LDL receptor

Question 1

what binds to the LDLR

Answer 1

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

Question 2

how does ApoB100 bind to the LDLR

Answer 2

by electrostatic interaction

Question 3

what is the LDL type A domain

Answer 3

The LDLR type A (or LA) domain is a
segment found on the LDLR that is
responsible for ligand binding

Question 4

structure of LA domain that makes it useful

Answer 4

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

Question 5

what happnes when apoB100 binds to the LDLR

Answer 5

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

Question 6

where does phosphorylation of LDLR occur

Answer 6

Phosphorylation occurs at the tyrosine
residue on a NPXY motif

Question 7

NPXY motif

Answer 7

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

Question 8

what does the phosphorylation of the tyrosine in the NPXY sequence lead to

Answer 8

The phosphorylation of the tyrosine in the NPXY sequence typically stimulates cell functions, such as receptor internalization

Question 9

Dephosphorylation of the NPXY motif
within the LDLR leads to

Answer 9

the recycling of
the LDLR back to the cell surface to pick
up more LDL

Question 10

LDL receptor family

Answer 10

all have the

YWTD B propellor
LA domain
EFG like domain

Question 11

what isthe EGF like domain

Answer 11

The epidermal growth factor (EGF)-like
domain is a component of several
secretory proteins and of the extracellular
region of several membrane proteins

Question 12

length of the EGF like domain

Answer 12

EGF-like domains are ~30-40 amino acids
in length, with 6 cysteines that form
disulfide bridges

Question 13

what is the function of the EGF like domain

Answer 13

Typically, the EGF-like domains act as a
structural component to a protein

Question 14

YWTD b propelor struture and aa sequence

Answer 14

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

Question 15

functions of the YWTD b propellor

Answer 15

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

Question 16

LDL receptor related protein 1 strutre

Answer 16

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

Question 17

ligand binding in LRP1

Answer 17

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

Question 18

what are some ligands that bind to LRP1

Answer 18

EL, LPL, HL

Question 19

LRP1 and LPL/HL

Answer 19

LRP1 directly binds LPL and HL

Question 20

human HL deficieny leads to

Answer 20

Human HL deficiency leads to elevated
chylomicron remnants and β-VLDL in the
bloodstream

Question 21

It is thought that cell surface LPL and HL
that capture lipoproteins can

Answer 21

‘bridge’ the
lipoproteins to LRP1

Question 22

how does LRP1 bind to remenants in the bloodstream

Answer 22

LRP1 binds remnant lipoproteins in the
bloodstream and removes them from the
bloodstream
– Binding of remnant lipoproteins is via apoE

Question 23

Deletion of LRP1 in animal models

Answer 23

embryonic lethal

Question 24

dleetion of apoE

Answer 24

Deletion of apoE leads to severe
atherosclerosis due to remnant lipoprotein
accumulation

Question 25

RCT

Answer 25

Reverse cholesterol transport (RCT) is
the process of removing non-hepatic
cholesterol for delivery to the liver and
ultimately for excretion from the body

Question 26

what does RCT require

Answer 26

the transfer of cholesterol
and PL from cells to apoA-I, preβ-HDL,
and mature HDL in the circulation – a
process called cholesterol efflux

Question 27

what is cholesterol efflux

Answer 27

Cholesterol efflux is the transfer of
cholesterol (and phospholipid) from cells
to apoA-I or various forms of HDL

Question 28

Cholesterol efflux to apoA-I is essential for

Answer 28

generating preβ-HDL and ‘mature’ HDL
– Efflux via ABCA1

Question 29

what is cholesterol efflux less essential for

Answer 29

Cholesterol efflux to ‘mature’ HDL also
occurs, but it is not essential
– Efflux via ABCG1 and SR-BI

Question 30

what is Needed for cholesterol efflux in vitro

Answer 30

– Macrophages & appropriate growth media
– Source of apoA-I and/or HDL
– Means to distinguish effluxed cholesterol from
macrophage & media cholesterol

Question 31

Macrophages & appropriate growth media involves what

Answer 31

– 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)

Question 32

Source of apoA-I and/or HDL involves what

Answer 32

– ApoA-I isolated by affinity chromatography
– HDL isolated by ultracentrifugation
– Plasma or sera (containing HDL and apoA-I)

Question 33

Means to distinguish effluxed cholesterol
from macrophage & media cholesterol involves what

Answer 33

Cells can be fed a labelled cholesterol:
* radiolabel (3H or 14C)
* stable isotope (2H or 13C)
* fluorescent (BODIPY)

Question 34

Why do cholesterol efflux
assays?

Answer 34

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)

Question 35

HDL cholesterol assocaition with CAD

Answer 35

no association

Question 36

Summary of steps in cholesterol
efflux assays

Answer 36

• 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

Question 37

how does HDL cholesterol change with HL and EL ko

Answer 37

increases the more -/- it becomes

Question 38

fractional efflux of cholesterol from lipase ko mice

Answer 38

increases in dko

Question 39

Options for cholesterol efflux assays

Answer 39

Acyl-CoA:cholesterol acyltransferase
(ACAT) inhibition:

nuclear LXR/RXR simulation

Question 40

nuclear LXR/RXR simulation

Answer 40

increases ABCA1 levels

Question 41

Acyl-CoA:cholesterol acyltransferase
(ACAT) inhibition:

Answer 41

prevents storage of labeled cholesterol as CE