Solubilization and reconstitution of dopamine D1 receptor from bovine striatal membranes: effects of agonist and antagonist pretreatment.
The bovine striatal dopamine D1 receptor was solubilized with a combination of sodium cholate and NaCl in the presence of phospholipids, following treatment of membranes with a dopaminergic agonist (SKF-82526-J) or antagonist (SCH-23390). The solubilized receptors were subsequently reconstituted into lipid vesicles by gel-filtration. A comparison of ligand-binding properties shows that the solubilized and reconstituted receptors bound [3H]SCH-23390 to a homogeneous site in a saturable, stereospecific and reversible manner with a Kd of 0.95 and 1.1 nM and a Bmax of 918 and 885 fmol/mg protein respectively for agonist- and antagonist-pretreated preparations. These values are very similar to those obtained for membrane-bound receptors. The competition of antagonists for [3H]SCH-23390 binding exhibited a clear D1 dopaminergic order in the reconstituted preparation obtained from either agonist or antagonist-pretreated membranes, except that (+)butaclamol was about four-fold more potent than cis-flupentixol in displacing [3H]SCH-23390 binding in preparation obtained from agonist-pretreated membranes compared to antagonist-pretreated membranes. The agonist/[3H]SCH-23390 competition studies revealed the presence of a high-affinity component of agonist binding in both the reconstituted receptor preparations. The number of high-affinity agonist binding sites, however, is 40-80% higher in reconstituted preparation obtained from antagonist-treated membrane compared to that obtained from the agonist-treated membrane. In both the preparations, 100 microM guanylylimidodiphosphate (Gpp(NH)p) completely abolished the high-affinity component of agonist binding compared to partial abolition in the native membranes, indicating a close association of a G-protein with the solubilized receptors. Whether the receptor was solubilized following agonist or antagonist preincubation of the membranes, the receptor-detergent complex eluted from a steric-exclusion HPLC column with an apparent molecular size of 360,000. Preincubation of the solubilized preparations with Gpp(NH)p had virtually no effect on the elution profile suggesting a lack of guanine nucleotide-dependent dissociation of G-protein receptor complex.
Srivastava
L K
LK
Department of Psychiatry, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada.
Ross
G M
GM
Bajwa
S B
SB
Mishra
R K
RK
D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001552:Benzazepines / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D019826:Cholic Acid; D002793:Cholic Acids; D003342:Corpus Striatum / Q000737:chemistry*; D015259:Dopamine Agents / Q000494:pharmacology*; D018492:Dopamine Antagonists; D018818:Fenoldopam; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D008081:Liposomes / Q000378:metabolism; D011954:Receptors, Dopamine / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D017447:Receptors, Dopamine D1; D012995:Solubility
D001552:Benzazepines; D002793:Cholic Acids; D015259:Dopamine Agents; D018492:Dopamine Antagonists; D008081:Liposomes; D011954:Receptors, Dopamine; D017447:Receptors, Dopamine D1; D006165:Guanylyl Imidodiphosphate; D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; D019826:Cholic Acid; D018818:Fenoldopam