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Solubilization and reconstitution of dopamine D1 receptor from bovine striatal membranes: Effects of agonist and antagonist pretreatment

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Abstract

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 thancis-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 highaffinity 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 obrained from the agonist-treated membrane. In both the preparations, 100 μM 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.

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Author notes

  1. Gregory M. Ross

    Present address: Beckman Instruments (Canada) Inc., L5T 1W5, Mississauga, Ontario, Canada

Authors and Affiliations

  1. Neuropharmacology Laboratory, Departments of Psychiatry and Biomedical Sciences, Faculty of Health Sciences, McMaster University, Health Sciences Centre - 4N52, 1200 Main Street West, L8N 3Z5, Hamilton, Ontario, Canada

    Lalit K. Srivastava, Samina B. Bajwa & Ram K. Mishra

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Srivastava, L.K., Ross, G.M., Bajwa, S.B. et al. Solubilization and reconstitution of dopamine D1 receptor from bovine striatal membranes: Effects of agonist and antagonist pretreatment. Neurochem Res 15, 647–657 (1990). https://doi.org/10.1007/BF00973757

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