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    • Since the cloning of ARs

    2023-05-25

    Since the cloning of ARs in the beginning of the 1990s, the efforts to characterize them have led to the accumulation of a substantial amount of experimental data. Decades of site-directed mutagenesis (SDM; Box 1) studies, in combination with pharmacological data and computational modeling, have paved the way for understanding receptor–ligand binding and activation signaling pathways. More recently, advances in membrane protein engineering and crystallography have sparked a surge of experimental GPCR structures [8], among which ARs have emerged as one of the most thoroughly characterized families: several structures are available for the inactive conformation of the human A2AAR bound to different chemotypes of orthosteric antagonists (Box 2) 9, 10, 11. Some of these reveal allosteric sites, like the binding site for the negative allosteric modulator (NAM) sodium ion (Na+) [12], or potential allosteric sites in the extracellular loop (EL) region [13]. Recently, the A1AR has been crystallized in complex with xanthine antagonists 14, 15. In addition, active-like (agonist-bound) structures of the A2AAR have been obtained with several adenosine derivatives 16, 17, 18, lately complemented with the first fully active conformation bound to an engineered G protein fragment [19]. The current structural information is further supplemented by data obtained from additional, complementary techniques (Box 1). These include nuclear magnetic resonance (NMR) 20, 21 or the mutagenesis/modeling combination known as biophysical mapping (BPM) [22]; a technique recently used to design A2AAR antagonists [23]. These different sources of experimental data can be integrated in computational models, which are used to elucidate the molecular determinants of ligand binding and receptor function [24]. In the ARs, protocols like proteochemometrics (PCM) [25] or free MK-4827 perturbation (FEP) 24, 26 have successfully filled the gap between affinity and structural data (Box 1). We herein review all the available mutagenesis data on the light of the structural information available for ARs. To do so, the existing SDM data were systematically collected from 80 individual publications and the resulting 2624 curated data points, of which 96% are from human receptors, were made available through the GPCRdb [27]. A comprehensive mapping of these data onto the available structural information provides an overview of ligand binding and activation events, which we present here in three sections: (i) orthosteric ligand binding; (ii) allosteric modulation; and (iii) receptor activation and G protein binding (Figure 1, Key Figure).
    Orthosteric Ligand Binding
    Extracellular Region: Ligand Kinetics and Receptor Architecture Mutations in the ELs do not only influence binding of orthosteric ligands, but may also play a role in ligand kinetics. In addition, this region plays a structural role through a series of cysteine bridges, and has been related to selectivity among certain receptor subtypes. Finally, mutational studies and lately crystal structures [11] suggest that this region might be the binding site of PAMs, in analogy to other GPCRs [51]. Here, we discuss the mutational data of the EL region in ARs.
    Sites for Allosteric Modulation in ARs Allosteric modulation of GPCRs is gaining acceptance as a new approach for drug development, since allosteric ligands typically display higher target selectivity compared to orthosteric ligands 3, 58. In ARs, two distinct receptor regions have been revealed as sites for allosteric modulation (Figure 3): the EL2 region and the sodium-binding pocket.
    Receptor Signaling and G Protein Binding The intracellular side of the TM bundle is more conserved within the GPCR superfamily than the extracellular domain [2]. This region undergoes the most pronounced conformational changes upon receptor activation and is implicated in the binding of the intracellular G protein. Four motifs play a major role here (Figure 4): NPxxY in TM7, the DRY motif (TM3), the ionic lock and the TDY triad, as observed in the G protein-bound crystal structure [19].