Translating biased signaling in the ghrelin receptor system into differential in vivo functions.

Biased signaling has been suggested as a means of selectively modulating a limited fraction of the signaling pathways for G-protein-coupled receptor family members. Hence, biased ligands may allow modulation of only the desired physiological functions and not elicit undesired effects associated with pharmacological treatments. The ghrelin receptor is a highly sought antiobesity target, since the gut hormone ghrelin in humans has been shown to increase both food intake and fat accumulation. However, it also modulates mood, behavior, growth hormone secretion, and gastric motility. Thus, blocking all pathways of this receptor may give rise to potential side effects. In the present study, we describe a highly promiscuous signaling capacity for the ghrelin receptor. We tested selected ligands for their ability to regulate the various pathways engaged by the receptor. Among those, a biased ligand, YIL781, was found to activate the Gαq/11 and Gα12 pathways selectively without affecting the engagement of ß-arrestin or other G proteins. YIL781 was further characterized for its in vivo physiological functions. In combination with the use of mice in which Gαq/11 was selectively deleted in the appetite-regulating AgRP neurons, this biased ligand allowed us to demonstrate that selective blockade of Gαq/11, without antagonism at ß-arrestin or other G-protein coupling is sufficient to decrease food intake.

Biased agonism and allosteric modulation of G protein-coupled receptor 183 - a 7TM receptor also known as Epstein-Barr virus-induced gene 2.

BACKGROUND AND PURPOSE: The GPCR Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) is activated by oxysterols and plays a pivotal role in the regulation of B cell migration during immune responses. While the molecular basis of agonist binding has been addressed in several studies, the concept of biased agonism of the EBI2 receptor has not been explored. EXPERIMENTAL APPROACH: We investigated the effects of the EBI2 endogenous agonist 7α,25-dihydroxycholesterol (7α,25-OHC) on G protein-dependent and -independent pathways as well as sodium ion allosterism using site-directed mutagenesis and functional studies. Moreover, we generated a homology model of the EBI2 receptor to investigate the structural basis of the allosteric modulation by sodium. KEY RESULTS: Residue N114, located in the middle of transmembrane-III at position III:11/3.35, was found to function as an efficacy switch. Thus, substituting N114 with an alanine (N114A) completely abolished heterotrimeric G protein subunit Gi α activation by 7α,25-OHC even though the specific binding of [3 H]-7α,25-OHC increased. In contrast, the N114A mutant was still able to recruit ß-arrestin and even had an enhanced potency (18.7-fold) compared with EBI2 wild type. Sodium had a negative allosteric effect on oxysterol binding that was mediated via N114, verifying the key role of N114. This was further supported by molecular modelling of the ion binding site based on a EBI2 receptor homology model. CONCLUSIONS AND IMPLICATIONS: Collectively, our data point to N114 as a key residue for EBI2 signalling controlling the balance between G protein-dependent and -independent pathways and facilitating sodium binding.

Molecular Mechanism of Action for Allosteric Modulators and Agonists in CC-chemokine Receptor 5 (CCR5).

The small molecule metal ion chelators bipyridine and terpyridine complexed with Zn2+ (ZnBip and ZnTerp) act as CCR5 agonists and strong positive allosteric modulators of CCL3 binding to CCR5, weak modulators of CCL4 binding, and competitors for CCL5 binding. Here we describe their binding site using computational modeling, binding, and functional studies on WT and mutated CCR5. The metal ion Zn2+ is anchored to the chemokine receptor-conserved Glu-283VII:06/7.39 Both chelators interact with aromatic residues in the transmembrane receptor domain. The additional pyridine ring of ZnTerp binds deeply in the major binding pocket and, in contrast to ZnBip, interacts directly with the Trp-248VI:13/6.48 microswitch, contributing to its 8-fold higher potency. The impact of Trp-248 was further confirmed by ZnClTerp, a chloro-substituted version of ZnTerp that showed no inherent agonism but maintained positive allosteric modulation of CCL3 binding. Despite a similar overall binding mode of all three metal ion chelator complexes, the pyridine ring of ZnClTerp blocks the conformational switch of Trp-248 required for receptor activation, thereby explaining its lack of activity. Importantly, ZnClTerp becomes agonist to the same extent as ZnTerp upon Ala mutation of Ile-116III:16/3.40, a residue that constrains the Trp-248 microswitch in its inactive conformation. Binding studies with 125I-CCL3 revealed an allosteric interface between the chemokine and the small molecule binding site, including residues Tyr-37I:07/1.39, Trp-86II:20/2.60, and Phe-109III:09/3.33 The small molecules and CCL3 approach this interface from opposite directions, with some residues being mutually exploited. This study provides new insight into the molecular mechanism of CCR5 activation and paves the way for future allosteric drugs for chemokine receptors.