ROLE OF INTERNALIZATION IN THE FUNCTIONAL REGULATION OF OPIOID & ORL-1 RECEPTORS

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To study the trafficking of mu, delta and ORL-1 receptors and their ligands following receptor activation. a). What is the cellular distribution and/or vesicular localization of mu receptors both in vitro and in vivo following various acute and chronic agonist treatments. In vitro, is the trafficking of the delta and ORL-1 receptors similar to that of the mu receptor? b) With the development and use of fluorescent opioid peptide ligands, can we study the trafficking of ligands and receptors be observed in real time. C) What are the kinetics of recover of surface opioid and ORL-1 receptors following agonist treatment both in vitro and in vivo? What are the processes involved in the recovery of surface receptor in vitro. 2. To identify the structural features of opioid ligands which are critical for triggering mu, delta,, and ORL-1 receptor sequestration and determine if any correlation exists between a ligand's ability to trigger internalization and its potency or efficacy for other receptor functions. a) What structural features of peptide and alkaloid ligands are required to induce sequestration of mu, delta and ORL-1 receptors in vitro. b) What is the relationship between a ligand's ability to trigger internalization and its binding affinity potency or efficacy in inhibition of cAMP accumulation and stimulation of GTPgammaS binding in mu, delta and ORL-1 receptors. c) Is an agonist's capacity to cause internalization the same in vivo as in vitro for mu, delta and ORL-1 receptors? 3. To determine the functional significance of sequestration of mu receptors. a. Is there a difference in desensitization or resensitization of the mu receptor in vitro between agonists that induce sequestration verses those that do not? b. Do mu agonists that induce internalization have the same effects on long-term potentiation (LTP) as those which do not? c. How do adaptational processes differ between wild type and internalization-deficient mutant mu receptors? d. What are the behavioral manifestations and pharmacological consequences of opioid treatment in a mouse expressing an internalization-deficient mu receptor? 4. To determine whether mu, delta, or ORL-1 receptor internalization can be observed under conditions which induce the endogenous release of opioid peptides. a. Can receptor sequestration be observed in the CNS following stress- induced analgesia (SIA) using the swim stress model? b. Does amygdala kindling result in receptor sequestration? c. Does stimulation of the perforant pathway of the hippocampus result in receptor sequestration?