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
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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 triskelions
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
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
Normal cell--------binding
Labeled-LDL
FH cell----------without binding
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
1973 in the laboratories of Michael Brown and Joseph
Goldstein of the University of Texas. interested in the
inherited condition familial hypercholesterolemia (FH).
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 clathrincoated 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 clathrinmediated 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
μ of AP2
Tyr residue
Dileucine signal σ of AP2
Tyrsine residue at 807
Clathrin coat β of AP2
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