The purpose of this K05 award application is to allow the principal investigator to focus his attention on his on-going research projects and to take periodic leaves of absence from his administrative and teaching commitments at University of Minnesota. The K05 award will allow PI to spend time in his collaborators' laboratories and pursue new or alternative approaches to his research goals. The career goal of PI has been the elucidation of the molecular mechanism of opioid tolerance and dependence. Tolerance and dependence to the repeated use of opioid drugs can be the consequences of the cellular compensatory responses to the signals being transduced by the receptors. Thus, it is of utmost importance to address an overall objective of PI's laboratory, i.e., the understanding of the mechanism in which neuronal cells could integrate the signals transduced by membrane receptors that utilize the same spectrum of second messenger systems. Our previous studies have demonstrated that the cloned opioid receptors coupled and activated the Gi/Go proteins with similar potencies. We have established also that there are distinct differences between mu and delta opioid receptor regulation of the same second messenger such as adenylyl cyclase. The probability exists for the involvement of cellular proteins other than the heterotrimeric G proteins in the opioid receptor signaling. Thus, it is our hypothesis that mu- and delta-opioid receptors utilize different G proteins for the regulation of the same effector, and that this is due to the subtle differences within the receptor domains involved in G protein interaction and activation. The opioid receptor signaling will involve the scaffolding of cellular proteins. By recruiting cellular proteins such as RGS to the proximity of the receptor signaling complexes, the amplitude and duration of the signals can be modulated. The extent of the signal will depend on the composition of the signaling complexes. Hence, in the current proposal, we will use the ecdysone mammalian-inducible expression system to alter the various G protein a subunit level so as to demonstrate the specific G protein involved in the mu- and delta-opioid receptor signaling. We will use the random saturation mutational analysis together with the Receptor Selection and Amplification Technology (RSAT) to pinpoint the domains involved in mu- and delta-opioid receptor-G protein interaction and activation. We will demonstrate the existing of opioid receptor signaling units, the signaling via protein scaffolding and will identify the cellular proteins involved in the scaffolding. We will then alter the contents of signaling units by the inducible expression system and examine the effects of such alteration on opioid receptor signaling. These studies should enhance our understanding of cellular regulation of the opioid receptor signaling.
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