 residues within the Galpha(t) Switch III and alpha4-helix alpha4/beta6 loop.


DISCUSSION

Folding and Functional State of Chimeras

Expression of a chimeric Galpha subunit incorporating sequences from Galpha, which is expressed in functional form in E. coli, has allowed us to express large amounts of functionally active Galpha(t)-like protein. Surprisingly, nearly 95% of Galpha(t) can be incorporated into the chimeric protein, and thus is permissive for proper folding. Two regions of Galpha(t) (residues 216-227 and 271-294, corresponding to inserts 2 and 3) contain 11 amino acid substitutions that lead to expression of insoluble protein when incorporated into the Galpha context. Another problem region between residues 237 and 270 decreases expression levels, presumably by perturbing the folding pathway in a more subtle way.

The soluble chimeric Galpha subunits are functional by several criteria. They are stably folded and soluble, they contain GDP and are able to undergo AlF(4) binding and assumption of an activated conformation measured by an increase in intrinsic fluorescence of Trp-207. The fact that the maximal intrinsic fluorescence change is similar to native alpha subunits suggests that both the inactive and active conformations are similar. With addition of increasing numbers of Galpha(t) residues, chimeric proteins become progressively more transducin-like in biochemical properties such as interaction with P and intrinsic rate of GDP release. In the presence of Gbeta(t), rhodopsin stimulated GDP/GTP exchange for Galpha(t), Galpha, and all chimeras as expected from reconstitution experiments(44, 45) . The stimulation was much more significant for Galpha(t), Chi6, and Chi9 than for Galpha and Chi2, which have significant spontaneous exchange. The fact that Gbeta(t) was strictly necessary to promote GDP/GTP exchange above spontaneous GDP release suggests that free alpha subunits have very little ability to interact on their own with activated receptors under conditions in which no beta contamination can occur.

Structural Basis and Functional Implications of Spontaneous GDP Release

The spontaneous GDP release rates of chimeric proteins varied dramatically, reflecting the known biochemical properties of the parent proteins. Galpha(t) has a low spontaneous release rate whose value varies in the literature (46) depending on how vigorously it has been separated from the potent catalyst of GDP release, activated rhodopsin(38) . Recombinant His(6)-Galpha showed a significant rate of intrinsic GDP release (K 0.05 min) similar to authentic NH(2)-terminally acylated and recombinant non-modified Galpha(39) , suggesting that NH(2)-terminal acylation or modification of the NH(2)-terminal sequence with the His(6) tag had no effect on its nucleotide binding properties. Insertion of the last 56 COOH-terminal amino acid residues of Galpha(t) into the Galpha context (Chi2), the region that contains only the TCAT guanine ring binding determinant, did not change the high rate of spontaneous GDP/GTP exchange of parent protein. However, the replacement of the NH(2)-terminal Galpha fragment 1-215 with that of Galpha(t) (Chi6), the region that contains all other GDP binding sites except the NKXD element, as well as the helical domain, resulted in a drastic decrease of GDP release rate. The presence of the additional Galpha(t) region encompassing residues 237-270 (Chi9) further decreased the rate of GDP release to very low values similar within our ability to measure it to native Galpha(t).

The structural basis for the different GDP off-rates is of great interest, since this is the rate-limiting step in G protein activation (47) . Galpha(t) and Galpha have quite similar overall folds in their activated, GTPS-bound forms (16, 19) ; the GDP-bound, inactive form of Galpha(t) has a very similar global fold, with differences in conformation concentrated around the beta and phosphates(17) . One rather striking difference between Galpha(t) and Galpha is the intimacy of packing of the two domains and, consequently, the buried nature of the guanine nucleotide, with Galpha having a slightly more open structure. In fact, the two domains of the structures are not superimposable unless one of the domains is shifted by a 5° angle ((19) ; Fig. 1C). Of course the binding of the guanine nucleotide in solution is a dynamic process, and the crystal structures, while providing a detailed framework for understanding the stereochemistry of guanine nucleotide binding, do not give complete insight into regions of protein flexibility that may contribute to GDP release. Second, there are probably many ways to affect the GDP release rate, as mutagenesis studies have shown(48, 49, 50) . The functional studies show a 15-fold decreased initial rate of spontaneous GDP release caused by replacing the Galpha(i) helical domain, along with alpha helices 1 and 2 and beta sheets 1-3 (residues 1-215), by that of Galpha(t) (Chi6). This suggests that the main determinants for GDP affinity are within this portion of the molecule and could be further delineated by future study.

The functional implications for the differences in spontaneous GDP release between different G proteins are quite dramatic. Since GDP release is the rate-limiting step for G protein activation, the spontaneous release rate should correspond to a tonic activation rate in the absence of activated receptors. The signaling cascades of each physiological system must adapt to or control the spontaneous activation properties of the particular G protein, which will effect receptor independent activation of channels or inhibition of adenylyl cyclase. The beta subunits can modulate this rate. In highly sensitive detection systems like the visual and olfactory systems, on the other hand, any spontaneous activation of any step in the cascade would increase the background noise and decrease sensitivity. This leads to the prediction that Galpha, which mediates olfactory activation of adenylyl cyclase, should have strikingly lower spontaneous GDP release than Galpha(s), which is >95% identical.

The chimeric approach allows expression and mutagenesis studies of Galpha(t), but cannot differentiate functions in regions that are highly conserved. Overall, the two proteins are quite homologous, and in regions of nucleotide binding or conformational switches this homology increases (Fig. 1A). The ``problem'' regions of Galpha(t) that do not support folding cannot be examined directly because they lead to insoluble protein. Site-directed mutagenesis of the 11 Galpha(t)-specific residues in these regions should help to define roles of these residues in folding or other functions.

G Protein Effector Interaction

GTP-bound Galpha subunits, upon release from the activated receptors and Gbeta, can regulate a wide variety of second messenger enzymes and ionic channels. These include adenylyl cyclase, PDE, phospholipase C, and K and Ca channels(4) . The interaction of Galpha(t) with PDE is well studied, resulting in detailed insight into how an activated GalphaGTP can activate its effector. In the presence of ROS membranes, two activated Galpha(t)GTP (Galpha(t)*) are able to bind to one effector moiety in the process of maximal PDE activation (51) . The first Galpha(t)* bound to PDE elicits 80-100% of its maximal activity, whereas the binding of the second Galpha(t)* adds little if at all to maximal PDE activity(52) . The Galpha(t)*-P-Palphabeta complex remains membrane-bound and appears to bind to the membranes more tightly than PDE alone(53) . In contrast to adenylyl cyclase, which is an intrinsic membrane protein, PDE is an extrinsic membrane protein, which can be removed from the membrane by low ionic strength. Soluble PDE can still be activated by Galpha(t), but with lower potency, and the details of the activation mechanism are different. Under these conditions, Galpha(t)GTP physically removes P from its complex with catalytic subunits Palphabeta, resulting in the formation of Galpha(t)*-P complex and an activated effector, Palphabeta(26, 27, 54) . Arshavsky and Bownds (55) have suggested that under conditions of light adaptation this mechanism could also function in the presence of ROS membranes. In this study, we have taken advantage of the ability to study Galpha(t)-P interaction in solution to map their interaction sites. Under in vivo dark-adapted conditions, however, Galpha(t) normally encounters P in complex with Palphabeta on the membrane, and future studies will be aimed at understanding the nature of this complex.

PDE is a potent inhibitor of PDE activity. The structural basis for this inhibition is a two-site interaction including a COOH-terminal inhibitory region and a central site for tight binding affinity; Galpha(t) perturbs both of these sites to cause PDE activation(29, 32) . Mutational analysis of P has revealed that positively charged residues located in the central region (Arg-24, Arg-25, Lys-29, and Lys-31) are involved in interaction with Palphabeta (42, 56, 57) , while 3 lysines at positions 41, 44, and 45 contact Galpha(t)GTP(42) . The interaction between the P-24-45 region and Galpha(t) has been studied using a photocross-linking approach(58) . The site of cross-linking of P fragment was localized to Galpha(t) region 306-310. It was suggested that the central P region may rather contact the alpha3-alpha3/beta5 region of Galpha(t) but reaches the covalent attachment site via the 12-Å cross-linker. The second, COOH-terminal Galpha(t) binding site of P is located within residues 63-76 and is adjacent to, but not overlapping with, the PDE inhibitory site encompassing residues 77-87 (32, 59) . Removal of 1, 2, or 3 residues from P's COOH terminus dramatically decreases the inhibitory potency of , leading to the idea that Galpha(t)GTP may cause PDE activation by displacing these residues(32) .

Galpha(t)GDP can interact with low affinity with PDE; however, its ability to activate effector has not been detected (40) . The recent report by Kutuzov and Pfister (60) that at high concentration Galpha(t)GDP may activate PDE (K(a) 50 µM) implies that at very high concentration an activating region of Galpha(t)GDP can activate PDE. GTP binding to Galpha(t) causes a conformational switch, leading to a 70-fold increased affinity for PDE. At least two putative regions of effector interaction on Galpha(t) have been identified. The synthetic peptide corresponding to residues 293-314 of Galpha(t) activates PDE (28) by coupling to a carboxyl-terminal fragment of P located within residues 46-87(29) . This region, corresponding to the surface exposed alpha4 helix and the alpha4/beta6 loop, does not change its conformation upon activation(16, 17) . The increased affinity of Galpha(t)GTP for PDE may serve to increase the local concentration of this activating region. The Switch II (15) and III regions (this work) interact with PDE only in their activated conformations. Coordinated interaction between these Galpha(t)GTP regions and PDE may be critical for effector activation.

Structural Basis for Effector Affinity and Specificity

The 200-fold difference in affinity of Galpha(t) and Galpha for PDE allowed us to dissect regions of Galpha subunits involved in effector interaction. The fact that some of the important regions, especially the switch regions, are very similar in the two proteins suggests a conserved switch-dependent surface that can not be analyzed by the chimeric approach. We were able to tease out the contributions of the switch regions somewhat since Galpha(t)bulletGDP still interacts with PDE, and the AlF(4)-dependent conformational switch in Galpha(t) led to a 70-fold increased affinity. Differentiation of the roles of the individual switch regions in effector interaction, however, requires a different approach.

Our studies define at least two effector binding surfaces, each of which is composed of several linear sites of Galpha(t). Galpha(t)GDP forms a low affinity complex with P through a major contact with the central region of PDE (K(d) 5.3 µM) and a weaker one with its COOH-terminal region (K(d) 25 µM). Galpha(t) activation increases its affinity to P approximately 70-fold, as a result of interaction with the conserved conformational switch regions. More than 10- and 30-fold increased affinity of the central and COOH-terminal P regions to activated Galpha(t) suggests that both P sites acquired new contacts with Galpha(t) switch regions.

Interaction Sites with P's Central Region

GalphaGDP does not interact with P; however, activated GalphaGDP-AlF(4) forms a low affinity complex (K(d) 2.4 µM) with the COOH-terminal region of P (K(d) 1.75 µM) through the conserved conformational switch regions. None of the chimeric proteins, except Chi9 and Chi10, were able to interact with P in the inactive state. Upon activation, their binding to P closely corresponded to the binding to P-63-87, and none of them bound to P 1-45. This allows us to conclude that the main interaction of Galpha(t)bulletGDP with P was between alpha(t) 237-270 and P 1-45. This region is highlighted in blue in Fig. 6.

A complementary study on binding of the synthetic peptide encompassing residues 232-259 of Galpha(t) to PLY (K(d) 4.3 µM) additionally proves the involvement of this region in effector interaction. Cunnick et al.(61) have found that synthetic peptide Galpha(t)-250-275, which contains a sequence corresponding to the alpha3/beta5 loop, was able to bind to P. The presence of this region establishes a basal low affinity binding for inactive Chi9 and Chi10, which is, however, 5-fold lower than for Galpha(t)GDP. Activation of Galpha(t) leads to a 10-fold higher affinity for the P central region, presumably involving switch regions.

Interaction Sites with P's COOH-terminal Region

The weak contact that Galpha(t)GDP has with P-63-87 (K(d) 25 µM) is enhanced by 30-fold in Galpha(t)*. Whereas GalphaGDP has no affinity for P, Galpha-GDP-AlF(4) interacts with this region only 3-fold less well than Galpha(t)-GTPS, clearly implicating the conserved GTP-dependent switch mechanism in interaction with P's carboxyl terminus. The conformational switch region is not completely conserved, and substituting Galpha(t) residues from the first half of the molecule, Switch III, and alpha4-alpha4/beta6 can marginally improve this interaction (from GalphaK(d) 1.75 to Chi10 K(d) 0.77). This suggests a composite binding site, which could involve organizing a complex binding site by the action of the conformational switch. An alternative possibility is that the structure of the sites is somehow disturbed in the chimeras affecting the affinity of interaction with P. The difference in affinity between the best chimera and Galpha(t) suggests that the chimeric proteins may still lack a contact point with P within the two problem regions. Site-directed mutagenesis should be able to address the issue.

It was shown earlier that synthetic peptide Galpha(t)-293-314 activates PDE (28) as well as binds to a COOH-terminal fragment of P located within residues 46-87(29) . Eight-fold weaker potency of PDE activation for this peptide (K(a) 8 µM) compared to that of Galpha(t)* (1 µM) in the absence of membranes implies that Galpha(t) region 293-314 is at least a part of an effector activating surface of Galpha(t). Our current data allow us to examine this surface in detail. The 293-314 region is exposed in Galpha(t)GDP and able to bind to the COOH terminus of P, however, very weakly. The approximately 10-fold lower affinity of this region as a part of the whole molecule (25 µM) to the COOH-terminal fragment of P compared to the affinity of Galpha(t)-293-314 alone (K(d) 2 µM, (29) ) may be explained by the greater accessibility of the flexible peptide for contact with P while separated from overall structural context of Galpha(t). Analysis of substituted peptides derived from Galpha(t)-293-314 for their ability to activate PDE has revealed that Asn-297, Val-301, Glu-305, Met-308, and Arg-310, located mostly on one exposed face of the alpha4 helix and the immediately adjacent portion of the alpha4/beta6 loop, are directly involved in Galpha(t)-P interaction(62) . We propose, based upon results obtained with the chimeras, that this activating region may form with Switch III an effective alignment for binding the P COOH-terminal region, resulting in 30-fold tighter contact and PDE activation. These two regions are colored pink in Fig. 6.

Chimera 5, which does not contain the 295-314 region but contains the amino-terminal half of Galpha(t) corresponding to the extra domain, the phosphate binding regions, and switches I and II, also binds to the COOH-terminal P region with similar affinity as chimeras 6 and 8. This suggests a composite binding site, which must contain at least two of three regions, since Chi8 with all three regions does not bind with higher affinity.

In the high affinity Galpha(t)-P complex, Erickson et al.(63) have recently estimated the distance between Cys-68 of P and Lys-267 of Galpha(t)GTPS to be 45 Å, based on the efficiency of energy transfer between these fluorescently labeled residues. In their proposed model of the Galpha(t)-P complex, the contacts of the COOH-terminal and central sites of P were assigned to Galpha(t) regions 106-116 and 300-310 (alpha4-alpha4/beta6), respectively. The involvement of the 106-116 region in interaction with P does not contradict our data that a P binding determinant is found within residues 1-215. However, our results prove the participation of the conformational switch regions of Galpha(t) in binding with the COOH-terminal and central sites of P as well as the existence of contact sites between the alpha3-alpha3/beta5 region of Galpha(t) and the central region of P. This contradicts the data of Erickson et al. The basis for differences in the two studies is not known. Hopefully, in the future, the crystal structure of the Galpha(t)-P complex should resolve this difference.

Fig. 6summarizes the effector-interacting surfaces we have described. The picture that emerges from this study and previous data is of a concerted set of mechanisms underlying: 1) selectivity of G protein effector interaction imparted by the alpha3 and alpha3-beta5 loop (blue region); 2) the formation by the GTP-dependent conformational switch of a composite effector surface, which increases affinity for the effector, without imparting high selectivity, because the switch must be extremely conserved among G proteins (Switch III, left pink region; Switches I and II, tan and purple regions); and 3) an ``activating region'' (right pink region; (28) ), which is highly specific for cognate effector(62) , but does not significantly contribute to the affinity of the interaction. We suggest that this activating region is essentially ``presented'' to the appropriate site on the effector by the switch-dependent and independent mechanisms (mechanisms 1 and 2).

Generality of Effector Activation Mechanisms

Our studies as well as several others implicate Galpha regions interacting with their effectors. Berlot and Bourne (30) have demonstrated using Galpha(s)/Galpha(i) chimeras that the regions of Galpha(s) corresponding to loops alpha2/beta4 (part of switch II), alpha3/beta5, and alpha4/beta6 are involved in adenylyl cyclase activation. The role of the divergent alpha3/beta5 loop in forming contacts with effector was additionally highlighted by Itoh and Gilman (31) on the basis of the importance of Trp-263, Leu-268, and Arg-269, located in this loop, in activation of adenylyl cyclase. The involvement of analogous regions of different Galpha proteins for interaction with different effectors combined with high sequence homology and very similar predicted structure suggests that the mechanisms of effector activation/inhibition by G proteins may have common structural features. Berlot and Bourne (30) were not able examine the basis of high affinity and selectivity of effector interaction in their study because both Galpha(s) and Galpha(i) interact with adenylyl cyclase. What the current study adds to our understanding is insight into the mechanisms of these important characteristics. We have shown that the selective high affinity of a G protein with its cognate effector is provided by the alpha3 helix and alpha3/beta5 loop (blue region), while a general increase in affinity for diverse effectors is provided by the GTP-dependent conformational switch. These ideas will be tested in future studies.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant 12092. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Physiology and Biophysics, College of Medicine, University of Illinois, 835 S. Wolcott Ave., M/C 901, Chicago, IL 60612-7342. Tel.: 312-996-7151; Fax: 312-996-1414.

(^1)
The abbreviations used are: PDE, phosphodiesterase; GTPS, guanosine 5`-3-O-(thio)triphosphate; PCR, polymerase chain reaction; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; HPLC, high performance liquid chromatography; ROS, rod outer segment(s).


ACKNOWLEDGEMENTS

We thank Navreena Gill for help with the computer graphics, Alex Spiess for peptide synthesis, Jeanne Millsap for preparation of Galpha(t) and Gbeta(t), Maurine Linder for Galpha expression vector, Melvin Simon for cDNA encoding bovine Galpha(t), and David Manning for preparation of Galpha(t)/Galpha-specific antibody 116.


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