p180, a novel recycling transmembrane glycoprotein with restricted cell type expression.
A 180-kilodalton (kDa) protein (p180) was identified among the antigens for a panel of monoclonal antibodies raised against human fibroblast cell surface proteins. Binding studies with 125I-Fab' fragments of an anti-p180 monoclonal antibody demonstrated that 10 to 30% of p180 was located on the plasma membrane and that the remaining 70 to 90% was on intracellular membranes. p180 was rapidly internalized from the cell surface at 37 degrees C, and kinetic analyses indicated that this was a constitutive process followed by the recycling of p180 back to the plasma membrane. Morphological studies demonstrated that on the cell surface p180 was concentrated in coated pits, whereas inside the cell it was found in endosomes as suggested by its colocalization with the transferrin receptor. Immunoblot analysis with a polyclonal antiserum raised against purified human protein showed that p180 has a restricted distribution with expression at high levels in fibroblast cultures and in tissues containing cells of mesodermal origin. A biochemical characterization of p180 showed it to be a transmembrane glycoprotein with an extracellular domain, which consists of approximately 30 kDa of complex oligosaccharides attached to at least 45 kDa of the protein core. The cytoplasmic domain of p180 was found to contain a serine residue(s) that was phosphorylated both in vivo and in vitro by activated protein kinase C. p180 was purified by subjecting solubilized membrane proteins from a human osteosarcoma cell line to immunoaffinity chromatography and gel filtration. The N-terminal sequence information obtained from the purified protein showed no homology to other known proteins. It was concluded that p180 may be a novel recycling receptor which is highly restricted in its expression to fibroblastlike cells.
Department of Biochemistry, Imperial College of Science and Technology, University of London, England.
C M
CM
Isacke
P
P
van der Geer
T
T
Hunter
I S
IS
Trowbridge
D000911:Antibodies, Monoclonal; D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis / Q000648:ultrastructure; D002846:Chromatography, Affinity; D004705:Endocytosis; D005455:Fluorescent Antibody Technique; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D007425:Intracellular Membranes / Q000032:analysis / Q000648:ultrastructure; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D012516:Osteosarcoma; D010750:Phosphoproteins / Q000302:isolation & purification; D011493:Protein Kinase C / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; D011493:Protein Kinase C