The allosteric vestibule of a seven transmembrane helical receptor controls G-protein coupling

Abstract

Seven transmembrane helical receptors (7TMRs) modulate cell function via different types of G proteins, often in a ligand-specific manner. Class A 7TMRs harbour allosteric vestibules in the entrance of their ligand-binding cavities, which are in the focus of current drug discovery. However, their biological function remains enigmatic. Here we present a new strategy for probing and manipulating conformational transitions in the allosteric vestibule of label-free 7TMRs using the M2 acetylcholine receptor as a paradigm. We designed dualsteric agonists as 'tailor-made' chemical probes to trigger graded receptor activation from the acetylcholine-binding site while simultaneously restricting spatial flexibility of the receptor's allosteric vestibule. Our findings reveal for the first time that a 7TMR's allosteric vestibule controls the extent of receptor movement to govern a hierarchical order of G-protein coupling. This is a new concept assigning a biological role to the allosteric vestibule for controlling fidelity of 7TMR signalling.

Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor

“…This new global analysis is superior to previous analyses (25,26,28,34) as it yields the allosteric affinity K B of a dualsteric ligand that has not been experimentally accessible before. Iper-6-naph displays partial M 2 AChR activation in relation to acetylcholine (ACh), which defines the maximal effect of the system (Fig.…”

Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics

“…Consistent with previous studies (5), the wild-type P1pal-19 did not compensate for deleterious mutations in the receptor i3 loop and therefore is not likely to activate G protein signaling by acting as a direct replacement for the intact receptor third intracellular loop. NMR spectra included total correlation spectroscopy with a mixing time of 40 ms, NOESY with a mixing time of 100 ms, and natural abundance 13 C heteronuclear single quantum coherence spectroscopy. Data were collected at 600 and 800 MHz at 25°C.…”

“…Cercopithecus aethiops kidney cells (COS7) and human embryonic cells (HEK293) were transfected with T7-PAR1 mutants using Lipofectamine (31) for inositol phosphate (InsP) and fluorescence assays and calcium phosphate for binding studies, and stable PAR1 Rat 1 cells (5) and Rat 1 PAR1-null cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in 5% CO 2 4 , 0.02% NaN 3 , 1.1 mM deuterated dithiothreitol (DTT), and 0.1 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid at pH 5.12. NMR spectra included total correlation spectroscopy with a mixing time of 40 ms, nuclear Overhauser effect spectroscopy (NOESY) with a mixing time of 100 ms, and natural abundance 13 C heteronuclear single quantum coherence spectroscopy. Data were collected at 600 and 800 MHz at 25°C.…”

“…Pepducin agonists are allosteric modulators that typically exhibit biphasic agonist and inhibitory activity on receptor-G protein signaling (5) and can favor receptor-G protein engagement of one class of G proteins over others, so-called biased agonism (11,12). Allosteric agonists and antagonists that bind at alternative sites to the orthosteric site (13) can provide certain advantages such as a greater GPCR subtype specificity and G protein-selective pathway engagement (14). Libraries of pepducins can be readily targeted to a GPCR of interest based on the sequences of the intracellular loops, including orphan GPCRs, and tested for allosteric activity by high throughput screening (15,16).…”

Discovery of a Novel Class of Negative Allosteric Modulator of the Dopamine D2 Receptor Through Fragmentation of a Bitopic Ligand

“…3,5 Such an approach has been successfully exploited to target GPCRs within the muscarinic acetylcholine receptor (mAChR) and adenosine receptor families. [6][7][8][9] In addition, recent studies have demonstrated that ligands that selectively target the M 1 and M 2 mAChRs may achieve their receptor-subtype-selectivity through a hitherto unappreciated bitopic mode of interaction. 10,11 One approach to explore the binding mode of bitopic ligands is to isolate both orthosteric and allosteric fragments from the parent structure.…”