There is only a limited understanding of the mechanisms of acute actions of opiolds and of chronic actions leading to the development of opioid tolerance and dependence. Morphine and related analgesics act at a family of G protein coupled opioid receptors. Agonist occupation of these receptors leads to activation of G protein and exchange of G alpha-bound GDP for GTP, followed by dissociation of G alpha and beta gamma subunits and interaction with downstream effectors. Action is terminated by the hydrolysis of G alpha-GTP to G alpha-GDP. RGS proteins are an important class of proteins that regulate G protein signaling (RGS proteins). These proteins are GTPase activation proteins (GAPs) and so alter the G protein cycle by increasing the rate of G alpha-bound GTP hydrolysis. The functional significance of these proteins in opioid-receptor-mediated processes is unknown.
The aim of this proposal is to define role(s) for RGS proteins in acute opioid signaling and in chronic effects following prolonged opoid exposure. There are over 20 RGS proteins and the basis for selectivity of action at particular receptor-G protein combinations is not known. To avoid the problem of working with RGS proteins that may not be the correct physiological partner for the opioid receptors, the studies will involve construction and expression of RGS-insensitive G alpha subunits that are insensitive to the GAP activity of all RGS proteins. In cells transfected with these RGS-insensitive G alpha subunits the acute (Low-Km GTPase, 35S GTP gammaS binding, adenylyl cyclase inhibition, intracellular [Ca2+]) and chronic (desensitization and supersensitivity) effects of opioids will be studied. In addition, the effects of opioid tolerance and withdrawal on the expression of RGS proteins will be investigated in cells and in rat central nervous tissue. The work will further define opioid signaling and suggest novel therapeutic targets for the treatment of pain and opiate drug abuse.
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