Identification and characterization of the dopamine (DA) transporter containing the cocaine binding site (cocaine receptor) in the CNS is a necessary step toward the development of efficacious treatment agents for cocaine abuse. This proposal will isolate and purify a putative DA transporter protein for protein sequencing using a GBR affinity chromatography method, and will identify its cDNA. The investigators have purified a glycoprotein of Mr 62 kDA which retains a pharmacologic profile suggestive of the DA transporter. Purification in sufficient yield for protein sequencing is now feasible. Western blots and immunoprecipitation studies of the polyclonal rabbit antibody against the protein suggests recognition of an epitope at a similar molecular weight. Screening of an expression library in phage lambda-gt11 from human substantia nigra using the polyclonal antibody has demonstrated 8 positive clones from an initial screen of 200,000 plaques. Rescreening of the library with the putative clones is presently in progress. Northern blots indicate positive signals localized to the substantia nigra using these clones. RACE PCR is now underway to obtain full length cDNA sequence utilizing primers from two sources 1) partially sequenced clones; 2) partial peptide sequence from the GBR-affinity purified protein.
Specific aims to be accomplished in this proposal are: 1a) complete pharmacologic characterization of the affinity purified DA transporter and reconstitution functional studies of the protein, 1b) protein sequencing; 2) full characterization of the rabbit polyclonal antibody including immunohistochemistry, 3) expression cloning of the DA transporter with subcloning and sequencing using primer extension to characterize inserts of subclones presently identified. Partial inserts will be used to rescreen the library to obtain full length clones. Full length cDNA specificity, which may be available from the initial subclones, will be confirmed using Northern hybridization and rescreening of the original expression library. RACE PCR will be used to generate 3'and 5' ends from substantia nigra RNA template. Expression of full coding sequence for DA transporter in a eukaryotic system such as COS cells which do not normally express cocaine binding, followed by pharmacologic and functional assessment of high affinity cocaine binding and dopamine uptake capability in the transformed COS cells. Expression in a Baculovirus system is planned to provide large quantities of transporter protein for protein sequencing and incorporation into liposomes for functional studies. Long range goals will include site directed mutagenesis and a survey of regional expression and ontogeny of brain mRNA levels coding for the cocaine receptor.
| Altman-Hamamdzic, S; Groseclose, C; Ma, J X et al. (1997) Expression of beta-galactosidase in mouse brain: utilization of a novel nonreplicative Sindbis virus vector as a neuronal gene delivery system. Gene Ther 4:815-22 |
