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.