This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Desensitization and internalization of the mu opioid G protein-coupled receptor are ligand dependent processes mediated by phosphorylation by G protein-coupled receptor kinases (GRKs) on multiple serine and threonine residues in the cytoplasmic tail. While endogenous peptides and methadone induce phosphorylation followed by receptor internalization into the cytoplasm, certain highly addictive drugs such as heroin and morphine differ significantly in their effects on phosphorylation, desensitization, and internalization. Some of these differences can be understood in terms of classical models of agonist efficacy. However, several lines of evidence suggest that there may be additional specificity in the regulatory effects of opiate drugs that are currently unexplained. The working hypothesis of the proposed studies is that opiate drugs, in addition to differing in relative efficacy for promoting G protein activation, produce different patterns of multiple phosphorylations in the mu opioid receptor, thereby 'encoding'some of the differences in cellular regulation observed in previous studies. The proposed studies will test this hypothesis using previously defined in vitro and cell-based systems to generate phosphorylated receptors under controlled conditions, followed by advanced protein mass spectrometry to precisely define patterns of receptor phosphorylation produced. The functional significance of putative agonist-specific differences in receptor phosphorylation will then be tested using transfected cells in which agonist-specific effects on opioid receptor regulation are known to occur. The proposed studies could provide significant new insight into mechanisms of opiate drug action and, more generally, may help extend our present understanding of partial agonism of GPCRs.