Characterization of the beta 2-microglobulin endocytic pathway in rat proximal tubule cells.
The uptake mechanism(s) of low-molecular-weight proteins by proximal tubule cells remains incompletely characterized. We utilized a biochemical and semiquantitative morphological approach to better characterize the endocytic pathway of an anionic protein, beta 2-microglobulin (beta 2M), in the rat proximal tubule. Indirect immunogold techniques revealed beta 2M was taken up via a classic receptor-mediated endocytic pathway. In vitro biochemical and morphological characterization of iodinated beta 2M and gold-conjugated beta 2M (gold-beta 2M) binding to isolated brush-border membrane vesicles (BBMV) documented specific and quantitatively similar binding interactions of the modified beta 2M with BBMV. Kinetic characterization of the in vivo endocytic pathway of gold-beta 2M was undertaken using microinfusion of individual tubules. beta 2M initially bound at the apical surface, was internalized into subapical coated vesicles and delivered to endosomal-like structures within 5 min, and, finally, was concentrated in lysosomal-like structures within 15 min. This uptake was inhibited by excess unconjugated beta 2M. In addition, we directly showed that uptake did not occur across the basolateral surface. Finally, by passing solubilized BBMV over beta 2M affinity columns we were able to isolate binding activity.
Sundin
D P
DP
Department of Medicine, University of Colorado School of Medicine, Denver.
Cohen
M
M
Dahl
R
R
Falk
S
S
Molitoris
B A
BA
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004705:Endocytosis; D006046:Gold; D007150:Immunohistochemistry; D007457:Iodine Radioisotopes; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D008871:Microvilli / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014018:Tissue Distribution; D001613:beta 2-Microglobulin / Q000493:pharmacokinetics*
D007457:Iodine Radioisotopes; D001613:beta 2-Microglobulin; D006046:Gold