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

Abstract

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 ␤ 2 -adrenoceptor agonists activate both G s and G i proteins, fenoterol, a full agonist of ␤ 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 ␤ 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.

Quantification of Functional Selectivity at the Human α1A-Adrenoceptor

“…There may be a steric effect of the ␣-ethyl group that compromises receptor conformational changes linked to G protein activation, without affecting receptor phosphorylation or arrestin binding. In cardiac myocytes, the S,R-isomer of fenoterol stimulates substantially higher activation of G␣ i2 than the R,R-isomer, whereas (R,R)-fenoterol produces higher activation of G␣s than (S,R)-fenoterol (Woo et al, 2009). This differential G s /G i coupling is apparent in functional assays including myocyte contractility and Erk1/2 phosphorylation, and the authors suggest that agonists selectively able to stimulate ␤ 2 -AR Gs coupling, without stimulating ␤ 2 -AR G i coupling or ␤ 1 -AR activation, may have considerable therapeutic benefit.…”

Cannabinoid Receptor Activation Correlates with the Proapoptotic Action of the β2-Adrenergic Agonist (R,R′)-4-Methoxy-1-Naphthylfenoterol in HepG2 Hepatocarcinoma Cells

“…The basis for the ligand-specific differences in pharmacological outcome lies in the interplay between the molecular structure of the agonist (Kahsai et al, 2011) and the cellular environment of the receptor. In the first instance, we have shown that the G s /G i selectivity of Fen is a function of molecular structure and stereochemistry, because Fen preferentially activated G S signaling in a cardiomyocyte contractility model, whereas (S,RЈ)-fenoterol and MNF activated both G S and G i proteins (Woo et al, 2009;Jozwiak et al, 2010).…”

Regulation of G protein subunit composition in cardiomyocytes: pharmacological implications

“…For example, in a seminal study, Michel Bouvier's group showed that the pharmacological profile of the β 1 AR with regard to cAMP accumulation is quite different from the profile with respect to the activation of extracellular signal-regulated kinase (ERK) pathways (Galandrin et al 2008). Similarly, the pharmacological profile of the recombinant β 2 AR with respect to coupling to G i and G s proteins in insect cell membranes is different (Wenzel-Seifert and Seifert 2000), and certain stereoisomers of β 2 AR ligands differ in their capacity to direct β 2 AR signaling towards G i versus G s proteins (Woo et al 2009;Seifert and Dove 2009). Coupling of a given GPCR to a specific G protein is not only influenced by the ligand bound to the receptor but also by the expression level of the receptor, the concentration of the G protein in the vicinity of the receptor, and the particular cell type examined (Kenakin 2007(Kenakin , 2009Schneider and Seifert 2010).…”

“…Accordingly, the role of G iα2 must be studied in greater detail. Moreover, ligand-specific β 2 AR coupling to G i and G s proteins has been observed in cardiomyocytes (Woo et al 2009). Furthermore, several therapeutically relevant βAR antagonists clinically used for the treatment of cardiovascular diseases including chronic heart failure, hypertension, and coronary heart disease constitute, in fact, not simple antagonists but rather ligands exhibiting biased signaling (Galandrin et al 2008;Rajagopal et al 2010).…”

“…Many βAR ligands have one or two chirality centers, but routinely, racemic mixtures are used in most studies (Seifert and Dove 2009), availability to researchers being the major reason. However, considering that fact that defined stereoisomers of βAR ligands stabilize unique receptor conformations and exhibit distinct signaling capacities (Seifert et al 2001;Woo et al 2009), it is highly important to examine pairs of chemically pure stereoisomers. Unfortunately, this is not trivial since for most important βAR ligands, only the potent and supposedly "active" eutomer is commercially available, whereas the less potent and supposedly "inactive" distomer is not.…”