Photoaffinity Labeling of the Human Dopamine Transporter UsingCocaine Analogs Pubblico
Henderson, Tamara Jackson (2008)
Published
Abstract
The photoaffinity ligand [125I]MFZ 2-24 has been used for labeling the human dopamine transporter (hDAT) to characterize the cocaine binding site. [125I]MFZ 2-24 has been shown to label a region within transmembrane domains (TM) 1-2 (Parnas et al., 2003). To further determine the site of photoincorporation, membranes from HEK 293 cells expressing hDAT were photolabeled and digested with trypsin. Following the digest, the labeled peptides were attached to the transmembrane domains. Residues R60 and R125 seem to be accessible to trypsin during the digest of the membrane preparation. Hydrolysis of these residues would cause the peptides formed during the digest to become displaced from the membranes and be found in the solution, which was shown to contain limited to no radioactivity. These results suggest that within TM 1-2 residues 1- 60 and 126-139 are not labeled by [125I]MFZ 2-24. This implies that the region between E61-R125 likely contains the radiolabeled residue. To further identify the labeled domain, [125I]MFZ 2-24 labeled hDAT was digested with enzymatic and chemical agents. The radiolabeled peptides were run on a high percentage gel and separated with HPLC, where fractions were collected. Radioactive fractions were subjected to subsequent enzymatic digestions and reanalyzed via HPLC. Shifts in the retention time of the labeled fragments suggested that the cleavage site of the corresponding enzyme was present in the initial peptide. The HPLC retention time of the peptide produced from a CNBr digest of hDAT suggests that the sequence PLFYM in TM2 contains the site of [125I]MFZ 2-24 incorporation. This is consistent with previous CNBr digests of hDAT, in which the peptide PLFYM in TM2 was thought to be labeled by [125I]MFZ 2-24 (Wirtz, 2004). Following HPLC separation of the peptides generated from a thermolysin digest of hDAT, the radiolabeled peptide was analyzed by Edman degradation. The second degradation cycle selectively released the most radioactivity, indicating that the second residue from the amino terminal end of the peptide is photolabeled. Considering the results from the CNBr digest of hDAT, HPLC analyses of a complete thermolysin digest and the Edman degradation results suggest that Y115 is possibly labeled by [125I]MFZ 2- 24.
Table of Contents
Chapter One: Introduction
Introduction to the Thesis
Dopamine Transporter: A Membrane Protein and Transporter
Membrane Protein
Methods Used for Studying Membrane Proteins
Transporters
Dopamine Transporter Function and Structure
Function of DAT
Structure of DAT
Cocaine and its Effect on DAT
Introduction to Cocaine
Effect of Cocaine on Monoamine Transporters
Reinforcing Effect of Cocaine on DAT
Binding of Cocaine and Other DAT Inhibitors
Understanding the Cocaine Binding Site in DAT
Photoaffinity Labeling
Introduction to Photoaffinity Labeling
Types of Photophores Used in Photoaffinity Labeling
Photoaffinity Labeling and DAT
Chapter Two: Methods
Cell Culture and Photoaffinity Labeling
Cell Culture and Membrane Preparation
Photoaffinity Labeling
Purification and Isolation of Radiolabeled hDAT
Immobilized Metal Affinity Chromatography
Gel Electrophoresis and Autoradiography
Western Blotting
Digestion of Radiolabeled hDAT
In Situ Digest of Radiolabeled hDAT
In-gel Digestion of Radiolabeled hDAT
Enzymatic Proteolysis with Trypsin
Enzymatic Proteolysis with Chymotrypsin
Enzymatic Proteolysis with Thermolysin
Chemical Cleavage with CNBr
Conversion of Homoserine Lactone to Homoserine
Separation and Digestion of Radiolabeled Peptides
HPLC and Monitoring of Radioactive Fractions
Peptide Digestion of Radioactive Fractions
Edman Degradation
Immobilization of Peptides and Manual Sequencing
Chapter Three: Results
Introduction to Results
Localization of Photoaffinity Labeled hDAT
The Affect Cocaine has on the Binding of hDAT
Photoaffinity Labels
Radioligands Label a Region Near or In the
Transmembrane Domains of wt hDAT
[125I]RTI-82 Incorporated Near or In the Transmembrane
Domains of x5C hDAT
CNBr Digest of [125I]MFZ 2-24 Labeled hDAT
Gel Electrophoresis of [125I]MFZ 2-24 Labeled hDAT CNBr Digestion
CNBr Effect on the Structure of Methionine
CNBr Effect on hDAT Radioligands
CNBr Digest of [125I]MFZ 2-24 Labeled hDAT
Chymotryptic Digest of [125I]MFZ 2-24 Labeled CNBr Peptide
Thermolysin Digest of [125I]MFZ 2-24 Labeled CNBr Peptide
Thermolysin Digest of [125I]MFZ 2-24 Labeled hDAT
HPLC Analysis of [125I]MFZ 2-24 Labeled hDAT
Thermolysin Digest
Thermolysin Digest of HPLC Fractions From Initial
Thermolysin Digest
Determination of [125I]MFZ 2-24 Labeled Residue
From a Thermolysin Digest
Chapter Four: Discussion
Introduction to the Discussion
Analysis of the Labeled Region of hDAT
The Effect of Cocaine on the Binding of hDAT Ligands
[125I]MFZ 2-24 Labels Near or In the Transmembrane Domains of wt hDAT
[125I]RTI-82 Labels Near or In the Transmembrane Domains of wt & x5C hDAT
Analysis of Radiolabeled CNBr Peptide
Mechanism By Which CNBr Cleaves Methionine
CNBr Digestion of hDAT
Chymotryptic Digest of CNBr Peptide
Thermolysin Digest of CNBr Peptide
Analysis of Thermolysin Digested hDAT
Thermolysin Digest of Labeled hDAT
Transmembrane Domains 1 & 2 of Neurotransmitter Transporters
References
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