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  • Based on the extracellular domain structures

    2024-04-15

    Based on the extracellular domain structures, we can infer that the dimer arrangement of GABAB receptor will undergo substantial changes upon receptor activation (Geng et al., 2013). First, agonist-stimulated closure of GABAB1 VFT would cause an upward rotation of its LB2 domain, coupled with an inward movement toward its GABAB2 counterpart. Second, the LB2 domain of GABAB2 subunit would also twist toward its GABAB1 counterpart, resulting in the formation of a novel interface that is sustained by polar contacts between the LB2 domains of both VFTs. The LB2-LB2 interaction allows GABAB2 VFT to stabilize the closed conformation of GABAB1 VFT, thus enhancing receptor activity. The importance of LB2-LB2 association is demonstrated by experiments where MDV3100 of a large glycan at this interface prevents agonist-induced receptor activation (Rondard et al., 2008). Furthermore, locking this LB2-LB2 interface through a disulfide bond is sufficient to confer constitutive activity to the receptor (Geng et al., 2013). These observations indicate that the LB2-LB2 interface found in the active-state GABAB1b VFT-GABAB2 VFT structure is physiologically relevant. However, the interface is not unbreakable, thereby allowing the receptor to return to its resting state. Structural observations suggest that GABAB receptor exists in a dynamic equilibrium between inactive and active conformations (Geng et al., 2013), like other GPCRs including β2-adrenergic receptor (Rosenbaum et al., 2011). Finally, the conformational changes reduce the distance between the C-termini of the two VFT modules. The decrease in the separation between VFTs is expected to be sensed by the TM domains.
    Orthosteric ligand recognition The ligand-binding subunit GABAB1 has its active and closed conformation stabilized by an agonist bound to VFT. In contrast, an antagonist would limit the receptor subunit to an inactive and open conformation. The open or closed configuration of GABAB1 VFT also determines whether the ligand in its interdomain crevice is exposed to the surrounding solvent, and could potentially influence the on- and off-rate of ligand binding. The orthosteric ligands of GABAB receptor are usually derivatives of GABA (Bowery et al., 2002, Froestl, 2010). Antagonists also feature a bulky substituent at either end that hinders closure of GABAB1 VFT (Geng et al., 2013) (Fig. 4A and B). Antagonists bind to LB1 domain via an interface composed primarily of hydrogen bonds. A majority of the antagonists studied, such as S-2-OH-saclofen, do not interact with the lower LB2 lobe at all. Two exceptions are CGP54626 and SCH50911, which manage to form direct contact with Trp278 on LB2 (Geng et al., 2013). Antagonists that maintain this interaction have stronger binding affinity than other candidates (Froestl, 2010, Geng et al., 2013). The role of LB2 in variable antagonist affinity makes it an important domain to consider when determining antagonist selectivity for pharmacological purposes. GABA and the clinical drug R-baclofen are among the best-understood GABAB receptor agonists (Bowery et al., 2002, Froestl, 2010). An agonist closes the groove of GABAB1 by interacting with both LB1 and LB2 domains of the VFT (Geng et al., 2013) (Fig. 4C and D). The two ends of an agonist are each held by a network of hydrogen bonds. An identical set of LB1 residues mediate the binding of agonists and antagonists. However, only agonists interact with both LB2 residues Tyr250 and Trp278. The difference between GABA and R-baclofen-bound GABAB1 is a complete flip of the indole ring found in Trp278, indicating that the orthosteric ligand-binding site of GABAB receptor exhibits plasticity and can accommodate various agonist structures without altering ligand affinity (Fig. 4E). The interdomain cleft of VFT is also the orthosteric ligand-binding site of all class C GPCRs (Kniazeff et al., 2011). These receptors have evolved an affinity for amino acids and their analogs (Bowery et al., 2002, Chandrashekar et al., 2006, Conigrave and Hampson, 2010, Conigrave and Ward, 2013, Froestl, 2010, Kniazeff et al., 2011, O'Hara et al., 1993). This is true even in the case of CaS receptor. Although it is traditionally held that Ca2+ is the principal agonist of CaS receptor, recent research shows that the VFT cleft of activated CaS receptor is solely occupied by an L-amino acid, L-Trp, instead of Ca2+ (Geng et al., 2016). Indeed, Ca2+ and L-amino acids serve as co-agonists to jointly trigger CaS receptor response (Geng et al., 2016).