Imbalances in dopamine (DA) receptor/G protein coupling dynamics are important in the onset and maintenance of several neuropathological diseases, such as schizophrenia, Parkinson's disease, drug abuse and attention deficit disorder. The D1-like receptors, D1 and D5, share similar structural, physiological and pharmacological homology. The functional attributes of these receptors in DA neurotransmission are largely unknown in diseased and normal states. We have shown that in transfected cells, these receptors can be functionally differentiated in that: D1 receptors couple to both G(s)alpha and G(o)alpha, while D5 couples to G(s)alpha and G(z)alpha. Moreover, D5 but not D1 receptors, inhibit phosphoinositide production. Moreover D1, but not D5, can inhibit adenyl cyclase activity, in the absence of receptor/G(s)alpha coupling. Through functional assays, we will examine the mechanism and functional consequences of D1 coupling to G(o)alpha, and D5 to G(z)alpha, in order to determine whether such coupling causes activation of alternate signaling pathways. Using progressively shorter synthetic peptides directed against specific amino acid motifs of intracellular loops of the D1 and D5 receptor, we will map the domains through which D1 couples to G(s)alpha/G(o)alpha and D5 to G(s)alpha/G(z)alpha. The ability of various peptides to block receptor/Galpha interactions will be examined through co-immunoprecipitation and functional assays. Deletion mutants will be constructed to demonstrate the participation of specific sites in receptor function. We will analyze the interactions between D1 and D5 receptors with their cognate G proteins, using a highly sensitive novel assay, fluorescence resonance energy transfer (FRET). Such FRET studies will enable us to determine in intact cells whether the receptors couple simultaneously to the two Galpha, or if such coupling occurs in a sequential manner. We will also examine interactions between synthetic peptides and G proteins, and determine whether receptor oligomerization is essential for dual coupling of D1 and D5 receptors to Galpha. A clear understanding of the mechanism and functional consequences of coupling of these receptors to different and diverse Galpha is important for defining the roles of these receptors in diseased and normal states, and may aid in the design of novel therapeutic treatments, to selectively activate or suppress specific signaling pathways.
Showing the most recent 10 out of 17 publications
