Opioid receptor regulates multiple effector systems in same cells with similar but distinct properties. Our previous findings indicate that the opioid agonist activates the Gi/Go proteins mediating the receptor functions with similar potencies. Hence, we hypothesize that integration of the opioid receptor signals must occur and this is accomplished by the formation of protein scaffolds or receptor signaling units. By recruiting different proteins to the receptor vicinity, e.g., beta-arrestin, Src, RGS, AGS etc., the magnitude and duration of the signals between effector systems could be modulated. During the past funding period, we have initiated studies to demonstrate our hypotheses. We examined the receptor domains involved in the G protein activation by mutational analyses and receptor chimera studies. Using adenoviruses to deliver the PTX-insensitive G-alpha subunits, we demonstrated the Get specificity in mediating opioid receptor functions. We have established the existing of signaling units by demonstrating the existence of heterodimeric receptor-G protein complexes and in identifying cellular proteins, e.g., the FK506 binding protein FKBP12 that interacted specifically with the carboxyl tail domains of MOR (mu-opioid receptor). The alteration in FKBP12 level could affect MOR regulation of intracellular Ca2+ homeostasis. Hence, in the current proposal, we will continue these on-going projects in order to determine and characterize the components of opioid receptor signaling units. We will use both the yeast two-hybrid and proteomic approaches to identify proteins that require multiple receptor domains for interaction. The receptor mutants that are defective in either signaling or trafficking will be used to characterize the roles of these proteins in opioid receptor functions. Over-expression of these proteins with adenoviruses, or the knockdown of these proteins levels in neuroblastoma N2A cells by siRNA approach will be carried out to determine their effects on two of the effectors regulated by opioid receptor, i.e., adenylyl cyclase and intracellular Ca2+ homeostasis. The alteration of these proteins levels in primary hippocampal cell cultures enriched in neurons expressing endogenous MOR will be carried out also. An inducible vector based siRNA silencing of these genes will be developed and used in our studies. These experiments are designed to address the mechanism in the neuronal integration of opioid receptor signals. Understanding of the mechanism will allow the eventual elucidation of the in vivo regulation of opioid receptor signaling during chronic agonist administration. ? ?
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