The overall goal of the research in our laboratories is to understand the mechanisms of alpha-2 adrenergic receptor (alpha2AR) signaling in enough detail to be able to intervene with ingenuity in a variety of pathophysiological states. The present proposal seeks to establish the in vivo functional relevance of several partial reactions that follow agonist occupancy: receptor phosphorylation, receptor binding to arrestin, and receptor endocytosis. In addition, we wish to understand the functional relevance of differing trafficking itineraries for the three alpha2AR subtypes and of alpha2AR interactions with 14-3-3 proteins and with spinophilin in vivo. These linked goals ultimately will be addressed by introducing alpha2AR structures with modified trafficking properties or altered partial reactions into the alpha2AR locus of the mouse, using Cre-loxP based homologous recombination strategies. However, to prioritize which mouse lines expressing mutant alpha2AR should be developed as well as to gain unprecedented insights concerning the structure-function relationships of alpha2AR in the context of native target cells, we will utilize stereotactic procedures to deliver adenoviral constructs encoding these various alpha2AR structures into the fourth ventricle of the brain of alpha2AAR """"""""knockout"""""""" mice. We will then evaluate the trafficking properties and the cellular functions elicited by these receptor structures in the locus ceruleus. For subsequently-developed homozygous mouse cell lines, we will evaluate alpha2AR suppression of Ca2+ currents and activation of K+ currents in the locus ceruleus and in superior cervical ganglion neurons. We will also evaluate a number of physiological parameters (including sedation, analgesia, lowering of blood pressure and suppression of epileptogenesis) and behavioral parameters, including measures of the efficacy of anti-depressant agents and indices for pre-existing """"""""depressive states"""""""". These proposed studies, representing a collaboration between the laboratories of Lee Limbird and Brian Kobilka, co- investigators in this study, represent the first effort to explore, in the context of native target cells and in vivo, the impact of partial reactions of alpha2 receptor signaling and of alpha2 receptor trafficking. We anticipate that the insights we obtain will inform the development of novel therapeutic strategies for a number of cardiovascular, neurological and behavioral disorders regulated by alpha2AR.
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