Characterization of the mu opioid receptor desensitization mechanisms in a heterologous expression system

grantor: University of TorontoEndogenous opioids play an important role in a variety of physiological processes and mediate their actions through at least three distinct subtypes of cell surface receptors: mu, kappa and delta. The mu opioid receptor is considered to be the major target for the production of analgesia and the development of drug tolerance and dependence. Receptor desensitization has been considered to play important role in opioid tolerance. However, understanding mu opioid receptor desensitization mechanisms using rat brain tissues or cell lines has been difficult because of the presence of multiple subtypes of opioid receptor. This thesis documents the mechanisms of desensitization of the mu opioid receptor expressed in Chinese hamster ovary cells without interference caused by the presence of other opioid receptor subtypes. In this cell line, the mu opioid receptor was desensitized after 1hr exposure to the opioid peptide, DAMGO, largely by the loss of receptors from the cell surface without affecting the affinity or proportion of agonist-detected high affinity sites. Using site-directed mutagenesis, the structural determinants for this agonist-induced functional desensitization was determined to be threonine 394 preceded by an acidic amino acid in the cytoplasmic tail, presumably serving as a primary phosphorylation site for G protein coupled receptor kinase. However, mu opioid receptor down-regulation with an agonist treatment appears to be governed by two distinct pathways. One was G protein-dependent, GRK-dependent, and the other was G protein-independent but tyrosine kinase-dependent. In addition, the mu opioid receptor was shown to be a direct substrate for protein tyrosine kinase. Unlike other prototypic G protein coupled receptor, G protein coupling to the mu opioid receptor was tighter and resistant to agonist-induced G protein uncoupling from the mu opioid receptor. The persistent G protein coupling property of the mu opioid receptor with agonist exposure is partially determined by the intracellular regions of the receptor with contributions from multiple transmembrane domains. Taken together, these findings establish that the agonist-induced mu opioid receptor desensitization is mediated by receptor down-regulation rather than G protein uncoupling from the receptor and that multiple domains of the receptor contribute to its tight coupling to G protein and resistance to agonist-induced dissociation.Ph.D