pH dependence of sulpiride binding to D2 dopamine receptors in bovine brain

D2 dopamine receptors from bovine brain (caudate nucleus and olfactory tubercle) have been solubilized using sodium cholate/NaCl and their glycoprotein properties studied in terms of their interaction with wheat-germ agglutinin-agarose (WGA-agarose). Under optimal conditions about 65% of the applied D2 dopamine receptors bound to WGA-agarose and could be eluted with N-acetylglucosamine. The ability of receptors to adsorb to the affinity column was shown to be dependent on the cholate and salt concentrations used. Digestion of the membrane bound D2 dopamine receptors with neuraminidase prior to solubilisation reduced the ability of the receptors to bind to WGA-agarose (50% of applied receptors bound) whereas digestion with N-acetylglucosaminidase did not significantly affect binding to WGA-agarose. Digestion with the two enzymes together resulted in a larger decrease in binding to WGA-agarose than was seen with the two enzymes alone (40% of applied receptors bound). Stepwise elution of bound receptors from the WGA-agarose columns using 2.5 mM- and 100-mM-N-acetylglucosamine showed that about 40% of the bound receptors interacted with WGA-agarose in a low-affinity manner, the remainder showing a high-affinity interaction. Neuraminidase treatment reduced the low-affinity population suggesting that the interaction of oligosaccharides bearing sialic acid with WGA-agarose is of lower affinity and that higher-affinity binding is via N-acetylglucosamine. These data are discussed in terms of the heterogeneity of carbohydrate moieties on the D2 dopamine receptors within a brain region. In all the tests applied here, however, receptors from caudate nucleus and olfactory tubercle behaved identically so their glycosylation patterns must be very similar.

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pH Dependence of Ligand Binding to D2 Dopamine Receptors

Biochemistry ◽

10.1021/bi00041a044 ◽

1995 ◽

Vol 34 (41) ◽

pp. 13635-13641 ◽

Cited By ~ 15

Author(s):

Ursula M. D'Souza ◽

Philip G. Strange

Keyword(s):

Ligand Binding ◽

Dopamine Receptors ◽

Ph Dependence ◽

D2 Dopamine Receptors

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Similarities in the glycosylation patterns of D2 dopamine receptors in different bovine brain regions

Biochemical Society Transactions ◽

10.1042/bst0151156a ◽

1987 ◽

Vol 15 (6) ◽

pp. 1156-1157

Author(s):

M. N. LEONARD ◽

P. G. STRANGE

Keyword(s):

Dopamine Receptors ◽

Brain Regions ◽

Bovine Brain ◽

D2 Dopamine Receptors

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Antisera specific for D2 dopamine receptors

Biochemical Journal ◽

10.1042/bj2890789 ◽

1993 ◽

Vol 289 (3) ◽

pp. 789-794 ◽

Cited By ~ 13

Author(s):

P L Chazot ◽

A J Doherty ◽

P G Strange

Keyword(s):

Dopamine Receptors ◽

Cho Cells ◽

Receptor Gene ◽

Bovine Brain ◽

Extracellular Loop ◽

Intracellular Loop ◽

D2 Dopamine Receptors ◽

Western Blots ◽

Recombinant Cho Cells ◽

Third Intracellular Loop

Antisera have been raised against two peptides from the sequence of D2 dopamine receptors: peptide 1 from the predicted second extracellular loop and peptide 2 from the predicted third intracellular loop. The antisera recognize specifically a 95 kDa band in Western blots of several bovine brain regions, which corresponds to the denatured D2 dopamine receptor, whereas in recombinant CHO cells expressing D2 dopamine receptors a 80 kDa band is seen. The antisera immunoprecipitate 10-20% of the D2 dopamine receptors from soluble preparations of bovine brain. The antisera recognize D2 dopamine receptors in immunofluorescence analyses of recombinant CHO cells bearing the receptor gene. The antisera directed against the third intracellular loop, but not those against the second extracellular loop, will interfere with the coupling of D2 dopamine receptors and G-proteins in bovine brain preparations.

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Heterogeneity of D2 dopamine receptors in different brain regions

Biochemical Journal ◽

10.1042/bj2480595 ◽

1987 ◽

Vol 248 (2) ◽

pp. 595-602 ◽

Cited By ~ 23

Author(s):

M N Leonard ◽

C A Macey ◽

P G Strange

Keyword(s):

Caudate Nucleus ◽

Dopamine Receptors ◽

Serotonin Receptors ◽

Brain Regions ◽

Bovine Brain ◽

Olfactory Tubercle ◽

Agonist Binding ◽

D2 Dopamine Receptors ◽

Substituted Benzamides ◽

Nucleotide Regulation

The binding of [3H]spiperone has been examined in membranes derived from different regions of bovine brain. In caudate nucleus, nucleus accumbens, olfactory tubercle and putamen binding is to D2 dopamine and 5HT2 serotonin receptors, whereas in cingulate cortex only serotonin 5HT2 receptor binding can be detected. D2 dopamine receptors were examined in detail in caudate nucleus, olfactory tubercle and putamen using [3H]spiperone binding in the presence of 0.3 microM-mianserin (to block 5HT2 serotonin receptors). No evidence for heterogeneity among D2 dopamine receptors either between brain regions or within a brain region was found from the displacements of [3H]spiperone binding by a range of antagonists, including dibenzazepines and substituted benzamides. Regulation of agonist binding by guanine nucleotides did, however, differ between regions. In caudate nucleus a population of agonist binding sites appeared resistant to guanine nucleotide regulation, whereas this was not the case in olfactory tubercle and putamen.

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