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Direct visualization of internalization and intracellular trafficking of dopamine-releasing protein-36aa.

Abstract
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.
AuthorsSean Smith, Victor D Ramirez
JournalNeuroendocrinology (Neuroendocrinology) Vol. 75 Issue 2 Pg. 98-112 (Feb 2002) ISSN: 0028-3835 [Print] Switzerland
PMID11867938 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)
CopyrightCopyright 2002 S. Karger AG, Basel
Chemical References
  • Glycoproteins
  • Gold Colloid
  • Intercellular Signaling Peptides and Proteins
  • Peptide Fragments
  • dopamine-releasing factor, rat
  • Tyrosine
  • Biotin
  • Protein Kinases
Topics
  • Amino Acid Sequence
  • Animals
  • Autocrine Communication (physiology)
  • Biotin
  • Endocytosis (physiology)
  • Glioma
  • Glycoproteins (genetics, pharmacokinetics)
  • Gold Colloid
  • Intercellular Signaling Peptides and Proteins
  • Mesencephalon (cytology)
  • Microscopy, Electron
  • Molecular Sequence Data
  • Peptide Fragments (genetics, pharmacokinetics)
  • Phosphorylation
  • Protein Kinases (metabolism)
  • Protein Transport (physiology)
  • Rats
  • Rats, Sprague-Dawley
  • Tumor Cells, Cultured (metabolism, ultrastructure)
  • Tyrosine (metabolism)

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