Allosteric ligands control the activation of a class C GPCR heterodimer by acting at the transmembrane interface

Abstract

G protein-coupled receptors (GPCRs) are among the most promising drug targets. They often form homo- and heterodimers with allosteric cross-talk between receptor entities, which contributes to fine tuning of transmembrane signaling. Specifically controlling the activity of GPCR dimers with ligands is a good approach to clarify their physiological roles and to validate them as drug targets. Here, we examined the mode of action of positive allosteric modulators (PAMs) that bind at the interface of the transmembrane domains of the heterodimeric GABAB receptor. Our site-directed mutagenesis results show that mutations of this interface impact the function of the three PAM tested. The data support the inference that they act at the active interface between both transmembrane domains, the binding site involving residues of the TM6s of the GABAB1 and the GABAB2 subunit. Importantly, the agonist activity of these PAMs involves a key region in the central core of the GABAB2 transmembrane domain, which also controls the constitutive activity of the GABAB receptor. This region corresponds to the sodium ion binding site in class A GPCRs that controls the basal state of the receptors. Overall, these data reveal the possibility of developing allosteric compounds able to specifically modulate the activity of GPCR homo- and heterodimers by acting at their transmembrane interface.

Cholesterol in Class C GPCRs: Role, Relevance, and Localization

“…All GPCRs in a lipid bilayer are not stand-alone molecules, rather they interact with other components of the bilayer e.g., lipids and sterols, most notably cholesterols [ 1 ]. Some members of GPCRs exist and function as monomers, especially within the class A GPCRs, while other GPCRs, mostly the class C GPCRs, form dimers [ 2 , 3 ], and oligomers with themselves or other GPCRs [ 4 ]. Most recently, several determined GPCR structures often existing as dimers, appear to indicate the presence of cholesterol.…”

G protein coupling and activation of the metabotropic GABAB heterodimer

“…These are called ago-PAMs 21 . It has been reported that ago-PAMs can cause the conformational changes in GB1-VFT and TMD observed upon agonist activation 22 , 23 . BHFF is a known ago-PAM, which is capable of causing the receptor activity alone 23 .…”

“…It has been reported that ago-PAMs can cause the conformational changes in GB1-VFT and TMD observed upon agonist activation 22 , 23 . BHFF is a known ago-PAM, which is capable of causing the receptor activity alone 23 . Since BHFF binds to the dimeric interface, it is speculated that it increases the chance for the GP engagement by stabilizing the active TM6-TM6 interface even without the agonist.…”

Probing GPCR Dimerization Using Peptides

“…Allosteric modulators usually have differing and enhanced physiochemical properties that ligands which solely bind to orthosteric binding sites may possess, which could aid in the design and development of ligands which target PPI surfaces. Studies have shown that targeting the heterodimeric gamma-aminobutyric acid B (GABA B ) receptor, using positive allosteric modulators (PAMs) that can bind at the interface of the transmembrane domains, can control the activation of this class C GPCR dimer ( 97 ). PAMs are allosteric modulators that enhance the agonist affinity for the receptor and negative allosteric modulators (NAMs) will inhibit the agonist affinity ( 98 ).…”

“…PAMs are allosteric modulators that enhance the agonist affinity for the receptor and negative allosteric modulators (NAMs) will inhibit the agonist affinity ( 98 ). The GABA B PAMs bind in the transmembrane interface which is conserved across most GPCRs, making these findings important for not only class C GPCRs, but for the whole superfamily ( 97 ). Importantly, the discovery of allosteric modulators is mostly limited to computational analysis, providing an obstacle for the development of these compounds.…”