Specific [3H]raclopride Binding to Neostriatal Dopamine D2 Receptors: Role of Disulfide and Sulfhydryl Groups

Overview

Journal Neurochem Res

Specialties Chemistry
Neurology

Date 1992 Aug 1

PMID 1379349

Citations 1

Authors

T A Reader

E Molina-Holgado

L Lima

S Boulianne

K M Dewar

Affiliations

Soon will be listed here.

Abstract

Receptor binding studies were performed in rabbit neostriatum (caudate-putamen) using the dopamine D2 antagonist [3H]raclopride. Treatment of the membrane preparations with the reducing agent L-dithiothreitol (L-DTT) as well as with the alkylating compound N-ethylmaleimide (NEM), produced dose-dependent decreases of specific [3H]raclopride binding; the IC50 values were of 3.1 and 1.2 mM, respectively. Saturation experiments showed that the reduction of disulfide (-S-S-) bonds by L-DTT (1 mM) decreased the number of binding sites, with only a slight increase in the affinity. On the other hand, alkylation of sulfhydryl (-SH) groups by NEM (1 mM) decreased both receptor number and affinity. The properties of the remaining binding sites were examined in competition curves with the physiological substrate dopamine and the dopaminergic antagonist (+)butaclamol. The IC50 values for (+)butaclamol in control and in L-DTT and NEM treated membranes were between 3.4 and 4.8 nM, with Hill coefficients (nH) of 1, indicating that the remaining binding sites conserved a high affinity for antagonist binding. In the case of dopamine, the curves were shallow (nH 0.45-0.64) and both compounds increased the IC50 from 0.7 microM (control) to 8 microM and 11 microM, for L-DTT and NEM respectively. Iterative analysis revealed that L-DTT produced a very important (greater than 60%) decrease in the number of high-affinity (RH) binding. After NEM, there was a decrease in both the number of (RH) and the affinity (KH) of the high-affinity binding sites, and in the affinity (KL) of the low-affinity sites. These results demonstrate the participation of -S-S- and -SH groups in the agonist conformation of the primary ligand recognition site of the dopamine D2 receptor. Alternatively, -S-S- and -SH groups could be related to the coupling of the primary ligand recognition protein with adenylate cyclase by means of an inhibitory type of G protein.

Citing Articles

Cdk5 phosphorylates dopamine D2 receptor and attenuates downstream signaling.

Jeong J, Park Y, Kim D, Lee S, Kwak Y, Lee S PLoS One. 2014; 8(12):e84482.

PMID: 24391960 PMC: 3877277. DOI: 10.1371/journal.pone.0084482.

References

Sunahara R, Niznik H, Weiner D, Stormann T, Brann M, Kennedy J . Human dopamine D1 receptor encoded by an intronless gene on chromosome 5. Nature. 1990; 347(6288):80-3. DOI: 10.1038/347080a0. View

Duman R, Terwilliger R, Nestler E, Tallman J . Sodium and potassium regulation of guanine nucleotide-stimulated adenylate cyclase in brain. Biochem Pharmacol. 1989; 38(12):1909-14. DOI: 10.1016/0006-2952(89)90488-7. View

Braestrup C, Andersen P . Effects of heavy metal cations and other sulfhydryl reagents on brain dopamine D1 receptors: evidence for involvement of a thiol group in the conformation of the active site. J Neurochem. 1987; 48(6):1667-72. DOI: 10.1111/j.1471-4159.1987.tb05721.x. View

Dewar K, MONTREUIL B, Grondin L, Reader T . Dopamine D2 receptors labeled with [3H]raclopride in rat and rabbit brains. Equilibrium binding, kinetics, distribution and selectivity. J Pharmacol Exp Ther. 1989; 250(2):696-706. View

Moxham C, Malbon C . Fat cell beta 1-adrenergic receptor: structural evidence for existence of disulfide bridges essential for ligand binding. Biochemistry. 1985; 24(22):6072-7. DOI: 10.1021/bi00343a007. View

McPherson G . Analysis of radioligand binding experiments. A collection of computer programs for the IBM PC. J Pharmacol Methods. 1985; 14(3):213-28. DOI: 10.1016/0160-5402(85)90034-8. View

Reader T, Briere R, Grondin L . Alpha-1 and alpha-2 adrenoceptor binding in cerebral cortex: role of disulfide and sulfhydryl groups. Neurochem Res. 1986; 11(1):9-27. DOI: 10.1007/BF00965161. View

Reader T, Boulianne S, Molina-Holgado E, Dewar K . Effects of monovalent cations on neostriatal dopamine D2 receptors labeled with [3H]raclopride. Biochem Pharmacol. 1990; 40(8):1739-46. DOI: 10.1016/0006-2952(90)90350-t. View

Stadel J, Lefkowitz R . Multiple reactive sulfhydryl groups modulate the function of adenylate cyclase coupled beta-adrenergic receptors. Mol Pharmacol. 1979; 16(3):709-18. View

10.

Parker R, WAUD D . Pharmacological estimation of drug-receptor dissociation constants. Statistical evaluation. I. Agonists. J Pharmacol Exp Ther. 1971; 177(1):1-12. View

11.

Sunahara R, Guan H, ODowd B, Seeman P, Laurier L, Ng G . Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1. Nature. 1991; 350(6319):614-9. DOI: 10.1038/350614a0. View

12.

Grandy D, Marchionni M, Makam H, Stofko R, Alfano M, Frothingham L . Cloning of the cDNA and gene for a human D2 dopamine receptor. Proc Natl Acad Sci U S A. 1989; 86(24):9762-6. PMC: 298581. DOI: 10.1073/pnas.86.24.9762. View

13.

Kebabian J, Calne D . Multiple receptors for dopamine. Nature. 1979; 277(5692):93-6. DOI: 10.1038/277093a0. View

14.

Creese I, Sibley D, Hamblin M, Leff S . The classification of dopamine receptors: relationship to radioligand binding. Annu Rev Neurosci. 1983; 6:43-71. DOI: 10.1146/annurev.ne.06.030183.000355. View

15.

Onali P, Olianas M, Gessa G . Characterization of dopamine receptors mediating inhibition of adenylate cyclase activity in rat striatum. Mol Pharmacol. 1985; 28(2):138-45. View

16.

Van Tol H, Bunzow J, Guan H, Sunahara R, Seeman P, Niznik H . Cloning of the gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine. Nature. 1991; 350(6319):610-4. DOI: 10.1038/350610a0. View

17.

Scheuhammer A, Cherian M . Effects of heavy metal cations, sulfhydryl reagents and other chemical agents on striatal D2 dopamine receptors. Biochem Pharmacol. 1985; 34(19):3405-13. DOI: 10.1016/0006-2952(85)90710-5. View

18.

Quennedey M, Bockaert J, Rouot B . Direct and indirect effects of sulfhydryl blocking agents on agonist and antagonist binding to central alpha 1- and alpha 2-adrenoceptors. Biochem Pharmacol. 1984; 33(24):3923-8. DOI: 10.1016/0006-2952(84)90002-9. View

19.

Dewar K, Reader T . Distribution of dopamine D1 and D2 receptors in rabbit cortical areas, hippocampus, and neostriatum in relation to dopamine contents. Synapse. 1989; 4(4):378-86. DOI: 10.1002/syn.890040413. View

20.

Kilpatrick B, De Lean A, Caron M . Dopamine receptor of the porcine anterior pituitary gland. Effects of N-ethylmaleimide and heat on ligand binding mimic the effects of guanine nucleotides. Mol Pharmacol. 1982; 22(2):298-303. View