Download Receptor-Mediated Endocytosis experiment pathway（II）

Receptor-Mediated Endocytosis
experimental pathway（II）
Wu Chenzi
Experiments about …
• The process of receptor binding
and internalization （内化作用）
• The LDL receptor
• Internalization signals
The process of
receptor binding and internalization
Technique — electron microscope
To visualize the process of receptor
binding and internalization, Michael Brown
and Joseph Goldstein (University of
Texas Medical School in Dallas) teamed
up with Richard Anderson, who had been
studying cellular structure with the
electron microscope.
Experimental pathway
• The group incubated fibroblasts（成纤维细胞）
from normal and FH subjects with LDL that
had been covalently linked（共价结合） to the
iron-containing protein ferritin（铁蛋白）.
• Because of the iron atoms, ferritin molecules
are able to scatter a beam of electrons and
thus can be visualized in electron microscope.
• A temperature of 4℃
Ligands（配体） can bind to the cell surface
but cannot be internalized（内化）, LDLferritin particles were seen to be bound to the
cell surface.
Found that …
1、The LDL particles were not randomly
scattered over the cell surface but were
localized to some segments of plasma
membrane —— coated pits.
2、Cells from patients with FH were found
to have a similar number of coated pits on
their surface, but no LDL-ferritin was bound
to these mutant cells.
• The results supported the proposal
that the mutant FH allele encoded
receptor that was unable to bind LDL.
• Subsequent electron microscopic studies
on the internalization of LDL-ferritin
revealed the endocytic pathway by which
these lipoprotein particles were
internalized
Postulated that …
• The rapid internalization of receptor-bound LDL
is strictly dependent on the localization of LDL
receptors in coated pits.
• If an LDL receptor failed to become localized
within a coated pit, it would be unable to
deliver its bound ligand to cellular lysosomes
and thus would be unable to affect cholesterol
（胆固醇） biosynthesis within the cell.
The LDL receptor
• J.D. mutation
At about this time, an LDL receptor with a
different type of mutation was discovered.
LDL receptors bearing this new defect bound
normal amounts of radioactively labeled LDL,
yet the receptor-bound lipoprotein failed to
internalized and consequently was not
delivered to cytoplasmic lysosomes for
processing.
• Structure of the LDL receptor
A transmembrane glycoprotein of 839
amino acids, with the 50 amino acids at
the c-terminal end of the protein
extending inward from the membrane as
a cytoplasmic domain
Its cytoplasmic domain was specifically
bound by a likely subunit of an AP
adaptor of the coated pits.
J.D. mutation
• Analysis of the J.D. mutant receptor
revealed that the protein contained a
single amino acid substitution: a tyrosine(酪
氨酸) residue normally located at position
807 was replaced by a cysteine(半胱氨酸).
This single alteration in amino acid
sequence obliterated the ability of the
protein to become concentrated in
coated pits.
J.D. mutant receptor
(unable to become localized in a cell’s coated pits)
Internalization signals
• Over the next few years, attention turned
to the amino acid sequences of the
cytoplasmic tails of other receptors that
become localized in coated pits.
Was there a common
internalization signal?
Two signals
• NPXY signal as in the LDL receptor
• YXXφ signal as in the transferrin（铁
转运
蛋白） receptor
Y — tyrosine
X — any amino acid
φ— an amino acid with a bulky hydrophobic
side chain （疏水侧链）
The YXXφ sequence
of the receptor
binds to the μ
subunit of the AP2
adaptors.
AP2 adaptor
complex binds to
the clathrin coat
by means of its
β subunit.
The interaction between
the adaptor and internalization signal
μ subunit（gray）
have tow
hydrophobic pockets
one that binds the tyr
and the other that
binds the bulky
hydrophobic side
chain of the
internalization signal
(purple).
Many Thanks