Thrombomodulin lacking the cytoplasmic domain efficiently internalizes thrombin via nonclathrin-coated, pit-mediated endocytosis.
Thrombomodulin (TM) is a transmembrane vascular endothelial cell receptor that is a cofactor in a major physiologically relevant natural anticoagulant system. We recently developed a cell model to examine one mechanism of regulation of TM cell surface expression and visually demonstrated that the receptor undergoes internalization predominantly via noncoated pits (Conway et al., 1992, J. Cell. Phys., 151:604-612). We have extended these studies to examine the role of the cytoplasmic domain of TM by deleting this region and expressing the truncated version of the molecule in COS cells (COS.Cyto.Del cells). Electron microscopy demonstrated internalization of gold-labeled anti-TM antibody or thrombin in a time- and temperature-dependent manner, similar to that seen with the wild-type transfected cells (COS.TM-CR). Endocytosis was characterized by initial surface clustering of gold particles, followed by aggregation into noncoated pits, early endosome formation, and, finally, entry into multivesicular bodies and lysosomes. There was a notable absence of gold particles in clathrin-coated pits and vesicles. The kinetics of binding and internalization of 125I-labeled ligand in COS.Cyto.Del cells was compared with that of COS.TM-CR cells and was not significantly different. These studies provide ultrastructural and quantitative data to indicate that TM efficiently undergoes endocytosis via nonclathrin-coated pits when the receptor is lacking the cytoplasmic domain. This finding suggests that there may be alternative regions of the molecule that mediate those signals necessary for internalization.
Conway
E M
EM
Department of Medicine, Toronto Hospital, University of Toronto, Ontario, Canada.
Nowakowski
B
B
Steiner-Mosonyi
M
M
D001483:Base Sequence; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D004705:Endocytosis; D005455:Fluorescent Antibody Technique; D006046:Gold; D008024:Ligands; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D013917:Thrombin / Q000378:metabolism*; D018180:Thrombomodulin / Q000235:genetics / Q000378:metabolism*
D002966:Clathrin; D008024:Ligands; D015345:Oligonucleotide Probes; D018180:Thrombomodulin; D006046:Gold; D013917:Thrombin