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

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

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

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 β-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.