Solubilization and reconstitution of the D-1 dopamine receptor: potentiation of the agonist high-affinity state of the receptor

Sidhu, Anita

doi:10.1021/bi00424a012

Cited by 18 publications

(49 citation statements)

References 29 publications

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“…2A); from the data, an association rate constant (k+,) of 3.05 X nM-' min-' was calculated ( Fig. 2A, inset) as described by Sidhu (1988). Bound [3H]NPY was displaced by unlabeled NPY in a time-dependent manner (Fig.…”

Section: Resultsmentioning

confidence: 94%

Characterization of Functional Neuropeptide Y Receptors in a Human Neuroblastoma Cell Line

Gordon

Kohout

Fishman

1990

Journal of Neurochemistry

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We identified receptors for neuropeptide Y (NPY) on an established human neuroblastoma cell line, SK-N-MC, which are functionally coupled to adenylate cyclase through the inhibitory guanine nucleotide-binding protein of adenylate cyclase, Gi. Intact SK-N-MC cells bound radiolabeled NPY with a KD of 2 nM and contained approximately 83,000 receptors/cell. Unlabeled porcine and human NPY and structurally related porcine peptide YY (PYY) competed with labeled NPY for binding to the receptors. NPY inhibited cyclic AMP accumulation in SK-N-MC cells stimulated by isoproterenol, dopamine, vasoactive intestinal peptide, cholera toxin, and forskolin. NPY inhibited isoproterenol-stimulated cyclic AMP production in a dose-dependent manner, with half-maximal inhibition at 0.5 nM NPY. Porcine and human NPY and porcine PYY gave similar dose-response curves. NPY also inhibited basal and isoproterenol-stimulated adenylate cyclase activity in disrupted cells. Pertussis toxin treatment of the cells completely blocked the ability of NPY to inhibit cyclic AMP production and adenylate cyclase activity. The toxin catalyzed the ADP-ribosylation of a 41-kDa protein in SK-N-MC cells that corresponds to Gi. The receptors on SK-N-MC cells appeared to be specific for NPY, as other neurotransmitter drugs, such as alpha-adrenergic, dopaminergic, muscarinic, and serotonergic antagonists, did not compete for either NPY binding or NPY inhibition of adenylate cyclase. Thus, SK-N-MC cells may be a useful model for investigating NPY receptors and NPY-mediated signal transduction.

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Section: Resultsmentioning

confidence: 94%

Characterization of Functional Neuropeptide Y Receptors in a Human Neuroblastoma Cell Line

Gordon

Kohout

Fishman

1990

Journal of Neurochemistry

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“…We had earlier demonstrated that, on solubilization and reconstitution of brain D-1 dopamine receptors into phospholipid vesicles, the agonist high-affinity sites were potentiated and comprised 40-50% ofthe total receptor population (21,22). These potentiated sites were due to increased coupling of receptor to G protein and were abolished by guanine nucleotide analogues.…”

Section: Resultsmentioning

confidence: 99%

“…The decreased ability ofDA-1 agonists to stimulate AC activity was not due to a defective AC enzyme per se but rather to a defective DA-l receptor-AC coupling mechanism (19). To investigate the mechanism for the defective DA-l dopamine receptor in the SHR and to develop a cell-free system (21,22) MO), whereas SM-2 Bio-Beads were from Bio-Rad Laboratories (Richmond, CA). Dulbecco's PBS (DPBS) was purchased from Gibco Laboratories (Grand Island, NY).…”

Section: Introductionmentioning

confidence: 99%

“…Proximal tubular membranes were solubilized by 1% sodium cholate, after pretreatment with 10 jM SKF R-38393, essentially as described before for rat striatal D-1 dopamine receptors (21,22). Briefly, membranes were suspended at 1-2 mg/ml in buffer A (50 mM Tris-HCI, pH 7.4, 120 mM NaCl, 5 mM KCI, 2 mM CaC12, and 1 mM MgCI2) in the presence of 10 jM SKF R-38393, a D-l selective agonist.…”

Section: Introductionmentioning

confidence: 99%

“…After a 15-20-min incubation on ice, the mixture was centrifuged at 31,300 g for 45 min. The clear supernatant obtained after high-speed centrifugation was stored frozen in small aliquots at -80°C (22,27).…”

Section: Introductionmentioning

confidence: 99%

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Persistent defective coupling of dopamine-1 receptors to G proteins after solubilization from kidney proximal tubules of hypertensive rats.

Sidhu¹,

Vachvanichsanong²,

José³

et al. 1992

J. Clin. Invest.

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The natriuretic effect of dopamine-1 (DA-1) agonists is reduced in spontaneously hypertensive rat (SHR), partly because of defective DA-1 receptor-adenylate cyclase (AC) coupling in renal proximal convoluted tubules. To investigate this defective coupling, DA-1 dopamine receptors from renal proximal tubules were solubilized and reconstituted into phospholipid vesicles.The binding of DA-1-selective ligand 1125I1SCH 23982 was specific and saturable, with no differences in receptor density or Kd between SHR and normotensive rats (Wistar-Kyoto rats; WKY). Competition experiments of the reconstituted DA-1 dopamine receptors in WKY with a DA-1-selective agonist, SKF R-38393, revealed the presence of high-(Kh = 350±209 nM) and low-affinity (K, = 70,500±39,500 nM) binding sites. 100 AM Gpp(NH)p abolished the agonist high-affinity sites, converting them to a low-affinity state (Ki = 33,650±10,850 nM). In SHR, one affinity site was noted (Ki = 13,800±500) and was not modulated by Gpp(NH)p (Ki = 11,505±2,295). The absence of guanine nucleotide-sensitive agonist high-affinity sites may explain the defective DA-1/AC coupling mechanism in the SHR. (J. Clin. Invest. 1992. 89:789-793.)

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

Srivastava

Ross²,

Bajwa

et al. 1990

Neurochem Res

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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.

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Solubilization and reconstitution of the D-1 dopamine receptor: potentiation of the agonist high-affinity state of the receptor

Cited by 18 publications

References 29 publications

Characterization of Functional Neuropeptide Y Receptors in a Human Neuroblastoma Cell Line

Characterization of Functional Neuropeptide Y Receptors in a Human Neuroblastoma Cell Line

Persistent defective coupling of dopamine-1 receptors to G proteins after solubilization from kidney proximal tubules of hypertensive rats.

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

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