During the past funding period, we have established that opioid agonists could induce the cloned opioid receptors to couple to all the Gi/Go proteins with similar proteins with similar potencies. We have established also that there are receptors to couple to all the Gi/Go proteins with similar potencies. We have established also that there are distinct differences between mu- and delta-opioid receptor activation of similar second messengers such as adenylyl cyclase, and other cellular proteins could be involved in the signaling of these opioid receptors. 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. It is also hypotheses that opioid receptor signaling is mediated via scaffolding of cellular proteins. By recruiting different cellular proteins such as RGS to the proximity of the receptor signaling complexes, the amplitude and duration of the signals can be modulated. Hence, in the current proposal, we will use the ecdysone mammalian-inducible expression system to alter the various G protein involved in the mu- and delta-opioid receptor signaling in the same clonal cell line. 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 the alter the contents of signaling units by the inducible expression system and examine the effects of such alteration on opioid receptor signaling. All these experiments are designed to address our overall objective of understanding the mechanism in which neuronal cells could integrate the signals transduced by the membrane receptors that utilize the same spectrum of second messenger systems. These proposed studies also could illuminate further the problem of tolerance and dependence since it is our hypothesis that tolerance and dependence development to the repeated use of the opioid drugs are the cellular compensatory responses to the signals being transduced. By modulating the amplitude and duration of the signals within the signaling units, it might be possible to alter the degree of tolerance and dependence.
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