Opioid receptors mediate the physiological actions of native opioid peptides and are the pharmacologic targets of highly addictive analgesic drugs. Individual subtypes of opioid receptor differ in their subcellular localization, serve specialized pre- and post-synaptic functions, and can undergo rapid endocytic membrane trafficking following ligand-induced activation. Thus opioid receptors are an important class of neural membrane proteins that, like synaptic vesicle and dense core vesicle membrane proteins, undergo regulated redistribution between the cell surface and specific endomembrane compartments. Mechanisms mediating regulated endocytosis of opioid receptors have been examined in some detail in non-neural cell types. However, little is known about mechanisms or pathways that mediate opioid receptor membrane trafficking in neurosecretory cells. Furthermore, while individual opiate drugs differ greatly in their ability to induce opioid receptor endocytosis in vivo when administered at clinically relevant doses, the physiological consequences of this regulated membrane trafficking are not understood. The Specific Aims of the proposed studies are to (1) Determine mechanisms mediating ligand-induced endocytosis of opioid receptors in PC12 cells and cultured neurons; (2) Characterize membrane pathway(s) mediating regulated endocytosis of opioid receptors in neurosecretory cells and cultured neurons; and (3) Create a transgenic mouse model to examine the physiological relevance of mu opioid receptor endocytosis to opiate drug action. These studies will provide fundamental new insight into membrane trafficking mechanisms that control the subcellular localization of opioid receptors in neurons, and will directly address the physiological relevance of these mechanisms to the in vivo effects of opiate drugs.