The glycosylation properties of D2 dopamine receptors from striatal and limbic areas of bovine brain

Abstract

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.

Purification of brain D2 dopamine receptor.

“…Therefore these bands could represent variants at the protein level (either different gene products or proteolytic variants) or glycosylation variants. We favour the latter explanation at present as we have shown that there are at least two different glycosylation variants of the receptor based on differential binding to WGA -agarose (Leonard et al, 1988).…”

Cellular colocalization of dopamine D1 and D2 receptors in rat medial prefrontal cortex

“…Even so, the images for the two receptor subtypes were both distributed around the outer perimeter of the cell body where only a limited amount of overlap in the composite confocal images could be observed. The dissimilar appearance and differential subcellular localization of D,-and D2like receptor binding fluorescence could be related to potential differences in glycosylation (Clagett-Dame and McKelvy, 1989;Jarvie et al, 1989;Leonard et al, 1988).…”

The dopamine D2 receptor is expressed in GH3 cells

“…Receptor solubilization was by a method modified from that of Leonard, Williamson & Strange (1988). Membrane pellets were stirred gently at 4°C in 10 volumes of chilled solubilization buffer (20 mM Hepes, 1 mM EDTA, 2m NaCl, 0.6%, w/v, sodium cholate and 1 mM PMSF) for 1 h and then centri¬ fuged at 100 000 g for a further 1 h. The supernatant receptor fraction was retained.…”