Amrat Patel

Developmentally regulated glycosylation of dopamine transporter

The dopamine transporter (DAT) in rat striatum was examined during postnatal development and aging after photolabeling with [125I]DEEP. The DAT-[125I]DEEP protein complex from adult rats (2 months) appeared as a broad diffuse band in SDS-PAGE gels with average apparent molecular mass of about 80,000 Da as previously found. However, the molecular mass was lower at birth (day 0) and at postnatal ages 4 and 14 days. In aged rats (104 weeks), the molecular mass was slightly higher than that found in young adults (60 days). In binding experiments with [3H]BTCP, there were age-related differences in Kd and Bmax with decreases in both Kd and Bmax found in aged rats. Treatment of photolabeled membranes with neuraminidase caused a reduction in DAT molecular mass, but age-related differences were maintained. Treatment with N-glycanase greatly reduced or eliminated the age-related differences. Several DAT peptide-specific polyclonal antibodies immunoprecipitated DAT-[125I]DEEP protein complex at different developmental ages. Taken together, these results suggest differential glycosylation of rat DAT occurs during postnatal development and aging; the increase is due to increases in the N-linked sugars rather than changes in either sialic acid content or the polypeptide.

Species differences in dopamine transporters : postmortem changes and glycosylation differences

Abstract: The apparent molecular masses of photoaffinity‐labeled dopamine transporters (DATs) from rat, human, dog, and primate kidney COS cells expressing the rat DAT1 cDNA differ. Sequences predicted from cDNA cloning reveal only one amino acid difference between the length of the rat and human DAT but one less site for potential N‐linked glycosylation in the human DAT. Possible posttranslational and postmortem bases for species differences in DAT molecular mass were explored. Rat DAT proteins from striata subjected to ∼5 h of postmortem delay modeled after the human postmortem delay process revealed small but consistent losses in apparent molecular mass and in cocaine analogue binding; the DAT molecular mass displayed no further losses for up to 30 h of model postmortem treatment. Degradative postmortem changes could thus contribute to molecular mass differences between rat and human DATs. Neuraminidase treatment reduced the apparent molecular mass of native rat DAT but not that of the rat DAT expressed in COS cells, suggesting that the sugars added to the DAT expressed in COS cells were different than those added to the rat brain striatal transporter. These differences could account for the somewhat higher Km values for expressed DAT cDNA in COS cells when compared with the wild‐type striatal transporter. These results are in accord with the differences in number of predicted N‐linked glycosylation sites between rat and human DATs and with cell‐type specificity in transporter posttranslational processing.

Microheterogeneity of dopamine transporters in rat striatum and nucleus accumbens

Previously we have shown that the [125I]DEEP-labeled dopamine transporter from the rat nucleus accumbens has a higher apparent molecular weight than that from striatum. The present study confirms and extends these observations. Experiments with nucleus accumbens showed [125I]-DEEP to specifically bind to a protein with an apparent molecular weight of 76 kDa and with the pharmacological properties of the dopamine transporter. In exoglycosidase studies, treatment with neuraminidase, but not alpha-mannosidase, reduced the apparent molecular weight of the dopamine transporter from both the striatum and nucleus accumbens; however, a difference in the apparent molecular weight was still observed. N-Glycanase treatment, on the other hand, did reduce the apparent molecular weight of the dopamine transporters from the two regions to a similar value, approximately 56 kDa. In radioligand binding studies examining the effect of partial deglycosylation on striatal dopamine transporters, neuraminidase did not affect specific [3H]WIN 35,428 binding at 4 and 40 nM concentrations. In conclusion, the present study demonstrates that the difference in the apparent molecular weight of the dopamine transporter from these two regions is due to a difference in glycosylation and that the dopamine transporter from both regions contains similar amounts of sialic acid in their carbohydrate structure. Furthermore, the present data also indicate that the polypeptide portion of the dopamine transporter from both regions could be the same gene product.

Expression of a single dopamine transporter cDNA can confer two cocaine binding sites.

RAOIOLABELED cocaine analogs can bind to low and high affinity sites on striatal dopamine transporters (DAT). Recently, a cDNA encoding a rat brain dopamine transporter pDAT1 has been cloned. COS cells transfected with the pDAT1 in a eukaryotic expression vector express both a high (KD = 3.4 nM) and low affinity (KD = 163.6 nM) cocaine binding sites, suggesting that both sites are provided by a single gene product.

A cocaine analog and a GBR analog label the same protein in rat striatal membranes

Because some evidence suggests that cocaine and GBR12935 bind to different sites, we utilized photoaffinity probes from both classes of compounds to see if they label the same protein. [125I]RTI-82 a cocaine analog, and [125I]DEEP, a GBR analog, labeled protein(s) showing the same molecular weight, a similar pharmacological profile and a similar sensitivity to neuraminidase.

Turnover of Rat Dopamine Transporter Protein in rDAT-LLC-PK1 Cells

We have determined the turnover of the dopamine transporter protein in a porcine kidney epithelial cell line, LLC-PK1, that stably expresses a rat dopamine transporter (rDAT-LLC-PK1). A cocaine analog, RTI-76 (3s-(p-chlorophenyl)tropan-2s-carboxylic acid-p-isothiocyanatophenylethyl ester), was used to block dopamine transporter binding irreversibly in intact cells, and the rate of reappearance of transporter binding was measured. RTI-76 inhibited [125I]RTI-55 binding to the dopamine transporter rapidly (within 10 min.) with an IC50 of 23.8 nM. After exposure to RTI-76, dopamine transporter binding reappeared in the cells over time with monoexponential kinetics with a half-life of 23 ± 1.6 h. Cell lines stably expressing DAT and irreversible binding ligands will be useful to study perturbations of transporter activity and their consequent effects on the transporter protein half-life.

Neurotransmitter Transporters: Is Glycosylation Necessary for Function?

A detailed description of the glycosylation of proteins is beyond the scope of this chapter. A brief outline of the process of glycosylation is described (Fig. 1). Emerging nascent polypeptide in the rough endoplasmic reticulum acquires a core of oligosaccharide from dolichol pyrophosphate catalyzed by glycosyltransferase (Hirschberg and Snider, 1987). This initial core of sugar residues (two N-acetylglucosamine(GlcNAc), nine mannose and three glucose residues) undergoes post-translational processing in endoplasmic reticulum and golgi to yield a final form of the oligosaccharide core. The specificity of sugar residues in the core is due to the presence of specific glycosyltransferases which are cell and tissue specific (Hubbard and Ivat, 1981; Kornfeld and Kornfeld, 1985).