Direct visualization of internalization and intracellular trafficking of dopamine-releasing protein-36aa.
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.
Smith
Sean
S
Neuroscience Program, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. smsmith2@uiuc.edu
Ramirez
Victor D
VD
D000595:Amino Acid Sequence; D000818:Animals; D019898:Autocrine Communication / Q000502:physiology; D001710:Biotin; D004705:Endocytosis / Q000502:physiology*; D005910:Glioma; D006023:Glycoproteins / Q000235:genetics / Q000493:pharmacokinetics*; D017979:Gold Colloid; D036341:Intercellular Signaling Peptides and Proteins; D008636:Mesencephalon / Q000166:cytology; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000493:pharmacokinetics*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000648:ultrastructure; D014443:Tyrosine / Q000378:metabolism
D006023:Glycoproteins; D017979:Gold Colloid; D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments; C058043:dopamine-releasing factor, rat; D014443:Tyrosine; D001710:Biotin; D011494:Protein Kinases