Induction of G protein-independent agonist high-affinity binding sites of D-1 dopamine receptors by beta-mercaptoethanol.
We have purified the D-1 dopamine receptor 8200-fold to 78% purity from rat striatal membranes. Critical to this purification was the N-ethylmaleimide (NEM)-mediated alkylation of all endogenous sulfhydryl groups, except those associated with the D-1 dopamine receptors, which were protected by the D-1 agonist SKF R-38393. Such NEM treatment of D-1 receptors abolished all agonist high-affinity binding sites of the receptors, but did not alter the antagonist binding properties. When NEM-treated D-1 receptors were affinity-purified by mercury-agarose columns, the pharmacological properties of these purified receptors were examined, after removal of beta-mercaptoethanol (beta ME), which was used for elution of receptors from the affinity column. Purified D-1 receptors displayed typical dopaminergic antagonist binding values; however, agonists bound to the purified receptors with only high-affinity binding values, despite the prior absence of high-affinity sites in crude soluble extracts of NEM-treated receptors. The agonist high-affinity binding of purified D-1 receptors was insensitive to modulation by the GTP analog Gpp(NH)p and occurred in the absence of any G proteins. These Gpp(NH)p-insensitive high-affinity sites appeared to be induced by beta ME, since similar high-affinity binding was also restored by beta ME to crude soluble and membrane-bound receptors, which had been pretreated with NEM. The ability of D-1 dopamine receptors to bind with high-affinity to agonists in the absence of functionally active G proteins may be an intrinsic property of the reduced state of D-1 dopamine receptors.
Sidhu
A
A
Department of Pediatrics, Georgetown University Children's Medical Center, Georgetown University, Washington, D.C. 20007, USA.
Kimura
K
K
Vachvanichsanong
P
P
D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003342:Corpus Striatum / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008623:Mercaptoethanol / Q000494:pharmacology*; D008970:Molecular Weight; D051381:Rats; D017207:Rats, Sprague-Dawley; D017447:Receptors, Dopamine D1 / Q000819:agonists / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D017447:Receptors, Dopamine D1; D006165:Guanylyl Imidodiphosphate; D008623:Mercaptoethanol; D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; D019204:GTP-Binding Proteins; D005033:Ethylmaleimide