The essential role for aromatic cluster in the β3 adrenergic receptor.

Aim: To explore the function of the conserved aromatic cluster F213(5.47), F308(6.51), and F309(6.52) in human β3 adrenergic receptor (hβ3AR).

Methods: Point mutation technology was used to produce plasmid mutations of hβ3AR. HEK-293 cells were transiently co-transfected with the hβ3AR (wild-type or mutant) plasmids and luciferase reporter vector pCRE-luc. The expression levels of hβ3AR in the cells were determined by Western blot analysis. The constitutive signalling and the signalling induced by the β3AR selective agonist, BRL (BRL37344), were then evaluated. To further explore the interaction mechanism between BRL and β3AR, a three-dimensional complex model of β3AR and BRL was constructed by homology modelling and molecular docking.

Results: For F308(6.51), Ala and Leu substitution significantly decreased the constitutive activities of β3AR to approximately 10% of that for the wild-type receptor. However, both the potency and maximal efficacy were unchanged by Ala substitution. In the F308(6.51)L construct, the EC(50) value manifested as a "right shift" of approximately two orders of magnitude with an increased E(max). Impressively, the molecular pharmacological phenotype was similar to the wild-type receptor for the introduction of Tyr at position 308(6.51), though the EC(50) value increased by approximately five-fold for the mutant. For F309(6.52), the constitutive signalling for both F309(6.52)A and F309(6.52)L constructs were strongly impaired. In the F309(6.52)A construct, BRL-stimulated signalling showed a normal E(max) but reduced potency. Leu substitution of F309(6.52) reduced both the E(max) and potency. When F309(6.52) was mutated to Tyr, the constitutive activity was decreased approximately three-fold, and BRL-stimulated signalling was significantly impaired. Furthermore, the double mutant (F308(6.51)A_F309(6.52)A) caused the total loss of β3AR function. The predicted binding mode between β3AR and BRL revealed that both F308(6.51) and F309(6.52) were in the BRL binding pocket of β3AR, while F213(5.47) and W305(6.48) were distant from the binding site.

Conclusion: These results revealed that aromatic residues, especially F308(6.51) and F309(6.52), play essential roles in the function of β3AR. Aromatic residues maintained the receptor in a partially activated state and significantly contributed to ligand binding. The results supported the common hypothesis that the aromatic cluster F[Y]5.47/F[Y]6.52/F[Y]6.51 conserved in class A G protein-coupled receptor (GPCR) plays an important role in the structural stability and activation of GPCRs.