Receptor-mediated endocytosis of albumin by kidney proximal tubule cells is regulated by phosphatidylinositide 3-kinase.
Receptor-mediated endocytosis of albumin is an important function of the kidney proximal tubule epithelium. We have measured endocytosis of [125I]-albumin in opossum kidney cells and examined the regulation of this process by phosphatidylinositide 3-kinase (PI 3-kinase). Albumin endocytosis was inhibited by both wortmannin (IC50 6.9 nM) and LY294002 (IC50 6.5 microM) at concentrations that suggested the involvement of PI 3-kinase in its regulation. Recycling rates were unaffected. We transfected OK cells with either a wild-type p85 subunit of PI 3-kinase, or a dominant negative form of the p85 subunit (Deltap85) using the LacSwitch expression system. Transfects were screened by immunoblotting with anti-PI 3-kinase antibodies. Under basal conditions, transfects demonstrated no expression of p85 or Deltap85, but expression was briskly induced by treatment of the cells with IPTG (EC50 13.7 microM). Inhibition of PI 3-kinase activity by Deltap85 was confirmed by in vitro kinase assay of anti-phosphotyrosine immunoprecipitates from transfected cells stimulated with insulin. Expression of Deltap85 resulted in marked inhibition of albumin endocytosis, predominantly as a result of reduction of the Vmax of the transport process. Expression of p85 had no significant effect on albumin uptake. The results demonstrate that PI 3-kinase regulates an early step in the receptor-mediated endocytosis of albumin by kidney proximal tubular cells.
Brunskill
N J
NJ
Department of Cell Physiology and Pharmacology, Leicester University Medical School, Leicester LE1 9HN, United Kingdom. njb18@le.ac.uk
Stuart
J
J
Tobin
A B
AB
Walls
J
J
Nahorski
S
S
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D004705:Endocytosis / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D007328:Insulin / Q000494:pharmacology; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007684:Kidney Tubules / Q000201:enzymology*; D007700:Kinetics; D009025:Morpholines / Q000494:pharmacology; D009893:Opossums; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D019000:Phosphotyrosine / Q000032:analysis; D011956:Receptors, Cell Surface / Q000502:physiology*; D012709:Serum Albumin / Q000378:metabolism*; D014162:Transfection / Q000235:genetics; D000077191:Wortmannin
D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D007328:Insulin; D009025:Morpholines; D011956:Receptors, Cell Surface; D012709:Serum Albumin; D019000:Phosphotyrosine; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D007544:Isopropyl Thiogalactoside; D019869:Phosphatidylinositol 3-Kinases; D000077191:Wortmannin