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

The first insight of structure in 1969

• Pig brain• Polygonal basketwork• Coating control infolding

Coated pits

SUGGESTING:

• coatings were an apparatus to control the infolding of the plasma membrane during vesicle formation

Biochemical nature in 1975

• Centrifugation• SDS-PAGE• 180KD

Clathrin triskelionsThe first studies of the biochemical nature of the vesicle coat were published in 1975 by Barbara Pearse of the Medical ResearchCouncil in Cambridge, England

SDS–polyacrylamide gel electrophoresis

Three heavy chain

Three light chain

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.

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.

Question

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

LDL’s relationship with with REDUCTASE

Postulation

• Interaction between cell and LDL

Labeled-LDL

Normal cell--------binding

FH cell----------without binding

Fibroblast cells from either a control subject (closed circles) ora patient with homozygous FH (open circles) were grown in dishes containingfetal calf serum

HMG CoA reductase activity

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

Receptor!!!

To visualize the process

• 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

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.

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

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

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) )

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

(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

Structure

YXXφ signal

Dileucine signal σ of AP2

Tyrsine residue at 807

μ of AP2

β of AP2

Tyr residue

Clathrin coat

New research

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

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

THANKS