The same mutation in Gsα and transducin α reveals behavioral differences between these highly homologous G protein α-subunits

Abstract

Mutating Arg-238 to Glu (R238E) in the switch 3 region of a transducin α (*Tα) in which 27 aa of the GTPase domain have been replaced with those of the α-subunit of the inhibitory G protein 1 (Gi1α), was reported to create an α-subunit that is resistant to activation by GTPγS, is devoid of resident nucleotide, and has dominant negative (DN) properties. In an attempt to create a DN stimultory G protein α (Gsα) with a single mutation we created Gsα–R265E, equivalent to *Tα–R238E. Gsα–R265E has facilitated activation by GTPγS, a slightly facilitated activation by GTP but much reduced receptor plus GTP stimulated activation, and an apparently unaltered ability to interact with receptor as seen in ligand binding studies. Further, the activity profile of Gsα–R265E is that of an α-subunit with unaltered or increased GTPase activity. The only change in Gsα that is similar to that in *Tα is that the apparent affinity for guanine nucleotides is decreased in both proteins. The molecular basis of the changed properties are discussed based on the known crystal structure of Gsα and the changes introduced by the same mutation in a *Tα (Gtα*) with only 23 aa from Gi1α. Gtα*–R238E, with four fewer mutations in switch 3, was reported to show no evidence of DN properties, is activated by GTPγS, and has reduced GTPase activity. The data highlight a critical role for the switch 3 region in setting overall properties of signal-transducing GTPases.

Heterotrimeric G proteins are molecular machines that transduce the signal generated by the binding of agonists to seven-transmembrane receptors into changes in the activity of effectors. Seven transmembrane receptors, also known as G protein-coupled receptors (GPCRs), and G proteins each constitute a family of structurally and functionally related molecules. The basic mechanism by which all GPCRs act is by catalyzing the exchange of GTP for GDP on the α-subunits of the trimeric G proteins. The binding of the nucleoside triphosphate promotes the dissociation of the so-far inactive trimer into an αGTP complex plus a βγ dimer, both of which are competent to interact and modulate the activity states of effectors (for recent reviews see refs. 1–3). A strict set of specificities exists that defines which of the 16 G protein α-subunits and which of the Gβγ dimers interact with which effector function. These specificity rules, which are best understood for α-subunits, dictate that the activated forms of stimultory G protein α (Gsα) stimulate adenylyl cyclases (ACs), the activated forms of Giα inhibit AC, and the rod and cone transducin α-subunits (Tαs) activate visual phosphodiesterase (PDE) in rod and cone photoreceptor cells, respectively. Differences in primary amino acid sequence among G protein α-subunits define their effector specificities. In support, studies of chimeric α-subunits have borne out the assumption that effector specificity resides in well defined topologically identified regions of α-subunits (cf. ref. 4).

In contrast to the easily understandable differences in effector specificity, structural features of α-subunits that define which of the many highly homologous GPCRs interacts with which α-subunit are largely unknown, and even less well, if at all, understood is how all agonist-occupied (activated) GPCRs promote the same nucleotide exchange reaction at their cognate G protein α-subunits. Hypotheses as to how nucleotide exchange comes about have in common the assumption that similar rate-limiting steps are facilitated, and that while the receptor–G protein interaction is determined by their appropriately differing interaction affinities, the kinetic steps that follow productive association of receptor to the trimeric G protein are likely to be very similar, if not the same.

Transducin, originally called “light-activated GTPase,” was not only the first signal-transducing G protein to be recognized to have an intrinsic GTPase activity, but also the first for which the αβγ nature of its subunit composition was uncovered and the first for which it was shown that GTP binding and activation was accompanied by an αβγ to GTPα plus βγ dimer subunit dissociation reaction (reviewed in ref. 2). In 1993, Tα became the first Gα-subunit for which a crystal-based model became available (5, 6). Structures for the α-subunit of the inhibitory G protein 1 (Gi1α) (7), the βγ dimer (8), and trimeric αβγ forms of transducin and Gi1 (9, 10) were reported shortly thereafter. The α-subunits were shown to be two-domain structures: an ≈180-aa GTPase domain, highly homologous to the smaller regulatory GTPase ras, and, inserted into ras's switch 1 effector sequence, a ≈120-aa six-helix helical domain (αA through αF) connected at each end to Switch 1 by linkers 1 and 2. Comparison of the structures of α-subunits in their inactive, GDP-bound forms (e.g., refs. 9–11) to those of their activated forms, occupied by either GTPγS (5, 12) or GMP-P(NH)P (13) led to the identification of three “switch” regions. Gα switch 1 and 2 are structurally homologous to the switch regions identified previously in ras (14). Switch 3 comprises the loop connecting the GTPase domain's fourth β-strand (β4) to its third α-helix (α3). The final α5-helix of α-subunits is somewhat shorter than ras α5 and includes at its C terminus 10 aa involved in receptor recognition (15). The guanine nucleotide binding site is formed by the GTPase domain, but is somewhat occluded by the closely juxtaposed helical domain.

Activation of Gα-subunits (as well as of ras and ras-like GTPases) involves the binding of both GTP and Mg²⁺ (Fig. 1). Binding of Mg²⁺ involves six coordination bonds of which two are contributed by GTP (one oxygen each of the β and γ phosphates, βO and γO) and two are provided by water oxygens locked in place by hydrogen bonds, one to the δO of an aspartic acid (Asp-223 in Gsα) and the other to an oxygen of the α-phosphate of the GTP. The last two coordination bonds are provided by oxygens of Gα amino acids: one of a Ser (Ser-54 of Gsα) and the other of a Thr (Thr-204 in Gsα).

Fig. 1.

Model of the 3D structure, based on coordinates taken from Protein Data Base ID code 1JCV, of the region of Gsα thought to be affected in the Gsα–R265E mutant. Mg and its six coordination bonds are shown in green; oxygen atoms of two water molecules (w), and relevant oxygens contributing to the coordination shell of Mg are shown in red, as are oxygens of Asp-223 and the α phosphate (αO), which stabilize by hydrogen bonds the coordinating water molecules. Some secondary structure features of Gsα are highlighted, as are the distances between atoms of Glu-50, Arg-258, and Arg-265 thought to interact by forming ion pairs.

Using a chimera between Tα and Gi1α (*Ta), which is better suited for expression in bacteria while preserving all known functions of Tα (4), as a model to explore the effects of mutations that would be predicted by the crystal structures to affect the interaction between the GTPase and the helical domains, Cerione and collaborators (16, 17) have recently tested how these mutations would correlate with changes in functionality. One such mutant, *Tα–R238E, which caught our attention as it could not be activated by GTPγS, was devoid of GDP or GTP, i.e., it was nucleotide-free, and behaved as a dominant negative (DN) when placed in an assay in which it could compete with activated WT *Tα for activation of PDE, the effector of transducin (17). R238 of *Tα lies in the Switch 3 domain of *Tα (Fig. 2).

Fig. 2.

Multiple amino acid sequence alignment comparing bovine transducin α (αt), human Gi1α (αi), the 394 variant of human Gsα (αs), and *Tα (chim αt-αi1). Amino acid sequences are compared with that of Gsα, where − denotes that the amino acid is identical to that in Gsα. A consensus line is shown for which an uppercase letter identifies the identity among all compared sequences, and − denotes that in at least one of the proteins the sequence differs by one or more amino acids. Secondary structure features (β-strands 1–6, α-helices 1–5 and A to G, switch 1, 2, and 3 regions, GTPase to helical domain linkers Lk1 and Lk2) are highlighted, as are sites of ADP-ribosylation by cholera (CTX) and pertussis (PTX) toxins and interactions with Mg. The alignment was generated with Accelrys GCG software using PILEUP to build the nn.msf file and BOXSHADE to render the figure.

There have been previous attempts to create DN forms of Gsα. The first was the attempt by Hildebrandt et al. (18) who introduced into Gsα the Ser-to-Asn mutation that confers DN properties to small ras-like GTPases. Although the Gsα–S54N so made did display DN properties [it was not activated by the combined actions of GTP and the β2-adrenergic receptor (β2AR] it exhibited strong spontaneous Gsα activity equivalent to 50% of the activity displayed by the fully activated WT Gsα. The most successful was Berlot's step-by-step approach, which culminated with the creation of a DN Gsα with seven mutations in three regions of the molecule (19). Here, we introduced into Gsα the mutation that converted *Tα into a DN, with the expectation that, with this single change, Gsα–R265, we might create a nucleotide-free α-subunit that would promote through its interaction with the β2AR the stabilization of the receptor in its high-affinity form for agonist and, because it would not bind GTP or GDP, that this interaction would be permanent, allowing the mutant Gsα to act as a DN element susceptible to be probed for defined conformations that in systems with transient GPCR interactions could not be observed.

Unexpectedly, however, the mutant Gsα, instead of being resistant to activation by GTPγS, was found to have a facilitated activation by guanine nucleotides and to interact well and reversibly with receptor. We report the properties of this mutant in comparison with the reported propertied of the cognate *Tα mutant.

Results and Discussion

Parallelism Between Signal Transduction by Transducin and Gs.

The activation of transducin α by receptor has features in common with those of Gsα and features that are unique. The progression of activation of transducin can be assessed by binding of GTPγS, changes in intrinsic Trp-207 fluorescence, proteolytic breakdown, and development of the ability to activate visual PDE, which occurs by interaction with and removal of inhibitory PDEγ from the αβγ₂ holoenzyme. On the receptor side, activation of transducin is promoted by illuminated MetaII rhodopsin, which has high affinity for nucleotide-free transducin and thereby promotes nucleotide exchange (for a review see ref. 20). Depending on the particular mode of reconstitution of the system from its components, one or the other of these parameters is measured to assess molecular properties of its components. In contrast, the progress of activation of the signal-transducing Gs protein is commonly followed by assessing its stimulation of AC in membranes of S49 cyc⁻ or cyc⁻kin⁻ cells (CK7 cells), as seen in response to added guanine nucleotide or aluminum fluoride or to activation of the β2AR in the presence of GTP.

In the present work we generated CK7 cells expressing Gsα–R265E, a mutation that is equivalent to the *Tα–R238E mutation found by Pereira and Cerione (17) to be resistant to activation by rhodopsin and GTPγ and studied its susceptibility to be activated by GTPγS, both without the cooperation of receptor stimulation (hormone-independent activation) and with simultaneous activation by receptor (hormone-stimulated activation of AC).

Gsα–R265E Is Susceptible to Hormone-Independent Activation by GTPγS and Hormone-Stimulated Activation by GTP.

Although the parental CK7 cells have no measurable AC activity under the conditions of our assay (data not shown), CK7 cells expressing human Gsα display a classical hormone-stimulated AC activity caused by reconstitution of the receptor–G protein–AC signal transduction pathway (Fig. 3). The reconstituted AC activity resembles that of the WT parental S49 cells (which express the native murine Gsα instead of the human protein) in that receptor-mediated stimulation almost absolutely depends on addition of a guanine nucleotide, and basal activities are very low (21). Compared with WT Gsα, the R265E mutant reconstituted an activity that in the presence of isoproterenol was reduced by 80% and with AlF₄⁻ was reduced by 70%. In contrast, activities measured in the presence of GTP were <2-fold higher, and with GTPγS were 2-fold higher than activities reconstituted with WT Gsα. Even so, isoproterenol still stimulated the AC activity mediated by the mutant Gsα between 2- and 3-fold. These results indicated that the mutant Gsα, unlike *Tα–R238E, is susceptible to activation by guanine nucleotides and is capable of transducing receptor signals into effector activation. The expression of the WT and mutant Gsα in the transformed cells appeared to be similar, as seen by ADP-ribosylation of a ≈45-kDa band in membranes of CK7 cells expressing the WT and the R265E forms of Gsα (data not shown). Although isoproterenol stimulates AC in membranes from Gsα–R265E expressing CK7 cells less than in membranes of WT Gsα-expressing cells, the EC₅₀ with which activation is obtained differs by <2-fold (Fig. 4), suggesting that the mutation does not interfere significantly with the signal transduction process. A similar result was obtained by assessing the stimulation of AC by PGE1 (Fig. 4).

Fig. 3.

Reconstitution of Gs-regulated AC activities by WT and R265E Gsα in S49 cyc⁻kin⁻ (CK7) cells. CK7 cells expressing the WT Gsα or the mutant Gsα–R265E were washed, suspended at 200,000 cells/10 μl in buffer containing 20 mM Na-Hepes (pH. 8.0), 50 mM NaCl, 1 mM DTT, and 1 mM EDTA, and analyzed for AC activity as described in Materials and Methods by incubating 10 μl of the cell suspensions for 20 min at 32°C in the presence of 10 μM GTP, 10 μM GTP plus 10 μM isoproterenol (GTP + ISO), 10 mM NaF plus 10 μM AlCl₃ (AlF₄⁻), or 100 μM GTPγS.

Fig. 4.

The activation of the mutant Gsα–R265E by GTP is impaired without a significant effect on the position of the dose–response relationship with which the agonist–receptor complex increases activation the G protein by the nucleotide. CK7 cells expressing Gsα (●) or the mutant Gsα–R265E (□) were washed and analyzed for AC activity by incubating 200,000 cells in the presence of 10 μM GTP and increasing concentrations of isoproterenol (Left) or PGE₁ (Right).

Activation of Gs by AlF₄⁻ requires GDP instead of GTP. To test whether the reduced stimulation of AC by AlF₄⁻ in membranes with the mutant Gsα might be casued by a reduction in affinity of the system for guanine nucleotides as suggested by the data obtained with *Tα, membranes from R265E-expressing cells were incubated at 4°C for 10 min with up to 5 mM GDP at a constant concentration of free Mg²⁺ of 5 mM and assayed at the same final concentration of Mg²⁺ after a 5-fold dilution into AC assay reagents. Even at 5 mM/1 mM GDP, the AlF₄⁻-stimulated activity was still reduced by 70–80% when compared with activity obtained under the same conditions in membranes with WT Gsα (data not shown). The reason for this decrease in the ability of the mutant Gs to be activated by the combination of AlF₄⁻ and GDP is unknown and was not explored further.

The data reported so far indicate that Gsα and *Tα differ markedly in their ability to be activated by guanine nucleotides and in their response to activated GPCR, i.e., light-activated rhodopsin vs. ligand-activated β2AR. Thus, a mutation that essentially inactivates *Tα as a signal-transducing molecule has very different effects on Gs, which when viewed from the guanine nucleotide point of view, results in facilitated activation by GTPγS instead of refractoriness to activation.

Mutant Gs–R265E Interacts With and Stabilizes the β2AR in a State of High Affinity for Agonist That is Indistinguishable from That Stabilized by WT Gs.

In addition to monitoring the interaction of receptor with the G protein by determining activation of the effector (PDE activity for Tα and AC activity for Gsα), the ability of a G protein to interact productively with receptor can be assessed by monitoring changes in affinity for agonist of its cognate receptor as seen by displacement of antagonist binding. As illustrated in Fig. 5, competitive inhibition curves of antagonist binding to the β2AR obtained by increasing concentrations of agonist are monophasic and of low affinity for agonist when membranes in which the receptor is embedded are devoid of Gsα (Fig. 5A), but are biphasic when membranes also contain Gs (Fig. 5 B and C). The biphasic nature of the displacement curves is the result of the coexistence of complexes of nucleotide-free Gs and agonist-occupied receptor, which display high affinity for the agonist, and the Gs-free receptor, which displays the same low affinity for agonist seen in for the bulk of the receptor in membranes without Gs.

Fig. 5.

WT Gsα and mutant R265E Gsα have similar ability to stabilize the β2AR of CK7 cells in its high agonist affinity state. Membranes (5–10 μg per incubation) from CK7/mock cells (A) or from CK7 cells expressing Gsα (B and C) or Gs–R265E (D and E) were incubated as described in Materials and Methods with 0.1 nM of ¹²⁵I-CYP and the indicated concentrations of unlabeled isoproterenol in the absence or presence of 100 or 500 μM GTP or GDP. Bound and free ligands were separated by the polyethylene glycol precipitation procedure described in Materials and Methods. A global fit of a two-state receptor model (lines) to the experimental results (symbols) was obtained by using an equilibrium dissociation constant of 0.16 nM for ¹²⁵I-CYP for the receptor–probe interaction, and the indicated equilibrium dissociation constants of the receptor states of high affinity (K_H) and low affinity (K_L) for the agonist isoproterenol. The resulting partition of the receptor between high-and low-affinity states, as percent of total in the high affinity state, is depicted for each experimental condition. The K_L and K_H values used for the global fit are the averages of values obtained by fitting each of the displacement data independently of the other displacement data. The results are representative of three independent such experiments with similar results. Values are means ± SD for the data set shown.

As seen by Scatchard analysis of specific ¹²⁵I-CYP binding to membranes of mock-infected and membranes from cells expressing Gsα or Gsα–R265E, the receptor abundance was between 0.8 and 1.3 pmol/mg membrane protein and had an equilibrium dissociation constant that varied between 0.14 and 0.18 nM in the presence of 5 mM MgCl₂. In agreement with previous studies, binding of the antagonist was unaffected by presence of guanine nucleotides (cf. refs. 22 and 23 and data not shown) and differed by <20% from one membrane preparation to another (data not shown). At 0.16 nM, equilibrium binding was reached within 5 min of incubation (data not shown). The data shown in Figs. 5 and 6 were obtained by incubating the reaction mixtures of CK7 membranes for 30 min at 32°C. Binding of isoproterenol to the β2AR was of an essentially monophasic nature. Expression of WT Gsα or R265E–Gsα changed agonist binding from monophasic to biphasic in agreement with a ternary complex model in which the β2AR exists in two states: one not associated with a G protein, identical to that found in Gs-deficient CK7 cell membranes, the other with a Gs-induced high affinity for agonist (21–24). The K_d values obtained for low and high agonist affinities did not differ significantly among membranes expressing one or the other Gs (WT or R265E) and were averaged, yielding values of 279 ± 55 and 3.0 ± 0.3 nM for the low agonist affinity K_d (K_L) and the high agonist affinity K_d (K_H), respectively. At the levels at which WT Gsα and mutant Gsα were expressed, the effectiveness with which they stabilized the receptor in its high-affinity state (R_H) was indistiguishable (47%; Fig. 5 B and D vs. C and E). Thus, WT and mutant Gs induce qualitatively and quantitatively the same high agonist affinity state of the receptor. However, they differed markedly in the concentration of nucleotide required to disengage from the receptor. This difference is best seen in the experiment of Fig. 6, which shows EC₅₀ values with which GTPγS and GDP disengaged the Gs from the receptor to be shifted by 35- and 52-fold when Gsα is compared with Gsα–R265E. Thus, the R265E mutation causes a significant reduction in the effectiveness with which guanine nucleotides act to cause the dissociation of the Gs–receptor complex.

Fig. 6.

Mutant Gs–R265E requires higher concentrations of nucleotides than the WT Gs to shift the CK7 cell β2AR from its state of high affinity for agonist to its state of low affinity for agonist. Membranes (15–20 μg protein per incubation) from CK7 cells expressing Gsα (A) or Gsα–R265E (B) were incubated in the absence of a nucleoside triphosphate regenerating system with 0.1 nM of ¹²⁵I-CYP, 50 nM isoproterenol, and the indicated concentrations of GDP (open symbols) or GTPγS (closed symbols). Note that the binding of ¹²⁵I-CYP to the membrane increases with the concentration of nucleotides because of the gradual transition of the β2AR from its high agonist affinity state to its low agonist affinity state. The EC₅₀ values reveal that the mutation causes marked a decrease in the affinity for nucleotide. The results are representative of two independent experiments.

A time-course study of the rate of activation of AC in membranes of CK7 cells expressing WT Gs as compared with Gs–R265E showed that the typical lag in activation of Gs in the absence of hormonal stimulation is shortened in membranes of Gsα–R265E-expressing cells (Fig. 7). The lag in AC activation by nonhydrolyzable GTP analogues correlates with slow binding of the analogues to G protein α-subunits, which has been shown to reflect a slow rate of dissociation of resident GDP from Gα-subunits (25). Taken together, our results are consistent with Gs–R265E having between 35- and 50-fold lowered affinities for GTP and GDP. In this respect the mutant Gs resembles the cognate mutant *Tα found by Pereira and Cerione (17) to be devoid of resident guanine nucleotide. It differs, however, in that the Gs mutant does interact productively with receptor and is activated by GTPγS, whereas the *Tα mutant does not. In agreement with a lower affinity for nucleotides, activation of the Gs–R265E–AC complex by GTPγS, in the presence of hormone required higher concentrations of the nucleotide for half-maximal effects than the WT counterpart (Fig. 8).

Fig. 7.

Kinetic analysis of AC stimulation reveals much faster activation of the Gs–R265E by GTPγS than that of WT Gs. Membranes from Gsα (A) and Gsα–R265E (B) expressing CK7 cells were tested for the rate of activation of AC by 100 μM GTPγ in the absence (○) and the presence of 100 μM isoproterenol (●). Assay mixtures (1.5 ml each) were incubated at 32°C, and formation of [³²P]cAMP was monitored by removing 50-μl aliquot at the indicted times. Note that at steady state the receptor-independent activation of Gs is similar to that of the mutant Gs.

Fig. 8.

Compared with WT Gs, the Gsα–R265E mutation requires a higher concentration of GTPγS for half-maximal activation in the presence of agonist-activated receptor and is very poorly activated by GTP. Membranes (10 μg protein per assay) from cells expressing Gsα (A and C) and Gsα–R265E (B and D) were tested for stimulation of AC activity by increasing concentration of GTPγS (C and D) in the presence (closed symbols) or absence (open symbols) of 100 μM isoproterenol. Note that the reduction in the shift in EC₅₀ for GTPγS activation of the mutant Gs in the presence of isoproterenol is in agreement with the data of Fig. 6, showing that activation of Gs proceeds with a marked lag, whereas that of the mutant Gs is much faster.

The fact that not only GTP but also GDP is competent in promoting the disengagement of Gs from the receptor is in agreement with the original 1970 finding that both GTP and GDP affect binding of glucagon to its receptor (26), which were extended to the turkey and S49 cell β-adrenergic receptors in 1980 (27). It would thus appear that the high agonist affinity receptor–G protein complex represents the receptor complexed with the nucleotide-free G protein, most likely trimeric because subunit dissociation requires occupancy of the Gsα by GTP. The fact that the β2AR forms a stable complex with nucleotide-free Gs and therefore has highest affinity for this form of the G protein may be a general property of GPCRs and underlies the mechanism by which they promote nucleotide exchange.

Not explained by our experiments is why, while GTPγS activates mutant Gs very well and the mutant Gs interacts productively with receptor, activation by GTP is minimal regardless of a cooperating receptor stimulation (Fig. 8B). One possibility not explored here is that the mutant may have a much higher intrinsic GTPase activity. The low abundance of Gsα in the transformed CK7 cells does not permit addressing this question at this time.

After many of the experiments shown here had been performed we became aware of an article by Barren at al. (28), in which the R238E mutation was introduced into a Tα–Gi1α chimera (Gtα*, chimera 8 in ref. 4) with four fewer amino acids from Gi1α, leaving switch 3 of Tα unaltered. R265E–Gtα* binds nucleotides, interacts productively with photoactivated rhodopsin exchanging GTPγS for GDP, and has a much lower GTPase activity. There are no indications that Gsα–R265E has an impaired GTPase (also see above). Such a feature would predict an increased activation effectiveness for GTP, instead of the decreased effectiveness seen in our experiments (Figs. 1 and 6B). It is clear that in comparison with Gsα the highly homologous transducin α is functionally very different. Moreover, the differing properties of R265E–*Tα and R265E–Gtα* highlight a critical role for the switch 3 region of G protein α-subunits.

On the Mechanism of the Changes Caused by the R265E Mutation.

Upon inspection of the 3D model of Gsα deduced from its crystal structure (e.g., Protein Data Base ID code 1JCV; Fig. 1), it becomes apparent that at a distance of 3.00 Å, Arg-265 and Glu-50 form an ionic pair. By inverting the polarity of the charge at the side chain of amino acid 265, the R265E mutation can be expected to disturb not only the positions of the side-chain atoms of Glu-50 but also the backbone peptide chain of the highly conserved α-subunit P-loop (sequence GAGESGK). The P-loop forms the pocket in which the phosphates of the guanine nucleotides are held. The negatively charged δ-carboxyl group of Glu-50 also interacts with the positively charged guanidino group of Arg-258 (distance of Arg-258 guanidino NH⁺ to Glu-50 Oε⁻ = 4.03 Å and Arg-258 Nε to Glu-50 Oε = 3.02 Å). In transducin α and Gi1α, Arg-258 is replaced with Val (Tα–V231) and Ala (Gi1α–Ala-231), respectively (see Fig. 2). It is likely that Gsα–R258 contributes to the stability of the P-loop. This stabilizing effect would be absent in the chimeric *Tα (Tα–Gi1α) protein and may be at the root of the differing impacts that mutating *Tα R238E and Gsα R265E have on the properties of the resulting mutant proteins.

Materials and Methods

Radioisotopes [α-³²P]ATP, [³H]cAMP, and [³²P]NAD⁺ were purchased from GE Healthcare-Amersham. [¹²⁵I]Iodocyanopindolol (¹²⁵I-CYP) was from NEN–PerkinElmer. Bovine gamma globulin, (−)isoproterenol, and PGE1 were from Sigma–Aldrich; polyethyleneglycol 6000 was from Calbiochem, and guanine nucleotides were from Boehringer-Ingelheim.

All other chemicals and biochemicals were of the highest purity commercially available and used without further purification.

Standard laboratory and recombinant DNA techniques were used to generate the R265E mutation in the cDNA coding for the 394-aa splice variant of the human Gsα (29). cDNAs were mutated by using the Quikchange II XL Site-Directed Mutagenesis kit from Stratagene. The WT and R265E Gsα subunits were stably expressed CK7 cells transformed by retroviral-mediated infection as described by Berlot (30). CK7 cells are derivatives of S49 thymoma cells that are cyc⁻ and kin⁻, i.e., deficient in Gsα activity and cAMP-dependent protein kinase activity. CK7 cell clones we grown in suspension, harvested, and used for preparation of purified membranes as described by Ross et al. (21), except that the membrane purification was stopped after obtaining the 43,000 × g pellet. Membranes were suspended in 20 mM Hepes-Na (pH 8.0), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, and 2 mM MgCl₂.

Cholera toxin was from List Biological Laboratories. Activation of the toxin and treatment of CK7 cell membranes were as described by Birnbaumer et al. (31). [³²P]ADP-ribosylation of membranes with cholera toxin followed by SDS/PAGE and visualization by autoradiography were as described by Hildebrandt et al. (32).

AC activities were measured as described by Iyengar et al. (33).

Specific binding of ¹²⁵I-CYP to the β2AR of CK7 cell membranes and separation of free ligand from bound ligand by the polyethyleneglycol precipitation method were as described by Abramowitz et al. (34). Specific binding was defined as the difference between ¹²⁵I-CYP bound in the absence and that bound in the presence of 0.4 μM unlabeled isoproterenol. A two-state model was used to fit by the least sum of the squares of differences method to the data according to the equation:

where RP* is the concentration of¹²⁵I-CYP specifically bound; P* is the concentration of the probe, ¹²⁵I-CYP; K_P is the equilibrium dissociation constant for the interaction of ¹²⁵I-CYP with the H and L states of the receptor; and the concentration of total receptor in the incubation = sum of concentrations of the receptor in the high-affinity, R_H, and low-affinity, R_L states that bind agonist H with high- and low-affinity equilibrium dissociation constants K_H and K_L, respectively. Fits of the model to the data were calculated by using the Solver routine of Microsoft Excel.

All experiments were repeated between two and four times. Figures show data that are representative of the data obtained in the repeats.