DESENSITIZATION OF DOPAMINE D-2 RECEPTORS

The objective of this research program is to determine the characteristics
and mechanisms of the regulation of dopamine D-2 receptors. It is crucial
that we understand the regulation of D-2 receptors, since idiopathic or
drug-induced changes in the density of dopamine receptors are thought to be
involved in the pathophysiology or treatment of psychiatric and movement
disorders such as schizophrenia, parkinsonism, and tardive dyskinesia. The
cloning of a cDNA for the D-2 receptor makes it possible to develop tools
that have not been available for the study of D-2 receptor regulation. In
particular, cell lines expressing a high density of D-2 receptors are being
created by transfecting cells with the cDNA clone, RGB-2. The aim of this
proposal is to develop several cell lines derived from various tissues and
to use these cells to determine the mechanisms of desensitization and
down-regulation of D-2 receptors. To achieve these goals, the following specific objectives will be met. 1)
Four new cell lines expressing D-2 receptors will be created by
transfecting cells derived from various tissues with either cDNA or genomic
DNA encoding the D-2 receptor. 2) Receptors on the cells will be
characterized by radioligand binding, by biochemical assays of adenylate
cyclase activity, and by photoaffinity labeling and gel electrophoresis. 3)
Conditions for treating cells will be optimized by assessing the
possibility of growth-dependent changes in D-2 receptor density. 4)
Desensitization of D-2 receptors will be evaluated using four measures of
rapid desensitization that assess coupling of receptors to G proteins and
subcellular localization of the receptors. 5) The effect on the density of
D-2 receptors of treating cells with D-2 receptor agonists will be
determined. The time course of agonist-induced loss of receptors will be
compared to that of desensitization of adenylate cyclase and to the
efficacy of the agonist for inhibition of adenylate cyclase. 6)
Agonist-induced changes in levels and transcription of D-2 receptor
messenger RNA will be quantified by Northern blot analysis and nuclear
runoff experiments.