Abstract Opioid and catecholamine receptors are key regulators of neurophysiology and behavior, and are important targets of therapeutic and abused drugs. These receptors all belong to the G protein-coupled receptor (GPCR) superfamily, the largest group of signaling receptors expressed in animals and a very important class of drug targets. GPCRs signal by allostery and are extensively regulated after ligand-induced activation by phosphorylation, endocytosis and interacting with a class of cytoplasmic adaptor proteins called arrestins. These regulatory processes are critically important to the actions of addictive drugs, which are typically administered repeatedly or over a prolonged period, and there is evidence for considerable diversity of the effects of drugs on these processes. The present research program is focused on elucidating the fundamental mechanistic basis of such selective regulation. In the previous funding period, we delineated drug-selective biochemical modes by which a functionally relevant GPCR kinase is recruited from the cytoplasm by opioid receptors. We also identified evidence for a discrete form of biased drug action determined by differences in the subcellular location of receptor activation. We also discovered a distinct mechanism of cellular arrestin regulation that defined by being independent of receptor phosphorylation. The proposed studies seek to extend this fundamental research effort with the goal of developing new understanding that can be leveraged for therapeutic benefit. Specifically, we propose to (1) Define an allosteric basis for agonist-selective phosphorylation of opioid receptors; (2) Delineate localization and trafficking of opioid-relevant adenylyl cyclase isoforms; and (3) Determine if β-arrestin has a phosphorylation-independent role in opioid drug discrimination.