Kinetic and morphological evidence for endocytosis of mammalian cell integrin receptors by using an anti-fibronectin receptor beta subunit monoclonal antibody.
Monoclonal antibody (mAb) 7E2.2, which recognizes the beta subunit of the hamster fibronectin receptor (FnR) (Brown, P.J., and Juliano, R. L. (1988) Exp. Cell Res. 177. 303), was used to examine the distribution of and to quantify the internalization of the FnR and possibly related integrins on adherent fibroblasts. Purified 7E2.2 IgG was iodinated and used in binding and internalization studies. Binding to Chinese hamster ovary cells was saturable with a Km of 0.3 nM and an estimated total number of cell surface beta subunits at 2 x 10(5) per cell. The FnR colocalized with fibronectin at cell adhesion contact sites and also was distributed evenly over the dorsal cell surface as discrete clusters. By using a direct immunocolloidal gold approach, the FnR was not associated with coated pits at 4 degrees C until internalization followed warming of the labeled cells to 37 degrees C. A proportion of the FnRs were endocytosed with a half-time of 6.5 min and, consistent with clathrin-mediated uptake, this was sensitive to hypertonic conditions. Receptor-immunocomplexes rapidly became localized within coated pits, small diameter tubules, and peripheral endosomes but the majority remained at the cell surface. At subsaturating concentrations of bound 7E2.2, approximately one-fourth of the total cell receptor population resided intracellularly at any one moment following steady-state; however, appreciable degradation of the iodinated mAb was not detected following accumulation for 4 h at 37 degrees C. These data showed that at least a portion of the FnR are endocytosed via a receptor-mediated pathway and suggested that these receptors do not immediately enter a degradative compartment.
Raub
T J
TJ
Drug Delivery Systems Research, Upjohn Company, Kalamazoo, Michigan 49001.
Kuentzel
S L
SL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis; D005260:Female; D007074:Immunoglobulin G / Q000276:immunology; D016023:Integrins / Q000276:immunology / Q000378:metabolism*; D010053:Ovary / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure*; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D016023:Integrins; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic