Unnatural amino acid receptor incorporation as a photoaffinity tool for GPCR heteromer signaling studies.

Abstract

Class A G protein-coupled receptors (GPCRs) have been reported by multiple groups to form receptor heteromers and serve as allosteric regulators of one another. Yet their mere existence has become a highly contested topic. We have employed a minimally invasive approach of introducing unnatural amino acids (UAAs), using amber codon suppression, at strategic positions predicted to form the heteromeric interface of the dopamine 2 (D2) and serotonin 2A (2A) receptor (R) heteromer that has been implicated in schizophrenia. Photoaffinity labeling (PAL) is a technique to investigate binding interactions by forming a covalent bond between two entities via irradiation of a photoactivatable group. UAAs can function as photoaffinity probes. By UV irradiation of the strategically incorporated UAA, the singlet carbene that is produced is crosslinked with the corresponding receptor to form a covalently bonded dimer. We show that when 4-azido-L-phenylalanine (AzF) replaces D2R(Y199) in TM5, trans-signaling to 2AR is potentiated upon UV irradiation, as does its interacting residue 2AR(F244) that is also in the TM5. Mutation of the nonlabeled D2R(Y199) or the D2R(Y199) to Leu abolishes the UV irradiation effect. These results suggest that the TM5-TM5 interface stabilizes the heteromer in the trans-signaling state. Our model of the heteromer interface predicts that 2AR(F244) also interacts with the D2R(L388) in TM6. Crosslinking the TM5-TM6 interface stabilizes the heteromer in the non-trans-signaling state. Results from this minimally invasive approach that support the predicted trans-signaling and non-trans-signaling interfaces lend more evidence the D2R and 2AR form functionally meaningful heteromers.