Purification and molecular cloning of the gene for a D1-dopamine receptor from the central nervous system.

摘要

Dopamine is a neurotransmitter of demonstrated importance in the processes of movement, thought, and emotion in the central nervous system and is involved in the pathophysiologies of Parkinson's disease, schizophrenia, and Tourette's syndrome. The effects of dopamine in the central nervous system are mediated by two distinct classes of G-protein coupled receptors termed D1 and D2 dopamine receptors.; The objective of my dissertation research was to investigate the molecular properties of the D1 class of dopamine receptors. This objective was pursued through the purification of the D1 receptor protein and the isolation and molecular cloning of the gene for an adenylyl cyclase coupled form of the D1 receptor which is expressed in human central nervous system.; To purify the receptor a biospecific affinity chromatography matrix was developed which was capable of enriching the D1 receptor 200-300 fold over soluble preparations with a recovery of 40%. The D1 receptor was purified to near homogeneity using sequential ion exchange, lectin, and sized exclusion chromatographies (6600 fold enrichment with a recovery of 8%). Final preparations had a specific activity of 11,000 pmol({dollar}sp3{dollar}H) SCH 23390 binding per mg of protein and were estimated to be at least 80-90% pure.; In order to isolate the gene for one of the D1 dopamine receptors, a human genomic library was screened using a partial length human D1 receptor cDNA probe. The gene for the D1 receptor is intronless within its coding region and is localized to the q34-35 locus of human chromosome 5. The cloned D1 receptor gene when expressed had the appropriate ligand binding pharmacology and was found to stimulate adenylyl cyclase but not phospholipase C. The message for this D1 dopamine receptor was found in the caudate, putamen, frontal cortex, and hippocampus, but not in substantia nigra, heart or kidney. These data indicate that we have isolated the gene for an adenylyl cyclase coupled subtype of the D1 dopamine receptor class which is expressed in the human central nervous system.