Solubilization and reconstitution of the D-1 dopamine receptor: potentiation of the agonist high-affinity state of the receptor.
The D-1 dopamine receptor was extracted from rat striatal membranes with sodium cholate and NaCl in the presence of a specific agonist and phospholipids. The soluble receptor then was reconstituted into phospholipid vesicles by further addition of phospholipids prior to detergent removal. Of the total membrane receptors, up to 48% were extracted and 36% were reconstituted into phospholipid vesicles. Yields were greatly reduced if the agonist was omitted or replaced with an antagonist. The solubilized and reconstituted D-1 receptors retained the pharmacological properties of the membrane-bound receptors, including the ability to discriminate between active and inactive enantiomers of specific agonists and antagonists. In this regard, the affinity of the reconstituted receptors for the D-1 specific antagonist 125I SCH 23982 was similar to that of the membrane-bound receptors with a Kd of 1.5 nM. Both the soluble and reconstituted forms of the D-1 receptor exhibited two affinity states for the D-1 specific agonist SK&F R-38393. In contrast to the low proportion of the receptors that had a high affinity for the agonists in striatal membranes (less than 6%), there was a dramatic increase following solubilization (22%) and reconstitution (40%). Similar results were obtained by using dopamine; the proportion of high-affinity sites increased from 4% (membrane-bound) to 48% (reconstituted) of the total receptor population. These high-affinity sites were coupled to G proteins, as guanyl nucleotides completely abolished them. Addition of guanyl nucleotides prior to solubilization or to reconstitution, however, had no effect on the subsequent yield of the reconstituted receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
Sidhu
A
A
Membrane Biochemistry Section, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Maryland 20892.
D000818:Animals; D001552:Benzazepines / Q000031:analogs & derivatives* / Q000378:metabolism; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D003342:Corpus Striatum / Q000378:metabolism*; D018492:Dopamine Antagonists; D007457:Iodine Radioisotopes; D007700:Kinetics; D008297:Male; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D011954:Receptors, Dopamine / Q000302:isolation & purification* / Q000378:metabolism; D017447:Receptors, Dopamine D1; D012995:Solubility
C049558:8-iodo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-ol; D001552:Benzazepines; D018492:Dopamine Antagonists; D007457:Iodine Radioisotopes; D011954:Receptors, Dopamine; D017447:Receptors, Dopamine D1