PROJECT SUMMARY 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. Even though receptor heteromers have been convincingly demonstrated in heterologous expression studies, their overexpression in these systems, a lack of evidence from high resolution structures of receptor heteromers, the lack of tools to probe their existence, and functional importance in vivo have fueled this skepticism. In this proposal, we employ 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 2A (D2) and serotonin 2A (2A) receptor (R) heteromer that has been implicated in schizophrenia, where antipsychotics targeting one or the other receptor form the current basis of pharmacotherapy. We employ a technique to covalently link interacting residues along the interface of the D2R/2AR heteromer in order to map the heteromer interface and stabilize the complex in the active or inactive state. 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 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 an azido-phenylalanine UAA 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. These results suggest that the TM5-TM5 interface stabilizes the heteromer in the active state. The crosslinked complex that is formed can then be further studied using western blot, fluorescence resonance energy transfer (FRET), and total internal reflection fluorescence (TIRF) microscopy to analyze association of the heteromer. Mass spectrometry (MS) will be used for accurate measurement of isotopically labeled probes to definitively prove receptor crosslinking at specific residues and to confirm formation of a heteromer. PALs with a distinct isotope pattern are similarly incorporated into receptors, and when prepared with the non-labeled UAAs in a 1:1 mixture, a mass spectrometric detection of proteins with the labeled probe can be applied. We propose to expand this overall approach to test whether crosslinking the TM5-TM6 interface of the two receptors stabilizes an interface that prevents trans-signaling, stabilizing the heteromer in the inactive state. This project is conducted at Northeastern University in the labs of Drs. Diomedes Logothetis (Pharmaceutical Sciences) and Roman Manetsch (Chemistry). This interdisciplinary project of molecular biophysics and medicinal chemistry will allow for rigorous trai...