Point mutations have proven to be powerful for the identification of structural amino acids involved in Gprotein coupled receptor function. However, the generation of appropriate mutants necessitates a preconceived hypothesis regarding which amino acids may be relevant. For a number of receptor functions it is difficult to assume a priori the involvement of particular amino acids and what properties of the amino acids might be important. This proposal aims to generate region-selective random mutational libraries of the mu-opioid receptor using PCR strategies. Initially these libraries will be used to screen for mutations that perturb mu-opioid receptor sequestration. Previously we have found that endogenous opioid peptides and a subset of alkaloids can induce sequestration of the mu-opioid receptor, whereas morphine and many clinically-used opiate drugs do not trigger receptor internalization, or do so only weakly. This suggests that agonist ligands which have similar effects on receptor-mediated signaling can have dramatically different effects on receptor trafficking.
The aim of the study is to use the random mutational libraries to understand the molecular basis for ligand-dependent receptor internalization. In the future, the results should help to clarify the significance of internalization in the development of opiate drug-induced tolerance and dependence.
