Over the years, the focus of this program has alternated between studies of various subtypes of adrenergic receptors. The present application focuses on alpha-1 subtypes rather than the beta-1 and beta-2 subtypes studied in the current period, because the aim is to explore the functional role of C-terminal splice variants that have been demonstrated for an alpha-1 subtype, but not for a beta subtype. The overall goal is to understand the functional role of GPCRs in signaling mechanisms that reach beyond the coupling to G proteins. From preliminary considerations, the investigator has identified interactions at the C terminus of the alpha subtypes as potential mediators of such signaling, and proposes to investigate a series of putative mediators including the neuronal immediate early gene product Homer, and the PDZ domain of neuronal NOS. The general hypotheses to be explored are: 1. that there are specific binding partners for C-terminal tails of each alpha-1 subtype; 2. that the subtypes have c-terminal splice variants with distinct binding partners; and 3. that these interactions with the various partners have important functional roles. To this end, the investigator plans to utilize fusion proteins of C-terminal tails and full-length receptors epitope-tagged at the N-terminus in order to pursue five Specific Aims: 1. The investigators will determine whether the neuronal immediate-early gene product Homer binds to a PPXXFR consensus binding motif in the alpha1D-AR tail and alters its function. 2. They will determine whether the PDZ domain of neuronal nitric oxide synthase binds to a GEEV consensus binding motif at the extreme C-terminus of the alpha1A-AR and alters its function. 3. They will identify novel binding partners for alpha1A, a1B, a1D-AR C-terminal tails using biochemical approaches and expressions library screening with tail fusion proteins. 4. They will identify, clone, and characterize splice variants of alphalambdapha1B and alpha1D-ARs. 5. They will use splice variants of a1A, a1B, and a1D-ARs as naturally occurring C-terminal variants to examine the functional roles of protein interactions at the C-terminal tails. These experiments will clarify the role of C-terminal interacting proteins in a1-AR function, and the significance of C-terminal splice variants.
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