Members of the large biogenic amine family of GPCRs are involved in several medically relevant physiological processes and, as such, are major therapeutic targets. Current pharmacological and transgenic techniques are unsuitable for detailed functional studies of a single receptor in a specific cell or tissue in vivo. The focus of this proposal is to develop novel receptor mutants which are selectively activated by a biologically-inert ligand. A """"""""designer"""""""" receptor used in combination with traditional pharmacology and transgenics approaches will reveal novel intra- and intercellular functions for the receptor. To this end, a yeast-based screen will be employed to identify acetylcholine muscarinic 3 receptor mutants that selectively respond to a foreign drug versus its native ligand. Upon initial characterization of drug potency and efficacy in yeast, the mutant receptor will be expressed in human cells to verify that activation of the designer receptor by the designer drug can initiate indigenous physiological responses, including PIP2 hydrolysis, ERK-1/2 phosphorylation and smooth muscle cell contraction. Finally, bioinformatics, including multi-receptor molecular modeling, will be used to rationally engineer pan-bioamine family designer receptors.
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