LDL receptor Flashcards
what binds to the LDLR
ApoB100 binds to the LDLR
– Only binds LDL, (thus not VLDL, β-VLDL, and
IDL)
* ApoB48 cannot bind to the LDLR
– Thus no binding of chylomicrons and
chylomicron remnants
how does ApoB100 bind to the LDLR
by electrostatic interaction
what is the LDL type A domain
The LDLR type A (or LA) domain is a
segment found on the LDLR that is
responsible for ligand binding
structure of LA domain that makes it useful
The LA domain within the LDLR has
specifically spaced negative amino acids
that electrostatically bind the positively
charged receptor binding domain on
apoB100 from LDL
what happnes when apoB100 binds to the LDLR
Upon binding of apoB100 to the LDLR, the
cytosolic portion of the LDLR becomes
phosphorylated
– This turns on a cell signaling cascade that
results in the internalization of a clathrin
coated membrane vesicle, carrying the LDLR
and LDL inside the cell
where does phosphorylation of LDLR occur
Phosphorylation occurs at the tyrosine
residue on a NPXY motif
NPXY motif
The NPXY motif is a 4 amino acid
sequence found on many membrane-
bound receptors on the cytosolic side of
the receptor
– Asn-Pro-Xaa-Tyr
what does the phosphorylation of the tyrosine in the NPXY sequence lead to
The phosphorylation of the tyrosine in the NPXY sequence typically stimulates cell functions, such as receptor internalization
Dephosphorylation of the NPXY motif
within the LDLR leads to
the recycling of
the LDLR back to the cell surface to pick
up more LDL
LDL receptor family
all have the
YWTD B propellor
LA domain
EFG like domain
what isthe EGF like domain
The epidermal growth factor (EGF)-like
domain is a component of several
secretory proteins and of the extracellular
region of several membrane proteins
length of the EGF like domain
EGF-like domains are ~30-40 amino acids
in length, with 6 cysteines that form
disulfide bridges
what is the function of the EGF like domain
Typically, the EGF-like domains act as a
structural component to a protein
YWTD b propelor struture and aa sequence
A propeller structure consisting of 6 blades
* Each blade is formed from a ~40 amino
acid sequence, starting and ending with a
cystine bridge, and containing the YWTD
(Tyr-Trp-Thr-Asp) sequence
* Each blade has 4 antiparallel β-strands
functions of the YWTD b propellor
varies
in many proteins
* The YWTD β-propeller function in the
LDLR is to act as a switch to release LDL
from the receptor in response to low pH
– The function of YWTD β-propellers within other LDLR family members is thought to be similar
LDL receptor related protein 1 strutre
The LDL receptor-related protein 1 (LRP1)
is the largest member of the LDLR family
at 600 kDa
* It is cleaved by a cellular proprotein
convertase (called furin) at the 8 th YWTD
β-propeller into an α-subunit (515 kDa)
and a β-subunit (85 kDa) that become
covalently linked together
ligand binding in LRP1
LRP1 has 4 distinct clusters for ligand
binding via LA domains
– There are several ligands that can associate
with LRP1 which are involved in different
processes
what are some ligands that bind to LRP1
EL, LPL, HL
LRP1 and LPL/HL
LRP1 directly binds LPL and HL
human HL deficieny leads to
Human HL deficiency leads to elevated
chylomicron remnants and β-VLDL in the
bloodstream
It is thought that cell surface LPL and HL
that capture lipoproteins can
‘bridge’ the
lipoproteins to LRP1
how does LRP1 bind to remenants in the bloodstream
LRP1 binds remnant lipoproteins in the
bloodstream and removes them from the
bloodstream
– Binding of remnant lipoproteins is via apoE
Deletion of LRP1 in animal models
embryonic lethal
dleetion of apoE
Deletion of apoE leads to severe
atherosclerosis due to remnant lipoprotein
accumulation
RCT
Reverse cholesterol transport (RCT) is
the process of removing non-hepatic
cholesterol for delivery to the liver and
ultimately for excretion from the body
what does RCT require
the transfer of cholesterol
and PL from cells to apoA-I, preβ-HDL,
and mature HDL in the circulation – a
process called cholesterol efflux
what is cholesterol efflux
Cholesterol efflux is the transfer of
cholesterol (and phospholipid) from cells
to apoA-I or various forms of HDL
Cholesterol efflux to apoA-I is essential for
generating preβ-HDL and ‘mature’ HDL
– Efflux via ABCA1
what is cholesterol efflux less essential for
Cholesterol efflux to ‘mature’ HDL also
occurs, but it is not essential
– Efflux via ABCG1 and SR-BI
what is Needed for cholesterol efflux in vitro
– Macrophages & appropriate growth media
– Source of apoA-I and/or HDL
– Means to distinguish effluxed cholesterol from
macrophage & media cholesterol
Macrophages & appropriate growth media involves what
– Primary cells: freshly isolated from blood
– Cell lines: ‘immortalized’ macrophages or
monocytes that can be differentiated into
macrophages
– Growth media: contains glucose and amino
acids, plus serum (that contains essential
growth factors)
Source of apoA-I and/or HDL involves what
– ApoA-I isolated by affinity chromatography
– HDL isolated by ultracentrifugation
– Plasma or sera (containing HDL and apoA-I)
Means to distinguish effluxed cholesterol
from macrophage & media cholesterol involves what
Cells can be fed a labelled cholesterol:
* radiolabel (3H or 14C)
* stable isotope (2H or 13C)
* fluorescent (BODIPY)
Why do cholesterol efflux
assays?
Tests the ability of macrophages, treated
with various agents, to be able to efflux
cholesterol
* Tests the ‘functionality’ of HDL from a
human subject or animal (treated with
various agents, or exhibiting a disorder)
HDL cholesterol assocaition with CAD
no association
Summary of steps in cholesterol
efflux assays
- Prepare cells and allow cells to settle
- Incubate cells 24h with labeled cholesterol
- Following labeling, incubate cells with BSA for 24h – allows for equilibration of labeled cholesterol
- Incubate cells for up to 8h in the presence of acceptors of cholesterol – apoA-I, HDL, sera
- Count cell and media label
how does HDL cholesterol change with HL and EL ko
increases the more -/- it becomes
fractional efflux of cholesterol from lipase ko mice
increases in dko
Options for cholesterol efflux assays
Acyl-CoA:cholesterol acyltransferase
(ACAT) inhibition:
nuclear LXR/RXR simulation
nuclear LXR/RXR simulation
increases ABCA1 levels
Acyl-CoA:cholesterol acyltransferase
(ACAT) inhibition:
prevents storage of labeled cholesterol as CE
