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Receptor-Mediated Endocytosis

Receptor-Mediated Endocytosis. Introduction. Question. How are the high-molecular- weight yolk （卵黄） proteins able to enter the oocyte( 卵母细胞 )?. The first research in 1964. Depressions on surface Bristly coat Invagination Forming coated vesicle. Coated Pits.

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Receptor-Mediated Endocytosis

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  1. Receptor-Mediated Endocytosis

  2. Introduction

  3. Question How are the high-molecular- weight yolk （卵黄）proteins able to enter the oocyte(卵母细胞)?

  4. The first research in 1964 • Depressions on surface • Bristly coat • Invagination • Forming coated vesicle

  5. Coated Pits

  6. The first insight of structure in 1969 • Pig brain • Polygonal basketwork • Coating control infolding

  7. Coated pits

  8. SUGGESTING: • coatings were an apparatus to control the infolding of the plasma membrane during vesicle formation

  9. Biochemical nature in 1975 • Centrifugation • SDS-PAGE • 180KD

  10. Clathrintriskelions The first studies of the biochemical nature of the vesicle coat were published in 1975 by Barbara Pearse of the Medical Research Council in Cambridge, England SDS–polyacrylamide gel electrophoresis Three heavy chain Three light chain

  11. Another research in 1973 • Examine cholesterol metabolism • HMG CoA reductase govern the biosynthesis of cholesterol • Fibroblast • LDL yellowish patches consisting of cholesterol deposits above the eyelids.

  12. Brief introduction about FH • familial hypercholesterolemia(FH) • People who were homozygous for the defective gene (the FHallele) had profoundly elevated levels of serum cholesterol (800 mg/dlvs. 200 mg/dl for a normal person), invariably developed severelyblocked (atherosclerotic) arteries, and usually died from heart attackbefore the age of 20.

  13. Question • How could lipoproteins affect the activity of HMG CoA reductase?

  14. LDL’s relationship with with REDUCTASE

  15. Postulation • Interaction between cell and LDL Normal cell--------binding Labeled-LDL FH cell----------without binding

  16. HMG CoA reductase activity Fibroblast cells from either a control subject (closed circles) or a patient with homozygous FH (open circles) were grown in dishes containing fetal calf serum

  17. 1973 in the laboratories of Michael Brown and Joseph Goldstein of the University of Texas. interested in the inherited condition familial hypercholesterolemia (FH).

  18. Receptor!!!

  19. LDL Covalently link to the iron-containing protein Normal fibroblast incubated with LDL-ferritin at 4℃ Close examination : LDL particles are not randomly scatter but localized to short segments To visualize the process

  20. LDL particles concentration 1977 Electron micrograph showing the binding of LDL to coated pits of human fibroblasts. The LDL is made visible by conjugating the particles to electron-dense, iron-containing ferritin.

  21. Results and conclusions LDL particles Coated pits -------similar Bind LDL -------Fail (FH mutation) Internalize -------Fail (J.D mutation) FH encode a receptor that was unable to bind LDL

  22. LDL receptor • The Low-Density Lipoprotein (LDL) Receptor is a mosaic protein that mediates the endocytosis of cholesterol-rich LDL • LDL receptor complexes are present in clathrin-coated pits (or buds) on the cell surface, which when bound to LDL-cholesterol via adaptin, are pinched off to form clathrin-coated vesicles inside the cell

  23. The O-linked sugar domain is thought to act mainly as a hydrophilic buffer zone that keeps bound lipoprotein particles away from the lipid bilayer of the plasma membrane. The NPxY motif in the cytoplasmic tail of the LDL receptor is required for clustering and internalization. Boxed references next to structural representations indicate the cited publication from which the respective structure has been reproduced. (Nature Structural Biology  8, 476 - 478 (2001) )

  24. AP2 adaptor complex • a multimeric protein that works on the plasma membrane to internalize cargo in clathrin-mediated endocytosis • a stable complex of four adaptins which give rise to a structure that has a core domain and two appendage domains attached to the core domain by polypeptide linkers

  25. (b) clathrin coat, AP2 adaptor, membrane receptors. PI(4,5)P2 - Internalization signals , Recruit AP2 to PM Each receptor is bound to a ligand being internalized. (c) Reconstruction of a clathrin cage containing 36 triskelions

  26. Structure YXXφ signal μ of AP2 Tyr residue Dileucinesignal σ of AP2 Tyrsine residue at 807 Clathrin coat β of AP2

  27. Newresearch • Scientist can label more proteins such as dynamin, AP2, in different colors.

  28. red-fluorescent LDL particle , a green-fluorescent clathrin-coated pit and its incorporation into a clathrin-coated vesicle, which becomes uncoated and moves into the cytoplasm

  29. THANKS

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