COMMUNICATION:
Communication between Switch II and Switch III of the Transducin alpha  Subunit Is Essential for Target Activation*

(Received for publication, March 18, 1997, and in revised form, July 1, 1997)

Qiubo Li and Richard A. Cerione Dagger

From the Department of Pharmacology, Veterinary Medical Center, Cornell University, Ithaca, New York 14853-6401

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Comparisons of the tertiary structures of the GDP-bound and guanosine 5'-O-(thiotriphosphate) (GTPgamma S)-bound forms of the alpha subunit of transducin (alpha T) indicate that there are three regions that undergo changes in conformation upon alpha T activation. Two of these regions, Switch I and Switch II, were originally identified in Ras, while Switch III appears to be unique to trimeric GTP-binding proteins (G proteins). We find that replacement of the Switch III region (aspartic acid 227 through asparagine 237) with a single alanine residue yields an alpha T subunit that fully binds and hydrolyzes GTP but no longer stimulates the activity of the cyclic GMP phosphodiesterase (PDE), the physiological target for transducin. We also show that changing glutamic acid 232 of alpha T to a leucine (E232L) had no effect on rhodopsin-stimulated GTP-GDP exchange nor on the GTP hydrolytic activity of alpha T. However, the GTPgamma S-bound form of the alpha TE232L mutant was unable to stimulate the activity of the cyclic GMP PDE. The lack of stimulation was not due to an inability of the alpha TE232L mutant to bind to the target. Taken together, these results indicate that glutamic acid 232 mediates a conformational coupling between Switch II and Switch III, which is essential for converting GTP-dependent G protein-target interactions into a stimulation of target/effector activity.


INTRODUCTION

GTP-binding proteins (G proteins) serve as molecular switches in a wide variety of biological response systems. Two large families, the Ras-related small G proteins and the trimeric G proteins, have received a great deal of attention because of their central role in signal transduction. The trimeric G proteins serve as intermediate signal transducers for seven-transmembrane-spanning (also called heptahelical or serpentine) receptors that are involved in responses to sensory, hormonal and neurotransmitter signals (1, 2). These G proteins consist of two functional units, the guanine nucleotide-binding alpha  subunit (Galpha ) and the beta gamma complex (Gbeta gamma ). The molecular switch capability of a trimeric G protein is mediated through the Galpha subunit, which cycles between the GDP-bound (inactive) and GTP-bound (active) states. A signal received from a receptor promotes the activation of a trimeric G protein by catalyzing the exchange of GTP for GDP on Galpha . This results in the dissociation of the GTP-bound Galpha from the Gbeta gamma complex, thereby enabling these subunits to regulate the activities of downstream target/effectors. GTP hydrolysis on the Galpha subunit promotes its re-association with Gbeta gamma , thus terminating the signal.

The vertebrate phototransduction system represents one of the best characterized G protein-coupled signaling cascades. In this system, the photoreceptor rhodopsin activates a trimeric G protein, transducin, generating a GTP-bound alpha  subunit (alpha T-GTP), which stimulates the target/effector enzyme, the cyclic GMP phosphodiesterase (PDE).1 The three-dimensional structures for alpha T in different guanine nucleotide-bound states, and more recently for the alpha T-beta gamma T holotransducin complex, have been solved by x-ray crystallography (3-6), as have the corresponding structures for the inhibitory GTP-binding protein of the adenylyl cyclase system, Gi1 (7-9). This structural information now provides the foundation for understanding the molecular basis of many aspects of G protein-mediated signaling.

Three distinct regions on trimeric G protein alpha  subunits have been shown to undergo conformational changes in response to GTP/GDP exchange (4, 5). Two of these regions, designated Switch I (Ser173 to Thr183 in alpha T) and Switch II (Phe195 to Thr215), are structurally analogous to the two conformationally-sensitive regions found in Ras (10) and EF-Tu (11), whereas the third region, designated Switch III (Arg227 to Arg238 in alpha T), is unique to the alpha  subunits of trimeric G proteins. The various available x-ray crystallographic structures of G proteins show that the conformational changes in Switch I and II are the direct result of GTP binding to residues within these regions. Specifically, the structural changes in Switch I are induced by the interaction of the gamma -phosphate of GTP with Thr177, while the changes in Switch II result from a hydrogen bond between Gly199 and the gamma -phosphate (4). However, Switch III does not directly contact GTP. Rather, it was shown to respond to Switch II through a series of polar interactions that were mediated and/or promoted by GTP-induced conformational changes in Switch II (4). At present, the functional role of the Switch III domain or the importance of its conformational coupling to Switch II is not known. The fact that the residues proposed to be responsible for this conformational coupling are conserved in all trimeric G protein alpha  subunits (4, 7) supports a critical role for Switch II-Switch III communication in some event associated with G protein activation, such as the GTP-mediated dissociation of rhodopsin and/or beta gamma T from alpha T or the GTP-dependent interaction of alpha T with the cyclic GMP PDE. In the present work, we have examined the importance of Switch III in G protein function and find that a conserved glutamic acid residue within the Switch III domain of alpha T is essential for the regulation of target/effector activity.


EXPERIMENTAL PROCEDURES

Purification of Retinal Proteins---Rod outer segments (ROS) were prepared as described previously (12, 13). Transducin and the cyclic GMP PDE were purified by exposing ROS to room light and repeated washings with 10 mM Hepes, pH 7.5, 6 mM MgCl2, 1 mM dithiothreitol (DTT), and 100 mM NaCl (isotonic buffer) and then with 10 mM Hepes, pH 7.5, 6 mM MgCl2, and 1 mM DTT (hypotonic buffer). The cyclic GMP PDE is released into the hypotonic wash and is further purified by hydroxyapatite chromatography (13). Transducin is released from ROS by washing with hypotonic buffer in the presence of 0.1 mM GTP or 0.1 mM GTPgamma S. The alpha T and beta gamma T subunit complexes are then resolved by Blue Sepharose chromatography (14).

Measurement of cGMP PDE Activity

Dark-adapted ROS membranes containing hydroxyapatite-purified cyclic GMP PDE were assayed for cyclic GMP hydrolysis by measuring proton release, as originally described by Yee and Liebman (15). The assays are performed at room temperature in a buffer containing 10 mM Hepes, pH 8.0, 60 mM KCl, 30 mM NaCl, 1 mM DTT, and 5 mM MgCl2, together with the protein components described in the legend to Fig. 3. The assays were initiated by the addition of cyclic GMP (5 mM), with the pH being recorded for 1-2 min at one determination/s. The PDE activity (nanomole/s) was calculated as the ratio of the slope of the pH change (millivolts) and the buffering capacity of the medium (millivolt/nmol) (16).


Fig. 3. Relative cGMP PDE activities of the bovine retinal alpha T, the purified recombinant wild type alpha T (alpha Twt), the purified alpha TDelta I2, and the purified alpha TE232L proteins. A, equal amounts of the different alpha T proteins were preloaded with GTPgamma S by ROS and beta gamma T and assayed for their abilities to stimulate cGMP hydrolysis by PDE. The rate of cGMP hydrolysis is measured as proton release per s and per nmol of PDE (100%, 800-1000 nmol of cGMP hydrolyzed per s and per nmol of PDE). None of the above alpha T proteins in the GDP-bound states was able to stimulate the PDE activity (data not shown). B, inhibition of the bovine retinal alpha T-stimulated PDE activity by the recombinant wild type alpha T and the alpha TE232 mutant. The GTPgamma S-bound retinal alpha T (20 nM) was incubated with 2 nM PDE in the presence of 20 nM recombinant wild type alpha T (column 2), 20 nM recombinant alpha TE232L mutant (column 3), or 40 nM recombinant alpha TE232L mutant (column 4) in either the GDP-bound (open bars) or GTPgamma S-bound state (hatched bars). The PDE activity stimulated by the bovine retinal alpha T in the absence of any competitor (column 1, solid bar) is set as 100% (800-1000 nmol of cGMP hydrolyzed per s and per nmol of PDE).
[View Larger Version of this Image (25K GIF file)]

Expression of Recombinant alpha T Subunits

The coding region of the bovine alpha T was amplified by the polymerase chain reaction using primers that create a 5'-end NdeI site and a 3'-end BamHI site. The polymerase chain reaction product was digested with NdeI and BamHI and ligated into pET15b (Novagen). The resultant vector was digested with NcoI and PstI to release the coding region of alpha T with its 5'-end fused in-frame to the hexa-His tag present in the vector pET15b. This released fragment was then blunt-ended using T4 DNA polymerase and ligated into pVL1393. To generate the alpha TDelta I2 deletion mutant (Asp227 through Asn237 replaced by a single Ala), and the alpha TE232L point mutant (Glu232 replaced by Leu), we employed a single-stranded DNA-based mutagenesis strategy (17), using synthetic oligonucleotide primers containing the indicated deletion or point mutation (alpha TDelta I2: 5'-CAGGCTCTCGTGCATTCGAGCGTAGGCGCTCAG-3'; and alpha TE232L: 5'-CACTTCGTCCTCGAGCACCAGC-3'). The pVL1393 vector carrying either the wild type, alpha TDelta I2, or alpha TE232L gene was introduced into Sf9 insect cells using the Baculogold transfection kit (PharMingen). The recombinant extracellular virus (rECV) was purified by a limiting dilution procedure (18). For production of the recombinant proteins, Sf9 insect cells were infected at 80% confluence with the purified rECVs at a multiplicity of infection of 5 and harvested typically 60 h post-infection. The His-tagged alpha T proteins were purified through Ni2+-nitrilotriacetic acid affinity chromatography following a protocol provided by Qiagen. The purified proteins were finally dialyzed against HMDN buffer (20 mM Hepes, pH 7.4, 5 mM MgCl2, 150 mM NaCl, and 1 mM DTT) containing 40% of glycerol and stored at -20 °C.


RESULTS AND DISCUSSION

The original finding that a third region on heterotrimeric G protein alpha  subunits undergoes structural changes upon GTP-GDP exchange (i.e. in addition to the Switch I and Switch II regions originally identified in Ras and EF-Tu (10, 11)) suggests that it may play a critical role in a GTP-dependent G protein function. To obtain experimental support for this suggestion, we examined the properties of an alpha T deletion mutant in which the entire Switch III domain (residues Asp227 through Asn237) was replaced by a single alanine residue. The deletion mutant, designated alpha TDelta I2, was expressed in Spodoptera frugiperda (Sf9) cells as a hexahistidine (His)-tagged fusion protein and purified by Ni2+ affinity chromatography. This results in a rapid and highly effective purification of the recombinant alpha T subunit, as shown in Fig. 1. The first three lanes in A show the Coomassie Blue-stained profiles for the alpha T subunit purified from bovine retina, the recombinant wild type His-tagged alpha T purified from Sf9 cells (which has a slightly slower mobility on SDS gels because of the His-tag), and the His-tagged alpha TDelta I2 mutant purified from Sf9 cells. B shows the corresponding Western blots that were obtained using a specific antibody raised against the carboxyl-terminal 10 amino acids of alpha T (16).


Fig. 1. Expression of the recombinant alpha T proteins in Sf9 insect cells. Aliquots of the native alpha T purified from bovine retina (Retinal alpha T), the recombinant wild type alpha T purified from Sf9 cells (alpha Twt), the alpha TDelta I2 purified from Sf9 cells (alpha TDelta I2), and the alpha TE232L mutant purified from Sf9 cells (alpha TE232L) were resolved on an SDS-polyacrylamide gel (12.5% acrylamide). The gel was either stained by Coomassie Brilliant Blue R-250 (A) or electroblotted onto the Immobilon membrane (Millipore) and probed with an antibody (AS/7) (16) raised against the carboxyl-terminal 10 amino acids of bovine alpha T (B).
[View Larger Version of this Image (42K GIF file)]

We first examined whether the deletion of the Switch III domain from alpha T affected rhodopsin- and beta gamma T-promoted [35S]GTPgamma S/GDP exchange. Fig. 2A shows that as has been documented previously (19, 20), when alpha T purified from bovine retina was added to urea-stripped ROS containing light-activated rhodopsin, there was a marked increase in [35S]GTPgamma S binding that was strongly stimulated by the addition of purified retinal beta gamma T. Virtually identical results were obtained with the Sf9-expressed, His-tagged wild type alpha T and the His-tagged alpha TDelta I2 deletion mutant. Likewise, the alpha TDelta I2 mutant was able to fully hydrolyze [gamma -32P]GTP (Fig. 2B). Taken together, the results presented in Fig. 2, A and B, indicated that the deletion of the Switch III domain did not impair the ability of alpha T to interact with rhodopsin nor with the beta gamma T subunit complex and that removal of Switch III did not interfere with the GTP-binding/GTP hydrolytic cycle of the G protein.


Fig. 2. GTP binding and GTPase activities of bovine retinal alpha T, the purified recombinant wild type alpha T (alpha Twt), the purified alpha TDelta I2, and the purified alpha TE232L. A, [35S]GTPgamma S binding activities of the alpha T proteins. An equal amount (100 nM, 4 µg of alpha T in a 100-µl reaction volume) of each of the alpha T proteins was mixed with 0.01 µM [35S]GTPgamma S and 2.0 µM cold GTPgamma S in the presence or absence of urea-stripped rod out segments (Ros) containing membrane-bound rhodopsin (29) and the retinal beta gamma T (100 nM). The binding of [35S]GTPgamma S was measured by filter assay and reported as nanomole of [35S]GTPgamma S bound per 100 nmol of the protein. B, relative GTPase activities of the different alpha T proteins. An equal amount (100 nM) of each of the alpha T subunits was incubated with beta gamma T (100 nM) and [gamma -32P]GTP in the presence or absence of ROS. The GTPase activities of the recombinant proteins are reported relative to that of the bovine retinal protein (defined as 100%; 150 pmol [32P]Pi released in 10 min).
[View Larger Version of this Image (27K GIF file)]

We then examined the ability of the alpha TDelta I2 mutant to functionally couple to the cyclic GMP PDE, by first loading the alpha TDelta I2 mutant with GTPgamma S (by incubation with ROS and bovine retinal beta gamma T) and then assaying cyclic GMP PDE activity, by measuring the H+ release that accompanies cyclic GMP hydrolysis. The results presented in Fig. 3A illustrate that the bovine retinal alpha T subunit and the recombinant wild type alpha T were essentially equivalent in their abilities to stimulate cyclic GMP hydrolysis. However, the alpha TDelta I2 deletion mutant was unable to stimulate PDE activity (relative to the basal activity measured in the absence of added alpha T). Thus, these results suggested that the integrity of the Switch III domain was essential for alpha T-mediated regulation of its target/effector enzyme.

An interesting possibility that was originally proposed following an examination of the x-ray crystallographic structure of the alpha T·GTPgamma S complex (3) was that the acidic amino acid residues, Asp233, Asp234, and Glu235 formed a potential binding site for a basic stretch of amino acids on the gamma PDE subunit. If this were the case, it would then explain why the deletion of the Switch III domain yields an alpha T subunit that is unable to stimulate effector activity. However, we have expressed and purified an alpha T mutant from Sf9 cells in which the three acidic amino acids were replaced by alanine residues and found that this triple mutant was fully active, not only in its ability to bind and hydrolyze GTP, but also in its ability to stimulate cyclic GMP PDE activity (data not shown).

This then led us to examine another possibility, namely that a conserved glutamic acid residue in the Switch III region, Glu232, is responsible for mediating the conformational communication between the Switch II and Switch III domains (4). To test this, we generated a mutant of alpha T in which a leucine residue was substituted for Glu232 (alpha TE232L) and expressed it in S. frugiperda (Sf9) insect cells as a hexahistidine (His)-tagged protein (see lane 4 in Fig. 1, A and B). Like the alpha TDelta I2 deletion mutant, we found that the alpha TE232L mutant was able to functionally couple to rhodopsin and/or beta gamma T, as read-out by its ability to undergo [35S]GTPgamma S/GDP exchange and GTP hydrolysis in a rhodopsin- and beta gamma T-dependent manner (Fig. 2, A and B). Moreover, just as was the case for the Switch III deletion mutant, the alpha TE232L mutant was unable to stimulate target/effector (PDE) activity (Fig. 3A), even when using amounts of the mutant that were in 10-fold excess relative to the retinal or recombinant wild type alpha T proteins (data not shown). Thus, the mutation of the single conserved Glu232 residue appeared to fully mimick the effects obtained upon the removal of the entire Switch III domain.

We used the alpha TE232L mutant to further examine the importance of Switch II domain-Switch III domain coupling in the stimulation of target/effector activity. We found that the inability of the alpha TE232L mutant to stimulate PDE activity cannot be attributed to its inability to bind to its PDE target. This was determined through competition experiments. Fig. 3B shows that like the GDP-bound wild type alpha T subunit (open bar in column 2), the GDP-bound form of the alpha TE232L mutant (open bars in columns 3 and 4) did not competitively inhibit the PDE stimulatory activity of the GTPgamma S-bound retinal alpha T subunit (shown as the solid bar in column 1). This was as expected, since the GDP-bound form of alpha T has only a weak affinity for the gamma PDE subunit. However, the GTPgamma S-bound form of alpha TE232L showed a dose-dependent inhibition (hatched bars in columns 3 and 4 in Fig. 3B), thus indicating that the alpha TE232L mutant can bind to gamma PDE in a GTPgamma S-dependent manner. The fact that the GTPgamma S-bound wild type alpha T subunit did not competitively inhibit the stimulatory activity of the retinal GTPgamma S-bound alpha T (column 2, hatched bar, in Fig. 3B) illustrates that the activated wild type alpha T subunit can fully substitute for the activated retinal alpha T. Moreover, these results demonstrate that the inhibitory effects are specific for the GTPgamma S-bound alpha TE232L mutant, such that the alpha TE232L molecule can act as a dominant-negative mutant.

Thus, GTPgamma S can both bind and induce the appropriate conformational changes within the alpha TE232L mutant that enable it to specifically interact with the target/effector molecule. This is further indicated by the results of limited trypsin treatment (Fig. 4). It has been well documented that trypsin treatment of the retinal alpha T subunit gives rise to defined proteolytic patterns that are absolutely dependent on the guanine nucleotide-bound state of alpha T (21, 22). Trypsin treatment of the GDP-bound wild type alpha T yields two stable fragments, an ~23-kDa fragment (shown in lane 2 under alpha Twt in Fig. 4) and an ~9-kDa fragment (not shown), whereas trypsin treatment of the GTPgamma S-bound wild type alpha T yields a stable 32-kDa (precursor) fragment (lane 3 under alpha Twt in Fig. 4). Based on the information provided from the tertiary structures for the different nucleotide forms of alpha T (4, 5), it is now clear that the protection afforded by GTPgamma S directly reflects a GTPgamma S-dependent conformational change that occurs within the Switch II domain and effectively moves the trypsin-sensitive Arg204 residue from a solvent-exposed environment to a less accessible position (by virtue of its interaction with Glu241). Thus, the protection against trypsin proteolysis afforded by GTPgamma S serves as a highly sensitive read-out for GTPgamma S-induced conformational changes within the Switch II domain and has frequently been used as a monitor for alpha T activation (23). The results presented in Fig. 4 (lanes 2 and 3 under alpha TE232L) show that GTPgamma S binding to the alpha TE232L mutant provides a similar protection against trypsin proteolysis, as observed with the wild type alpha T subunit. Therefore, the mutation of Glu232 neither perturbs GTPgamma S binding nor the GTPgamma S-induced conformational alteration of Switch II. However, mutation of Glu232, while preserving the GTP-dependent binding of the alpha T subunit to the cyclic GMP PDE, completely uncouples this binding from target/effector stimulation.


Fig. 4. Trypsin digestion of the recombinant wild type alpha T and the alpha TE232L mutant. Five µg of the wild type alpha T subunit (alpha Twt) or the alpha TE232L mutant either alone (lane 2) or preloaded with GTPgamma S by ROS and beta gamma T (lane 3) was incubated with trypsin (0.5 µg) at room temperature for 30 min. The digestion mixtures as well as the alpha T subunits before trypsin digestion (lane 1) were boiled in Laemmli buffer (30) and subjected to SDS-polyacrylamide gel electrophoresis (15% acrylamide) and immunoblotting with an anti-alpha T antibody (16).
[View Larger Version of this Image (32K GIF file)]

The location of Glu232 in the loop connecting the beta 4 strand and alpha 3 helix of alpha T places it in a prime position to couple conformational transitions between Switch II and Switch III. In particular, x-ray crystallographic analysis shows that upon GTPgamma S binding, Glu232 is engaged in direct interactions with Arg201 and Arg204 of Switch II and in a water-mediated interaction with Gly199 of Switch II (5). Given our findings, we conclude that the conformational coupling between Switch II and Switch III is responsible for converting a primary binding interaction between activated alpha T and its target (gamma PDE), perhaps involving residues in Switch II (24) or in other regions of alpha T (25-27), into a secondary stimulatory interaction between the gamma PDE subunit and the alpha 4-beta 6 residues 305-314 of alpha T (28). Moreover, these results indicate that Glu232 plays an essential role in mediating this conformational coupling, thereby translating alpha T-target (PDE) interactions into a specific regulatory event. The fact that this glutamic acid residue is conserved in all trimeric Galpha subunits further suggests that it plays a fundamental role in converting target binding into target/effector regulation in a wide variety of G protein-coupled signaling pathways.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant EY06429.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Tel.: 607-253-3888; Fax: 607-253-3659.
1   The abbreviations used are: PDE, phosphodiesterase; ROS, rod outer segment(s); DTT, dithiothreitol; GTPgamma S, guanosine 5'-O(thiotriphosphate).

ACKNOWLEDGEMENTS

We acknowledge Al Berger, Jon Erickson, and Rohit Mittal for helpful discussions and other assistance during the course of these studies. We also thank Cindy Westmiller for her expert technical assistance.


REFERENCES

  1. Lefkowitz, R. J., and Caron, M. G. (1988) J. Biol. Chem. 263, 4993-4996 [Free Full Text]
  2. Gilman, A. G. (1987) Annu. Rev. Biochem. 56, 615-649 [CrossRef][Medline] [Order article via Infotrieve]
  3. Noel, J. P., Hamm, H. E., and Sigler, P. B. (1993) Nature 366, 654-663 [CrossRef][Medline] [Order article via Infotrieve]
  4. Lambright, D. G., Noel, J. P., Hamm, H. E., and Sigler, P. B. (1994) Nature 369, 621-628 [CrossRef][Medline] [Order article via Infotrieve]
  5. Sondek, J., Lambright, D. G., Noel, J. P., Hamm, H. E., and Sigler, P. B. (1994) Nature 372, 276-279 [CrossRef][Medline] [Order article via Infotrieve]
  6. Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319 [CrossRef][Medline] [Order article via Infotrieve]
  7. Coleman, D. E., Berghuis, A. M., Lee, E., Linder, M. E., Gilman, A. G., and Sprang, S. R. (1994) Science 265, 1405-1412 [Medline] [Order article via Infotrieve]
  8. Mixon, M. B., Lee, E., Coleman, D. E., Berghuis, A. M., Gilman, A. G., and Sprang, S. R. (1995) Science 270, 954-960 [Abstract]
  9. Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058 [Medline] [Order article via Infotrieve]
  10. Pai, E. F., Kabsch, W., Krengel, U., Holmes, K. C., John, J., and Wittinghofer, A. (1989) Nature 341, 209-214 [CrossRef][Medline] [Order article via Infotrieve]
  11. Jurnak, F. (1985) Science 230, 32-36 [Medline] [Order article via Infotrieve]
  12. Gierschik, P., Simons, C., Woodward, C., Somers, R., and Spiegel, A. (1984) FEBS Lett. 172, 321-325 [CrossRef][Medline] [Order article via Infotrieve]
  13. Phillips, W. J., Trukawinski, S., and Cerione, R. A. (1989) J. Biol. Chem. 264, 16679-16688 [Abstract/Free Full Text]
  14. Pines, M., Gierschik, P., Milligan, G., Klee, W., and Spiegel, A. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4095-4099 [Abstract]
  15. Yee, R., and Liebman, P. A. (1978) J. Biol. Chem. 253, 8902-8909 [Medline] [Order article via Infotrieve]
  16. Cerione, R. A., Kroll, S., Rajaram, R., Unson, C., Goldsmith, P., and Spiegel, A. M. (1988) J. Biol. Chem. 263, 9345-9352 [Abstract/Free Full Text]
  17. Kunkel, T. A., Roberts, J. D., and Zakour, K. A. (1987) Methods Enzymol. 155, 367-382
  18. Goswami, B. B., and Glazer, R. I. (1991) BioTechniques 5, 626-630
  19. Wessling-Resnick, M., and Johnson, G. L. (1987) J. Biol. Chem. 262, 3697-3705 [Abstract/Free Full Text]
  20. Wessling-Resnick, M., and Johnson, G. L. (1987) J. Biol. Chem. 262, 12444-12447 [Abstract/Free Full Text]
  21. Fung, B. K. K., and Nash, C. R. (1983) J. Biol. Chem. 258, 10503-10510 [Abstract/Free Full Text]
  22. Bigay, J., Deterre, Ph, Pfister, C., and Chabre, M. (1987) EMBO J. 6, 2907-2913 [Abstract]
  23. Hurley, J. B., Simon, M. I., Teplow, D. B., Robishaw, J. D., and Gilman, A. G. (1984) Science 226, 860-862 [Medline] [Order article via Infotrieve]
  24. Faurobert, E., Otto-Bruc, A., Chardin, P., and Chabre, M. (1993) EMBO J. 12, 4191-4198 [Abstract]
  25. Cunnick, J., Twamley, C., Udovichenko, I., Gonzalez, K., and Takemoto, D. J. (1994) Biochem. J. 297, 87-91 [Medline] [Order article via Infotrieve]
  26. Erickson, J. W., Mittal, R., and Cerione, R. A. (1995) Biochemistry 34, 8693-8700 [Medline] [Order article via Infotrieve]
  27. Mittal, R., Erickson, J. W., and Cerione, R. A. (1996) Science 271, 1413-1416 [Abstract]
  28. Rarick, H. M., Artemyev, N. O., and Hamm, H. E. (1992) Science 256, 1031-1033 [Medline] [Order article via Infotrieve]
  29. Yamanaka, G., Eckstein, F., and Stryer, L. (1985) Biochemistry 24, 8094-8101 [Medline] [Order article via Infotrieve]
  30. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]

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