The principle objective of this grant application is to delineate the mechanisms underlying modulation of D-1 receptor function and activity, at the molecular level. The research will test our central hypothesis that different tissue contain D-1 receptors, which couple differentially to multiple effectors, resulting in different physiological and pharmacological properties. The multiple signal transducing ability of D-1 receptors may be a result of these receptors coupling to not only Gs but also to Gi. Such couplings to multiple G proteins may also be important in understanding the mechanisms by which D-1 receptors modulate D-2 dopamine receptor function. The studies outlined in this grant will help define the role of D-1 dopamine receptors in diseases such as schizophrenia, Parkinson's disease, depression and drug addiction. Using the tools we developed for such studies, the D-1 dopamine receptors from rat retina, rat striatum and human SK-N-MC cells will be solubilized and purified. These receptors will be analyzed and compared with respect to their pharmacological properties, glycosylation, resolution into two subpopulations after elution from affinity columns and regulation of agonist high-affinity binding sites. The significance of glycosylation in physiological function and the linkage of D-1 receptors to phosphoinositide metabolism, will be tested in SK-N-MC cells. The ability of D-1 receptors from all three tissue sources to couple to different G proteins will be measured, using methods we developed, by reconstituting purified receptors with exogenous purified G proteins will be determined. The specificity and affinity of coupling for Gi protein subtypes will be assayed by reconstituting receptors with isolated Gil, Gi2 and Gi3. By reconstituting D-1 receptors with exogenous effectors of adenylate cyclase, the ability of these purified receptors to mediate physiological activity can be measured. The inactivation of Gi and Go by pertussis toxin-treatment of these effector systems prior to reconstitution, will permit an analysis of the impact of these G proteins in dampening D-1-mediated activation of adenylate cyclase. The ability of D-1/D-2 receptors to interact in striatal membranes will be examined by photoaffinity labeling and radioligand binding methods. The role of G proteins in promoting such interactions will be tested by reconstitution of crude soluble receptors with specific G proteins. The ability of D-1/D-2 receptors to interact, under these conditions, will be measured. Such studies will demonstrate whether specific G proteins mediate such D-1/D-2 interactions in membranes.
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