COCAINE RECEPTOR--BIOCHEMICAL AND MOLECULAR STUDIES

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The goal of this project is to elucidate the biochemical properties of the cocaine receptor or the dopamine transporter. We have previously shown that the dopamine transporter in the rat striatum and nucleus accumbens are heterogeneous in molecular weight. When the transporters were treated with N-glycanase, the molecular weight of both transporters were reduced significantly and the apparent differences were either absent or minimized. This suggests that the factor in heterogeneity is differences in glycosylation although it is not possible to rule out other sources of heterogeneity as well. We have also prepared anti- peptide antibodies to the dopamine transporter and some of these transporter antibodies are useful for immunoprecipitation and western blotting. There are species differences in the cloned CDNAS for dopamine transporters. Accordingly, we examined the molecular weight of transporters, post-mortem changes as well as glycosylation differences among the different species. The molecular weights differed as one would expect based on differences predicted by the CDNA sequences. Dopamine transporters bind cocaine and its analogues with two sites of differing affinities. This has raised the issue of whether or not there are more than one type of dopamine transporter or whether heterogeneity due possibly to glycosylation could account for the different sites. Accordingly, a CDNA for the dopamine transporter was transfected into COS cells and, these cells expressed two binding sites for cocaine. These data indicate that the heterogeneous binding occurs from a single protein which must be modified in different ways. We have examined the distribution of dopamine transporter MRNA in brain by in situ hybridization analysis. We find that cell groups found in the midbrain contain high levels of MRNA while cell groups outside of the midbrain contain very low levels of MRNA for the transporter. We have made substantial progress in understanding the nature of the dopamine transporter protein, how it is processed and where it is produced.