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Dissertation Information for Anh Thi Pham

NAME:
- Anh Thi Pham

DEGREE:
- Ph.D.

DISCIPLINE:
- Chemistry

SCHOOL:
- Emory University (USA) (2006)

ADVISORS:
- Joseph B. Justice Jr.

COMMITTEE MEMBERS:
- Vincent P. Conticello
- Dale E. Edmondson

MPACT Status: Fully Complete

Title: Photoaffinity labeling of the human dopamine transporter with RTI-82

Abstract: The yeast expression system Pichia was explored for its ability to overexpress DAT. A pcDNA3.1 plasmid carrying hexahistidine tagged hDAT at the N-terminal was cloned into pPic3.5K and subsequently transformed into the yeast strain GS115 and KM71. The C-terminal hexahistidine hDAT construct was cloned in a similar manner except the pPic3.5K plasmid was modified to incorporate a PentaHis-Ser-Asn-Ser-hexahistidine sequence. Pichia strains KM71 and GS115 were transformed with N- and C-terminal histidine tagged hDAT. A 2.3 kb fragment, correlating to hDAT, was observed in the Pichia strains KM71 and GS115. The PCR product sequence showed homology between the Pichia clones and the N-terminal histidine tagged construct. This suggested both the integration of the hDAT insert, and dopamine transporter DNA sequence coding into the yeast genome. The dopamine transporter functionality was assessed with [ 3 H]WIN 35,428 radioligand binding. The results obtained show no specific binding of the ligand---suggesting the expressed hDAT is nonfunctional. The DAT could be detected by Coomassie blue and silver staining. These amounts are sufficient for mass spectrometric studies and HPLC method development. Studies were undertaken to generate functional hexahistidine tagged hDAT. Histidine and FLAG tagged hDAT constructs were prepared; the C- and N-terminal terminal constructs were created by ligating the hDAT cDNA into the pcDNA3.1 myc his vector or peDNA3.1 his b vector respectively. Plasmid p3XFLAGCMV-10 was used to create the N-terminal FLAG vectors. COS 7 cells were utilized to assay the expression and functionality of both the C-terminal and N-terminal histidine tag constructs. RDEV and radioligand binding analysis indicated that the C-terminal histidine tagged hDAT was not functionally expressed. RDEV transport assays did not show dopamine uptake, and radioligand binding did not indicate hDAT specific binding of [ 3 H]WIN 35,428. Immunoblot analysis did indicate that the construct was being produced. The N-terminal histidine hDAT, the N-terminal FLAG hDAT, and the N-terminal FLAG/histidine hDAT constructs were shown to be functional, and displayed dopamine uptake. To prepare greater hDAT material HEK 293 cell cultivation on polymer spheres microcarriers was explored; however, recoveries were low. Alternatively, more robust approaches may need to be developed. Additionally, these systems proved difficult to purify, with no single approach proving satisfactory. Research was undertaken to localize RTI-82, the photoaffinity analog of cocaine, in the human dopamine transporter (hDAT) labeling. This would help to identify and characterize the active site for cocaine binding within the dopamine transporter. Previous work has localized the region of incorporation to transmembrane domains 4-6 in rat DAT (Vaughan et al., 1996). To proceed, functional hDAT was constructed with incorporation of a 3X FLAG and 6X histidine epitope at the N-terminus for purification of [ 125 1] RTI-82 photolabeled hDAT. Following photolabeling, hDAT membranes were solubilized, purified on an immobilized metal affinity column (IMAC), and separated by SDS-PAGE. Photolabeled hDAT bands were digested with trypsin, chymotrypsin or cyanogen bromide and separated on a high percentage gel. Bands corresponding to labeled hDAT peptides were analyzed by HPLC, TLC, and mass spectrometry. A 3kDa band was obtained from chymotryptic digestion. Mass spectral analysis identified two peptides: a 2644 Da fragment, whose amino acid sequence was consistent with a region in the N terminus, and a 2391 Da fragment, whose sequence K260-L281 comprised a portion of the second intracellular loop and a substantial region of transmembrane domain 5 (TM 5). The 2391 Da peptide sequence has a methionine, M272 at its midpoint. Therefore, CNBr, which cleaves proteins at Met residues, was used to digest. Additionally, [ 125 1] RTI-82 tryptic and chymotryptic hDAT peptide conjugated to sequelon aryl amine disks showed that the label resides on the C-terminus of the methionine cleavage site. The cyanogen bromide digests could not be analyzed by mass spectroscopy; however, SDS-PAGE could be used to identify bands migrating at approximately 10 kDa. TLC analysis was found to support the presence of methionine in the cyanogen bromide digests. For example, cyanogen bromide digestion of [ 125 I]RTI-82 labeled chymotryptic or tryptic peptide revealed methionine. Thus, the 2391 Da chymotryptic peptide and 10 kDa band possessed similar sequences. The data obtained are consistent with cleavage of M272 of TM 5 and M371 of TM 7. These results suggest that the region of RTI-82 incorporation is within a narrow region of TM 5 from P273 to L281.

MPACT Scores for Anh Thi Pham

A = 0
C = 0
A+C = 0
T = 0
G = 0
W = 0
TD = 0
TA = 0
calculated 2008-03-16 16:02:30

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