This is a revised application based on data showing that the first full D1 dopamine agonist dihydrexidine that we developed caused profound acute antiparkinsonian effects in primates. Recent data indicate that some, but not all, full D1 agonists produce marked tolerance when administered repeatedly. We also have shown that D1 dopamine receptor agonists of similar efficacy can differ dramatically in desensitization liability. Using a series of rigid Dl agonists, we shall determine if selective conformations are promoted by different full Dl agonists, and whether these conformations represent different, if overlapping, receptor states compared to those that promote G protein coupling. First, we shall study differences in how novel D1 agonists functionally desensitize hemagglutinin (HA)-tagged human D1 receptors (HA-D1R) in C-6 glioma cells as affected by the extent and/or pattern of GRK activity. The rate and extent of D1 receptor phosphorylation by different agonists, and the effects of dominant negative GRK mutants, will be determined. Molecular and pharmacological tools will be used to assess the relative roles of endogenous GRKs and PKA in such desensitization. We also shall determine the cellular response of GRK to agonist exposure by assessing its translocation and interaction with G-beta-gamma complements. Second, we shall map the phosphorylated residues of the hD1R receptor. Patterns of agonist-induced receptor phosphorylation induced by GRKs and second-messenger-kinases (e.g., PKA) will be assessed by mutating subsets of serines and threonines in HA-hDl-C-6 cells. We also shall overexpress HA-D1R with and without various GRKs in HEK293 cells. The phosphorylated HA-hD1 receptor will be isolated by immunoprecipitation, cleaved with protease, and sequenced by MALDVToF, Ion Trap, and/or nano-ESI-mass spectrometry. Finally, we shall examine the relationship between GRK activity and arrestin binding in the initiation of G protein uncoupling and functional desensitization in the HA-hD1 C-6 system. The complement of Q-arrestins will be determined, and alterations in high affinity binding of GRKs and arrestins to D1 receptors measured following exposure to select D1 agonists. The loss of receptor-G-protein coupling from binding of labeled GTP-analogs will be assessed to correlate receptor uncoupling with levels of )-arrestin binding. The necessity of 5-arrestin(s) in functional desensitization of the HA-hD1 receptor in C-6 cells will be studied using dominant-negative mutants, and the relation between GRK and p-arrestins assessed using tools developed in Aim 1 studies. These studies of beta-arrestin binding and recruitment will provide another functional endpoint in the delineation of the molecular mechanisms of D1 receptor desensitization.
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