Mutations inducing divergent shifts of constitutive activity reveal different modes of binding among catecholamine analogues to the beta(2)-adrenergic receptor.

Department of Neuroscience, University of Rome, 'Tor Vergata', Rome, Italy.
British Journal of Pharmacology (Impact Factor: 4.99). 05/2002; 135(7):1715-22. DOI: 10.1038/sj.bjp.0704622
Source: PubMed

ABSTRACT 1. We compared the changes in binding energy generated by two mutations that shift in divergent directions the constitutive activity of the human beta(2) adrenergic receptor (beta(2)AR). 2. A constitutively activating mutant (CAM) and the double alanine replacement (AA mutant) of catechol-binding serines (S204A, S207A) in helix 5 were stably expressed in CHO cell lines, and used to measure the binding affinities of more than 40 adrenergic ligands. Moreover, the efficacy of the same group of compounds was determined as intrinsic activity for maximal adenylyl cyclase stimulation in wild-type beta(2)AR. 3. Although the two mutations had opposite effects on ligand affinity, the extents of change were in both cases largely correlated with the degree of ligand efficacy. This was particularly evident if the extra loss of binding energy due to hydrogen bond deletion in the AA mutant was taken into account. Thus the data demonstrate that there is an overall linkage between the configuration of the binding pocket and the intrinsic equilibrium between active and inactive receptor forms. 4. We also found that AA mutation-induced affinity changes for catecholamine congeners gradually lacking ethanolamine substituents were linearly correlated to the loss of affinity that such modifications of the ligand cause for wild-type receptor. This indicates that the strength of bonds between catechol ring and helix 5 is critically dependent on the rest of interactions of the beta-ethanolamine tail with other residues of the beta(2)-AR binding pocket.


Available from: Tommaso Costa, Jun 02, 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The concepts of functional selectivity and ligand bias are becoming increasingly appreciated in modern drug discovery programs, necessitating more informed approaches to compound classification and, ultimately, therapeutic candidate selection. Using the β2AR as a model, we present a proof of concept study that assessed the bias of 19 β-adrenergic ligands, including many clinically used compounds, across four pathways (cAMP production, ERK1/2 activation, calcium mobilization and receptor endocytosis) in the same cell background (HEK293S cells). Efficacy-based clustering placed the ligands into five distinct groups with respect to signaling signatures. In some cases, apparent functional selectivity originated from off-target effects on other endogenously expressed adrenergic receptors, highlighting the importance of thoroughly assessing selectivity of the responses before concluding receptor-specific ligand-biased signaling. Eliminating the non-selective compounds did not change the clustering of the 10 remaining compounds. Some ligands exhibited large differences in potency for the different pathways, suggesting that the nature of the receptor-effector complexes influences the relative affinity of the compounds for specific receptor conformations. Calculation of relative effectiveness (within pathway) and bias factors (between pathways) for each of the compounds, using an operational model of agonism, revealed a global signaling signature for all of the compounds, relative to isoproterenol. Most compounds were biased toward ERK1/2 activation over the other pathways, consistent with the notion that many proximal effectors converge on this pathway. Overall, we demonstrate a higher level of ligand texture than previously anticipated, opening perspectives for the establishment of pluridimensional correlations between signalling profiles, drug classification, therapeutic efficacy and safety.
    Molecular pharmacology 12/2013; 85(3). DOI:10.1124/mol.113.088880 · 4.12 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The α1A-AR is thought to couple predominantly to the Gαq/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gαq coupling-defective variants of α1A-AR, as well as a combination of Ca2+ channel blockers, uncovered cross-talk between α1A-AR and β2-AR that leads to potentiation of a Gαq-independent signaling cascade in response to α1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α1A-AR. α1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α1A-AR. Iso induced signaling at α1A-AR was further interrogated by probing steps along the Gαq /PLC, Gαs and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α1A-AR, and CHO_α1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca2+ mobilization that could be blocked by α1A-AR selective antagonists. The kinetics of Iso induced Ca2+ transients differed from typical Gαq- mediated Ca2+ mobilization, lacking both the fast IP3R mediated response and the sustained phase of Ca2+ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gαq.
    PLoS ONE 01/2015; 10(1):e0115701. DOI:10.1371/journal.pone.0115701 · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The β2-adrenergic receptor (β2-AR), a G protein-coupled receptor (GPCR), is a physiologically important transmembrane protein that is a target for drugs used for treatment of asthma and cardiovascular diseases. Study of the first steps of ligand recognition and the molecular basis of ligand binding to the orthosteric site is essential for understanding the pharmacological properties of the receptor. In this work we investigated the characteristic features of the agonist association–dissociation process to and from the different conformational forms of β2-AR by use of advanced molecular modeling techniques. The investigation was focused on estimating the free energy profiles (FEPs) corresponding to the process of a full agonist ((R,R)-fenoterol) and an inverse agonist (carazolol) binding and unbinding to and from β2-AR. The two different conformational forms of β2-AR, i.e. active β2-AR–PDB: 3P0G and inactive β2-AR–PDB: 2RH1 were included in this stage of the study. We revealed several significant qualitative differences between FEPs characteristic of both conformational forms. Both FEPs suggest the existence of three transient binding sites in the extracellular domain of β2-AR. Comparison of the residues surrounding these transient binding sites in both β2-AR states revealed the importance of the aromatic residues F194, H932.64, H2966.58, and H178 (extracellular part of β2-AR) in the early stages of the binding process. In addition, slightly different exit and entry paths are preferred by the ligand molecule in the extracellular part of β2-AR, depending on the conformation of the receptor. Electronic supplementary material The online version of this article (doi:10.1007/s00249-015-1010-4) contains supplementary material, which is available to authorized users.
    European Biophysics Journal 03/2015; 44(3). DOI:10.1007/s00249-015-1010-4 · 2.47 Impact Factor