Dopamine D2 receptors, the targets of all antipsychotic drugs currently available, have been reported to form dimeric and/or oligomeric complexes with many other receptors. These complexes and their functional properties are of great interest given the possibility of developing novel therapeutics that selectively target distinct receptor complexes, with the potential of reduced side-effects. Nevertheless, there is still great debate about whether these and other class A G protein-coupled receptor (GPCR) complexes even exist, as well as their potential physiological relevance. Of the methods capable of detecting receptor interactions in living cells, single-molecule imaging techniques offer the distinct advantage of potentially revealing the stochastic and dynamic behaviors of individual molecules in real time, without the population and time averaging inherent in ensemble measurements. Specifically, single-molecule fluorescence resonance energy transfer (smFRET) is a robust tool for reporting interaction distances less than 10 nm, but has rarely been used in mammalian cells, and never with GPCRs, due to multiple experimental challenges. We have now developed robust methods for imaging, tracking, and quantifying smFRET of GPCRs in living cells. In parallel, using pulsed-interleaved excitation fluorescence cross- correlation spectroscopy (PIE-FCCS) we can robustly explore interactions at much higher levels of receptor expression. While we find that the class A mu opioid receptor is a monomer over a 1000-fold range of receptor expression, our preliminary results show that the D2R does in fact transiently interact in the membrane when expressed at high densities. Furthermore, using PIE-FCCS we see strong evidence for heteromeric interactions of D2R and adenosine A2A receptor, a heteromeric pair shown to exhibit substantial functional crosstalk. We propose to study the dynamics of these interactions and to identify modulators of their kinetics, as tools to probe the potential role of homodimers and heterodimers in vivo. We will also extend our studies to the D2R-NMDA receptor (NMDAR) heteromer, a complex that we have now shown to be upregulated both in rodent and human brain exposed to psychostimulants that elevate extracellular dopamine levels. Although the mechanism is unknown, there is evidence that D2R activation leads through increased D2R-NMDAR to inhibition of NMDAR currents and reduced neuronal excitability, suggesting a novel molecular target for integration of dopamine signaling and glutamatergic signaling, both of which have been implicated in schizophrenia and psychosis. We thus propose the following specific aims to expand our lab’s ongoing multi-level approach to understanding dopamine receptor structure and function both in vitro and in vivo. Aim 1: Determine the dynamics of dopamine D2R homo- and Class A GPCR hetero-dimerization in living cells and in brain slices and identify modulators of these dynamics to probe their functional eff...