receptor-G Protein Interaction Research Articles

Protein engineering approaches to determine adenosine receptor-G protein interactions.

The adenosine receptor subfamily of G protein-coupled receptors is an important family of membrane receptors that modulate blood pressure and more recently have been implicated in cancer, neurodegenerative diseases, and diabetes, making them a significant fraction of drug discovery efforts. Adenosine A2A receptor (A2AR), one of four subfamily members, has a longer C-terminus than the other adenosine receptor subtypes, which may contribute to its exceptional trafficking to the plasma membrane, but its flexibility has been attributed to protein-protein interactions. Chimeric A1 and A3 receptors with A2AR C-termini showed improved localization to the plasma membrane and are functionally active, with greater than three-fold higher yields than previously reported from other heterologous expression systems. Interestingly, these chimeras are able to bind to their native G proteins, suggesting the specificity of interaction does not rely on the C-terminus. To investigate this further using purified A2AR and Gαs, we determined the affinity and binding rates via surface plasmon resonance (SPR). Together with in vivo signaling data, our results suggest that loss of downstream signaling for the truncation may result from a reduced GPCR-G protein complex interaction, which may be important for interpretation of structure-based drug discovery screening. Current studies are focused on the effect of lipid nanodiscs on A2AR-G protein interactions.

Age-related changes in CB1 receptor expression and function and the behavioral effects of cannabinoid receptor ligands

Cannabinoid use has increased among aging individuals. However, little information on age-related differences in the behavioral effects of these agents is available. To explore potential differences in the behavioral effects of cannabinoids, we determined effects of Δ9-tetrahydrocannabinol (THC, 1–10 mg/kg) or rimonabant (0.3–3.2 mg/kg) on operant fixed-ratio responding (FR10) for food in young adult (6 months) and aged (29 months) rats. THC dose-dependently decreased responding for food. Rimonabant alone had little or no effect on responding up to 1.0 mg/kg, but disrupted responding following a 3.2 mg/kg dose. Rimonabant (1.0 mg/kg) partially antagonized response disruption by THC. These effects were similar in young adult and aged rats. However, aging has been reported to change the neurobiology of cannabinoid CB1 receptors. To confirm our rats exhibited such differences, we assessed CB1 receptor binding sites and function in six subcortical (caudate, nucleus accumbens CA1, and CA2/CA3), and three cortical regions (medial prefrontal, temporal, entorhinal) in young adult (6 months) or aged (26 months) male Lewis rats using quantitative autoradiography. CB1 receptor binding sites were reduced in cortical, but not subcortical brain regions of aged rats. CB1 receptor function, at the level of receptor-G protein interaction, was not different in any region studied. Results indicate that down-regulation of CB1 receptor binding sites observed in cortical regions of aged rats was not accompanied by a commensurate decrease in CB1 receptor-stimulated [35S]GTPγS binding, suggesting a compensatory increase in receptor function in cortical areas. Together, our results provide additional evidence of age-related changes in central CB1 receptor populations. However, the functional compensation for decreased CB1 receptor binding may mitigate changes in behavioral effects of cannabinoids. With the rising use of cannabinoid-based therapeutics among aging populations, further evaluation of age-related changes in the cannabinoid system and the impact of these changes on effects of this class of drugs is warranted.

NanoLuc (NLuc) complementation assay elucidates role of specific G‐proteins in GPR88 signaling

GPR88 is an orphan G protein-coupled receptor (GPCR) expressed at high levels in the striatum and is a target for the development of therapeutics to treat multiple neuropsychiatric disorders. While a few small-molecule synthetic agonists have been reported, no synthetic antagonists or endogenous ligands have been identified. Typical 2nd messenger, cell-based assays for agonist and antagonist identification and signaling pathway elucidation rely on complex downstream events, with interpretation of results complicated from effects of transfected receptor overexpression and interference from endogenous receptors and proteins. The NLuc complementation assay, based on an engineered luciferase from the deep-sea shrimp, Oplophorus gracilirostris, can monitor receptor-G protein interaction in live cells and profile compounds according to the G proteins they activate. The synthetic agonists, 2-PCCA [(1R,2R)-2-(pyridin-2-yl)cyclopropane carboxylic acid], and 2-OMPP [(2R)-N-(2-hydroxy-1-(4-((2-methylpentyl)oxy)phenyl)ethyl)-2-phenylpropanamide] are suspected to activate GPR88 via a Gαi-coupled signaling pathway. In this study we show the activation of GPR88 by 2-PCCA and 2-OMPP inhibited isoproterenol induced cAMP production in transiently transfected GPR88 HEK-293T cells with relative EC50 values of 250 nM and 1.0 μM, respectively, consistent with prior reports. The EC50 value for PCCA in HEK-293T/GPR88 transients is five times that in RH-7777 cells with COS-7 cells showing no cAMP response. These results highlight the importance of the cellular background in engineered systems. Additionally, the NLuc complementation assay shows PCCA receptor binding triggers activation of several G-proteins including Gαi1(EC50= 760 nM), Gαi0 (EC50=3.3μM) and GαS (EC50=4.5 nM) indicating the effects of varying G-protein expression in different cell lines. In combination with our computational endeavors, 2nd messenger and the NLuc complementation assays are valuable tools in our research as we develop high-throughput screening processes and probe signaling pathways in search of GPCR based therapeutics.

G protein-coupled receptor-G protein interactions: a single-molecule perspective.

G protein-coupled receptors (GPCRs) regulate many cellular and physiological processes, responding to a diverse range of extracellular stimuli including hormones, neurotransmitters, odorants, and light. Decades of biochemical and pharmacological studies have provided fundamental insights into the mechanisms of GPCR signaling. Thanks to recent advances in structural biology, we now possess an atomistic understanding of receptor activation and G protein coupling. However, how GPCRs and G proteins interact in living cells to confer signaling efficiency and specificity remains insufficiently understood. The development of advanced optical methods, including single-molecule microscopy, has provided the means to study receptors and G proteins in living cells with unprecedented spatio-temporal resolution. The results of these studies reveal an unexpected level of complexity, whereby GPCRs undergo transient interactions among themselves as well as with G proteins and structural elements of the plasma membrane to form short-lived signaling nanodomains that likely confer both rapidity and specificity to GPCR signaling. These findings may provide new strategies to pharmaceutically modulate GPCR function, which might eventually pave the way to innovative drugs for common diseases such as diabetes or heart failure.

CAMP guided his way: a life for G protein-mediated signal transduction and molecular pharmacology-tribute to Karl H. Jakobs.

Karl H. Jakobs, former editor-in-chief of Naunyn-Schmiedeberg's Archives of Pharmacology and renowned molecular pharmacologist, passed away in April 2018. In this article, his scientific achievements regarding G protein-mediated signal transduction and regulation of canonical pathways are summarized. Particularly, the discovery of inhibitory G proteins for adenylyl cyclase, methods for the analysis of receptor-G protein interactions, GTP supply by nucleoside diphosphate kinases, mechanisms in phospholipase C and phospholipase D activity regulation, as well as the development of the concept of sphingosine-1-phosphate as extra- and intracellular messenger will presented. His seminal scientific and methodological contributions are put in a general and timely perspective to display and honor his outstanding input to the current knowledge in molecular pharmacology.

Preassociation between the 5-HT7 serotonin receptor and G protein Gs: molecular determinants and association with low potency activation of adenylyl cyclase.

According to early models of GPCR signaling, G proteins only interact with activated receptors. However, some GPCRs were shown to assemble with G proteins before receptor activation, in accordance with more recent models. Previously, we found that the 5-HT7 receptor, as opposed to the 5-HT4 receptor, was preassociated with Gs, but the molecular determinants for this interaction are still elusive. In a series of chimeric 5-HT7 receptors with intracellular segments from 5-HT4, we determined the receptor-G protein interaction by performing antibody-immobilized fluorescence recovery after photobleaching and fluorescence resonance energy transfer. We identified the intracellular loop 3 and C-tail of the 5-HT7 receptor to be responsible for the preassociation with Gs, and we further delineated the TM5 extension in the intracellular loop 3 and helix 8 in the C-tail as the molecular determinants. These chimeric exchanges converted the 5-HT7 receptor into a collision-coupled receptor that recruited G proteins only upon agonist activation, whereas reciprocal exchanges converted 5-HT4 to a preassociated receptor. The 5-HT7 receptor displayed 2-component agonist-induced Gs signaling with high and low potency. In addition, the same segments were involved in low-potency signaling and preassociation. The correspondence between Gs preassociation and low-potency Gs signaling is a novel aspect of GPCR pharmacology.-Ulsund, A. H., Dahl, M., Frimurer, T. M., Manfra, O., Schwartz, T. W., Levy, F. O., Andressen, K. W. Preassociation between the 5-HT7 serotonin receptor and G protein Gs: molecular determinants and association with low potency activation of adenylyl cyclase.

Single-molecule imaging reveals receptor-G protein interactions at cell surface hot spots.

G-protein-coupled receptors mediate the biological effects of many hormones and neurotransmitters and are important pharmacological targets. They transmit their signals to the cell interior by interacting with G proteins. However, it is unclear how receptors and G proteins meet, interact and couple. Here we analyse the concerted motion of G-protein-coupled receptors and G proteins on the plasma membrane and provide a quantitative model that reveals the key factors that underlie the high spatiotemporal complexity of their interactions. Using two-colour, single-molecule imaging we visualize interactions between individual receptors and G proteins at the surface of living cells. Under basal conditions, receptors and G proteins form activity-dependent complexes that last for around one second. Agonists specifically regulate the kinetics of receptor-G protein interactions, mainly by increasing their association rate. We find hot spots on the plasma membrane, at least partially defined by the cytoskeleton and clathrin-coated pits, in which receptors and G proteins are confined and preferentially couple. Imaging with the nanobody Nb37 suggests that signalling by G-protein-coupled receptors occurs preferentially at these hot spots. These findings shed new light on the dynamic interactions that control G-protein-coupled receptor signalling.

Coding GPCR-G protein specificity.

G protein-coupled receptors (GPCRs) are ubiquitous gatekeepers of cellular response and signal predominantly by recruitment and activation of G proteins. In a recent paper in Nature, Flock et al. use large-scale bioinformatics to build a model of GPCR-G protein selectivity and an interactive database to interrogate potential receptor-G protein interactions.

Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms

We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand binding, receptor activation, and receptor-G protein interaction. We compared the effects of these mutations on the action of two atypical M1 functionally preferring agonists (N-desmethylclozapine and xanomeline) and two classical non-selective orthosteric agonists (carbachol and oxotremorine). Mutations of D105 in the orthosteric binding site and mutation of D99 located out of the orthosteric binding site decreased affinity of all tested agonists that was translated as a decrease in potency in accumulation of inositol phosphates and intracellular calcium mobilization. Mutation of D105 decreased the potency of the atypical agonist xanomeline more than that of the classical agonists carbachol and oxotremorine. Mutation of the residues involved in receptor activation (D71) and coupling to G-proteins (R123) completely abolished the functional responses to both classical and atypical agonists. Our data show that both classical and atypical agonists activate hM1 receptors by the same molecular switch that involves D71 in the second transmembrane helix. The principal difference among the studied agonists is rather in the way they interact with D105 in the orthosteric binding site. Furthermore, our data demonstrate a key role of D105 in xanomeline wash-resistant binding and persistent activation of hM1 by wash-resistant xanomeline.

A Kinetic Model of GPCRs: Analysis of G protein Activity, Occupancy, Coupling and Receptor‐State Affinity Constants

G protein-coupled receptors, upon agonist binding, accelerate the exchange of GDP for GTP in G proteins coupled to them. The activated G protein interacts with effector proteins to mediate diverse biological functions. We present a kinetic, quaternary complex model, based on a system of coupled linear first-order differential equations that accounts for GTPase activity, ligand binding to receptor, and guanine nucleotide binding to G protein. We solved the model numerically to predict the extents of G protein activation, receptor occupancy by ligand, and receptor coupling that result from varying the ligand concentration, presence of GDP and/or GTP, the ratio of G protein to receptor, and the equilibrium constants governing receptor pre-coupling and constitutive activity. We also simulated responses downstream from G protein activation using a transducer function. Our model shows that agonist-induced G protein activation can occur with either a net decrease or increase in total receptor-G protein coupling. We also demonstrate that affinity constants of the ligand for both the active and inactive states of the receptor can be derived to a close approximation from analysis of simulated responses downstream from receptor activation. Finally, we show that the concentration of GDP greatly affects the potency of agonists for stimulating the binding of non-hydrolyzable GTP analogs. Our analysis relates to agonist-stimulated [35S]GTPγS binding measurements and the interpretation of receptor-G protein interactions using fluorescence techniques. Finally, it validates our prior methods for estimating the active state affinity constants of ligands.

A kinetic model of GPCRs: analysis of G protein activity, occupancy, coupling and receptor-state affinity constants

Context: G protein-coupled receptors are vital macromolecules for a wide variety of physiological processes. Upon agonist binding, these receptors accelerate the exchange of GDP for GTP in G proteins coupled to them. The activated G protein interacts with effector proteins to implement downstream biological functions. Objective: We present a kinetic, quaternary complex model, based on a system of coupled linear first-order differential equations, which accounts for the binding attributes of the ligand, receptor, G protein and two types of guanine nucleotide (GDP and GTP) as well as for GTPase activity. Methods: We solved the model numerically to predict the extents of G protein activation, receptor occupancy by ligand and receptor coupling that result from varying the ligand concentration, presence of GDP and/or GTP, the ratio of G protein to receptor and the equilibrium constants governing receptor pre-coupling and constitutive activity. We also simulated responses downstream from G protein activation using a transducer function. Results: Our model shows that agonist-induced G protein activation can occur with either a net decrease or increase in total receptor-G protein coupling. In addition, we demonstrate that affinity constants of the ligand for both the active and inactive states of the receptor can be derived to a close approximation from analysis of simulated responses downstream from receptor activation. Discussion and conclusion: The latter result validates our prior methods for estimating the active state affinity constants of ligands, and our results on receptor coupling have relevance to studies investigating receptor-G protein interactions using fluorescence techniques.

G Protein Activation: A Dynamic Process

The Gα subunit of heterotirmeric G Proteins is responsible for regulating intracellular signals through guanine nucleotide binding and release, the catalytic rate limiting step. Recent studies have shown interactions of Gα with Guanine nucleotide Exchange Factors (GEFs), including non-receptor-like GEFs, cause dynamic structural changes which facilitate substrate release. Although receptor-G Protein interactions are well known a lack of understanding of non-receptor-G protein interactions still exists, namely Gα interactions with the non-receptor GEF Ric-8A. A 10-to-100 fold decrease in nucleotide exchange rate for Ric-8A compared to receptors has been shown, which begs the question: What is the cause for the decrease between receptor and non-receptor GEF activity? To answer this, the dynamic and structural interactions of Gα-Ric-8A have been probed by fluorescence anisotropy and single-molecule Forster Resonance Energy Transfer (smFRET). Conformational changes in the ms times-scale of inter and intra-domain states and increase segmental motions of the switch (I-III) regions seem to play a role in substrate release.

Ligands Raise the Constraint That Limits Constitutive Activation in G Protein-coupled Opioid Receptors

Using a cell-free bioluminescence resonance energy transfer strategy we compared the levels of spontaneous and ligand-induced receptor-G protein coupling in δ (DOP) and μ (MOP) opioid receptors. In this assay GDP can suppress spontaneous coupling, thus allowing its quantification. The level of constitutive activity was 4-5 times greater at the DOP than at the MOP receptor. A series of opioid analogues with a common peptidomimetic scaffold displayed remarkable inversions of efficacy in the two receptors. Agonists that enhanced coupling above the low intrinsic level of the MOP receptor were inverse agonists in reducing the greater level of constitutive coupling of the DOP receptor. Yet the intrinsic activities of such ligands are identical when scaled over the GDP base line of both receptors. This pattern is in conflict with the predictions of the ternary complex model and the "two state" extensions. According to this theory, the order of spontaneous and ligand-induced coupling cannot be reversed if a shift of the equilibrium between active and inactive forms raises constitutive activation in one receptor type. We propose that constitutive activation results from a lessened intrinsic barrier that restrains spontaneous coupling. Any ligand, regardless of its efficacy, must enhance this constraint to stabilize the ligand-bound complexed form.

New Insights into Structural Determinants for Prostanoid Thromboxane A2 Receptor- and Prostacyclin Receptor-G Protein Coupling

G protein-coupled receptors (GPCRs) interact with heterotrimeric G proteins and initiate a wide variety of signaling pathways. The molecular nature of GPCR-G protein interactions in the clinically important thromboxane A2 (TxA(2)) receptor (TP) and prostacyclin (PGI(2)) receptor (IP) is poorly understood. The TP activates its cognate G protein (Gαq) in response to the binding of thromboxane, while the IP signals through Gαs in response to the binding of prostacyclin. Here, we utilized a combination of approaches consisting of chimeric receptors, molecular modeling, and site-directed mutagenesis to precisely study the specificity of G protein coupling. Multiple chimeric receptors were constructed by replacing the TP intracellular loops (ICLs) with the ICL regions of the IP. Our results demonstrate that both the sequences and lengths of ICL2 and ICL3 influenced G protein specificity. Importantly, we identified a precise ICL region on the prostanoid receptors TP and IP that can switch G protein specificities. The validities of the chimeric technique and the derived molecular model were confirmed by introducing clinically relevant naturally occurring mutations (R60L in the TP and R212C in the IP). Our findings provide new molecular insights into prostanoid receptor-G protein interactions, which are of general significance for understanding the structural basis of G protein activation by GPCRs in basic health and cardiovascular disease.

Extracellular Loop II Modulates GTP Sensitivity of the Prostaglandin EP3 Receptor

Unlike the majority of G protein-coupled receptors, the prostaglandin E(2) (PGE(2)) E-prostanoid 3 (EP3) receptor binds agonist with high affinity that is insensitive to the presence of guanosine 5[prime]-O-(3-thio)triphosphate (GTPγS). We report the identification of mutations that confer GTPγS sensitivity to agonist binding. Seven point mutations were introduced into the conserved motif in the second extracellular loop (ECII) of EP3, resulting in acquisition of GTP-sensitive agonist binding. One receptor mutation W203A was studied in detail. Loss of agonist binding was observed on intact human embryonic kidney 293 cells expressing the W203A receptor, conditions where high GTP levels are present; however, high affinity binding [(3)H]PGE(2) was observed in broken cell preparations washed free of GTP. The [(3)H]PGE(2) binding of W203A in broken cell membrane fractions was inhibited by addition of GTPγS (IC(50) 21 ± 1.8 nM). Taken together, these results suggest that the wild-type EP3 receptor displays unusual characteristics of the complex coupled equilibria between agonist-receptor and receptor-G protein interaction. Moreover, mutation of ECII can alter this coupled equilibrium from GTP-insensitive agonist binding to more conventional GTP-sensitive binding. This suggests that for the mutant receptors, ECII plays a critical role in linking the agonist bound receptor conformation to the G protein nucleotide bound state.

Receptor-G Protein Interaction Studied by Bioluminescence Resonance Energy Transfer: Lessons from Protease-Activated Receptor 1

Since its development, the bioluminescence resonance energy transfer (BRET) approach has been extensively applied to study G protein-coupled receptors (GPCRs) in real-time and in live cells. One of the major aspects of GPCRs investigated in considerable details is their physical coupling to the heterotrimeric G proteins. As a result, new concepts have emerged, but few questions are still a matter of debate illustrating the complexity of GPCR-G protein interactions and coupling. Here, we summarized the recent advances on our understanding of GPCR-G protein coupling based on BRET approaches and supported by other FRET-based studies. We essentially focused on our recent studies in which we addressed the concept of preassembly vs. the agonist-dependent interaction between the protease-activated receptor 1 (PAR1) and its cognate G proteins. We discussed the concept of agonist-induced conformational changes within the preassembled PAR1-G protein complexes as well as the critical question how the multiple coupling of PAR1 with two different G proteins, Gαi1 and Gα12, but also β-arrestin 1, can be regulated.

Disease-causing Mutation in PKR2 Receptor Reveals a Critical Role of Positive Charges in the Second Intracellular Loop for G-protein Coupling and Receptor Trafficking

Prokineticins are a pair of signal factors involved in many physiological processes by binding to two closely related G-protein-coupled receptors, PKR1 and PKR2. Recently, mutations in prokineticin 2 (PK2) and PKR2 are found to be associated with Kallmann syndrome and/or idiopathic hypogonadotropic hypogonadism, disorders characterized by delayed puberty and infertility. However, little is known how PKRs interact and activate G-proteins to elicit signal transduction. In the present study, we took advantage of one disease-associated mutation (R164Q) located in the second intracellular (IL2) loop of PKR2, to investigate the role of IL2 loop in the cell signaling, G-protein binding and receptor trafficking. R164Q mutant PKR2 showed normal cell surface expression and ligand binding capacity. However, the PKR2 signaling was abolished by R164Q mutation. We demonstrated that R164Q mutation disrupted the interaction of IL2 loop to the Gα(q), Gα(i), and Gα(16)-proteins. A positive-charged amino acid at this position is required for proper function, and the signaling efficacy and potency depend on the net amount of positive charges. We also demonstrated that the interactive partner of Arg-164 may localize in the C-terminal five residues of Gα(q)-protein. A series of mutation analysis indicated that the basic amino acids at the C terminus of IL2 loop may function cooperatively in GPCRs. Furthermore, R164Q mutation also results in minimal ligand-induced endocytosis of PKR2. As many GPCRs share structural homology in the C terminus of IL2 loop, our findings may have general application in understanding structure and function of GPCRs.

G Proteins in Reverse Mode: RECEPTOR-MEDIATED GTP RELEASE INHIBITS G PROTEIN AND EFFECTOR FUNCTION

Active G protein-coupled receptors activate heterotrimeric Galphabetagamma proteins by catalyzing the exchange of GDP by GTP at the Galpha subunit. A paradoxical attenuation of G protein-activated inwardly rectifying potassium channels (GIRK) upon stimulation of native cells with high concentrations of agonist is known. However, a deactivation of activated G proteins by active receptors has not been experimentally studied in intact cells. We monitored GIRK currents and G(o) protein activation by means of fluorescence resonance energy transfer (FRET) in parallel. The results suggested that GIRK currents were paradoxically attenuated due to an inactivation of G(o) proteins by active alpha(2A)-adrenergic receptors. To study the mechanisms, G protein activation and receptor-G protein interactions were analyzed as a function of nucleotide type and nucleotide concentrations by means of FRET, while controlling intracellular nucleotides upon permeabilization of the cell membrane. Results suggested a receptor-catalyzed dissociation of GTP from activated heterotrimeric Galphabetagamma. Consequently, nucleotide-free G proteins were sequestrated in heterotrimeric conformation at the active receptor, thus attenuating downstream signaling in an agonist-dependent manner.

Inhibition of Heterotrimeric G Protein Signaling by a Small Molecule Acting on Gα Subunit

The simultaneous activation of many distinct G protein-coupled receptors (GPCRs) and heterotrimeric G proteins play a major role in various pathological conditions. Pan-inhibition of GPCR signaling by small molecules thus represents a novel strategy to treat various diseases. To better understand such therapeutic approach, we have characterized the biomolecular target of BIM-46187, a small molecule pan-inhibitor of GPCR signaling. Combining bioluminescence and fluorescence resonance energy transfer techniques in living cells as well as in reconstituted receptor-G protein complexes, we observed that, by direct binding to the Galpha subunit, BIM-46187 prevents the conformational changes of the receptor-G protein complex associated with GPCR activation. Such a binding prevents the proper interaction of receptors with the G protein heterotrimer and inhibits the agonist-promoted GDP/GTP exchange. These observations bring further evidence that inhibiting G protein activation through direct binding to the Galpha subunit is feasible and should constitute a new strategy for therapeutic intervention.

Stereochemistry of an Agonist Determines Coupling Preference of β2-Adrenoceptor to Different G Proteins in Cardiomyocytes

A fundamental question regarding receptor-G protein interaction is whether different agonists can lead a receptor to different intracellular signaling pathways. Our previous studies have demonstrated that although most beta(2)-adrenoceptor agonists activate both G(s) and G(i) proteins, fenoterol, a full agonist of beta(2)-adrenoceptor, selectively activates G(s) protein. Fenoterol contains two chiral centers and may exist as four stereoisomers. We have synthesized a series of stereoisomers of fenoterol and its derivatives and characterized their receptor binding and pharmacological properties. We tested the hypothesis that the stereochemistry of an agonist determines selectivity of receptor coupling to different G protein(s). We found that the R,R isomers of fenoterol and methoxyfenoterol exhibited more potent effects to increase cardiomyocyte contraction than their S,R isomers. It is noteworthy that although (R,R)-fenoterol and (R,R)-methoxyfenoterol preferentially activate G(s) signaling, their S,R isomers were able to activate both G(s) and G(i) proteins as evidenced by the robust pertussis toxin sensitivities of their effects on cardiomyocyte contraction and on phosphorylation of extracellular signal-regulated kinase 1/2. The differential G protein selectivities of the fenoterol stereoisomers were further confirmed by photoaffinity labeling studies on G(s),G(i2), and G(i3) proteins. The inefficient G(i) signaling with the R,R isomers is not caused by the inability of the R,R isomers to trigger the protein kinase A (PKA)-mediated phosphorylation of the beta(2)-adrenoceptor, because the R,R isomers also markedly increased phosphorylation of the receptor at serine 262 by PKA. We conclude that in addition to receptor subtype and phosphorylation status, the stereochemistry of a given agonist plays an important role in determining receptor-G protein selectivity and downstream signaling events.