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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS029685-01A1
Application #
3416556
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1992-06-03
Project End
1995-03-31
Budget Start
1992-06-03
Budget End
1993-03-31
Support Year
1
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Georgetown University
Department
Type
Schools of Medicine
DUNS #
049515844
City
Washington
State
DC
Country
United States
Zip Code
20057
Sidhu, A; Niznik, H B (2000) Coupling of dopamine receptor subtypes to multiple and diverse G proteins. Int J Dev Neurosci 18:669-77
Sidhu, A; Olde, B; Humblot, N et al. (1999) Regulation of human D1 dopamine receptor function and gene expression in SK-N-MC neuroblastoma cells. Neuroscience 91:537-47
White, B H; Kimura, K; Sidhu, A (1999) Inhibition of hormonally induced inositol trisphosphate production in Transfected GH4C1 cells: A novel role for the D5 subtype of the dopamine receptor. Neuroendocrinology 69:209-16
Uh, M; White, B H; Sidhu, A (1998) Alteration of association of agonist-activated renal D1(A) dopamine receptors with G proteins in proximal tubules of the spontaneously hypertensive rat. J Hypertens 16:1307-13
Sidhu, A; Kumar, U; Uh, M et al. (1998) Diminished expression of renal dopamine D1A receptors in the kidney inner medulla of the spontaneously hypertensive rat. J Hypertens 16:601-8
White, B H; Sidhu, A (1998) Increased oxidative stress in renal proximal tubules of the spontaneously hypertensive rat: a mechanism for defective dopamine D1A receptor/G-protein coupling. J Hypertens 16:1659-65
Sidhu, A (1998) Coupling of D1 and D5 dopamine receptors to multiple G proteins: Implications for understanding the diversity in receptor-G protein coupling. Mol Neurobiol 16:125-34
Sidhu, A; Kimura, K; Uh, M et al. (1998) Multiple coupling of human D5 dopamine receptors to guanine nucleotide binding proteins Gs and Gz. J Neurochem 70:2459-67
Sidhu, A; Banta, M; Uh, M et al. (1998) Photoaffinity labeling of D1 and D5 dopamine receptors. Neuroscience 82:1095-101
Sidhu, A; Uh, M; Sela, S et al. (1997) Molecular and structural differences between rat brain D-1 and renal DA-1 dopamine receptors. Neurosci Res 29:1-8

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