Document Detail

Direct visualization of internalization and intracellular trafficking of dopamine-releasing protein-36aa.
MedLine Citation:
PMID:  11867938     Owner:  NLM     Status:  MEDLINE    
Dopamine-releasing protein (DARP) is a potent regulator of dopamine (DA) release known to be involved in the development of rat catecholaminergic systems. In the present study, we examined the internalization and transport of DARP-36aa, a synthetic peptide derived from the N-terminal sequence of DARP, in rat C6 glioma cells. A colloidal gold DARP-36aa conjugate (DARP-36aa: AU) and biotinylated DARP-36aa were employed to visualize internalization and intracellular transport of DARP-36aa. Electron microscopy demonstrated that DARP-36aa: AU was rapidly incorporated into C6 glioma cells. Internalization via clathrin-coated pits and vesicles was clearly observed followed by transport and sorting of DARP-36aa: AU into multivesicular bodies, tubulo-vesicular endosomes, and lysosomes. Internalization of DARP-36aa: AU was also examined in primary mesencephalic cell cultures where a similar pattern of internalization and transport via clathrin-coated pits and vesicles was observed. Fluorescence microscopy using a biotinylated DARP-36aa/avidin-rhodamine conjugate revealed that DARP-36aa is diffusely distributed on the plasmalemma prior to internalization at 4 degrees C. Following a 30-min incubation at 37 degrees C DARP-36aa was concentrated in the cytosol, particularly in areas surrounding cellular projections and the perikaryon. Immunocytochemical studies employing biotinylated DARP-36aa and an anti-clathrin heavy chain antibody demonstrated that DARP-36aa and clathrin colocalize during DARP-36aa internalization. We also observed a marked increase in tyrosine phosphorylation of a 45-kD protein in response to DARP-33a stimulation in C6 glioma cells. Genistein, a specific tyrosine kinase inhibitor, significantly inhibited DARP-36aa: AU internalization and transport in C6 glioma cells. These findings suggest that tyrosine kinase activity may result in DARP-36aa receptor-mediated endocytosis.
Sean Smith; Victor D Ramirez
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Neuroendocrinology     Volume:  75     ISSN:  0028-3835     ISO Abbreviation:  Neuroendocrinology     Publication Date:  2002 Feb 
Date Detail:
Created Date:  2002-02-27     Completed Date:  2002-07-19     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  0035665     Medline TA:  Neuroendocrinology     Country:  Switzerland    
Other Details:
Languages:  eng     Pagination:  98-112     Citation Subset:  IM    
Copyright Information:
Copyright 2002 S. Karger AG, Basel
Neuroscience Program, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
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MeSH Terms
Amino Acid Sequence
Autocrine Communication / physiology
Endocytosis / physiology*
Glycoproteins / genetics,  pharmacokinetics*
Gold Colloid
Mesencephalon / cytology
Microscopy, Electron
Molecular Sequence Data
Peptide Fragments / genetics,  pharmacokinetics*
Protein Kinases / metabolism
Protein Transport / physiology*
Rats, Sprague-Dawley
Tumor Cells, Cultured / metabolism,  ultrastructure
Tyrosine / metabolism
Reg. No./Substance:
0/Glycoproteins; 0/Gold Colloid; 0/Peptide Fragments; 0/dopamine-releasing factor, rat; 55520-40-6/Tyrosine; 58-85-5/Biotin; EC 2.7.-/Protein Kinases

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