|
GREAT EXPERIMENTS
The discovery of the LDL receptor: Clues to receptor-mediated endocytosis
Joseph Goldstein and Michael Brown
|
|
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
|
|
..
|
|
In 1972 the two of us, working as newly
appointed Assistant Professors in Medicine at the University of Texas
Southwestern Medical Center in Dallas, set out to unravel a human
genetic disease called familial hypercholesterolemia (FH). In patients
with FH, the concentration of cholesterol in blood is elevated many
times above normal, and heart attacks occur early in life. FH is
inherited as an autosomal dominant trait. When we began our work 30
years ago, the underlying defects in about 35 recessive disorders were
known. All of these are deficiencies in enzymes (e.g., phenylketonuria,
Lesch-Nyhan syndrome) or alterations in hemoglobin (e.g., sickle cell
anemia; β-thalassemia). Because no
diseases with dominant enzyme defects had been elucidated, we suspected
that the basic defect in FH would not be an enzyme deficiency. Rather,
we hypothesized that FH would be a regulatory defect resulting from a
failure of end-product repression of cholesterol synthesis. The
possibility excited us because genetic defects in feedback regulation
had not been observed, and we hoped that study of this disease might
expose a fundamental regulatory mechanism.
|
|
..
|
|
Cholesterol is an essential component of
the plasma membrane of animal cells, where it maintains the barrier
function between cells and environment, modulates fluidity, and creates
raft-like structures that facilitate the activities of signaling
molecules. Cholesterol is also the precursor for the manufacture of all
steroid hormones and bile acids, and it plays a crucial role in the
formation of the myelin sheath that surround axons. In the bloodstream
of humans and vertebrate animals, cholesterol is transported in
lipoprotein particles. In the 1950s and 1960s, physiologists delineated
the two major cholesterol-carrying lipoprotein particles, low density
lipoprotein (LDL) and high density lipoprotein (HDL); epidemiologists
observed that elevated concentrations of LDL predispose to heart
attacks, whereas elevated amounts of HDL are protective; and physicians
learned that excess circulating cholesterol in FH patients is contained
in LDL, not HDL.
|
|
..
|
|
Our approach to unraveling the genetic
defect in FH was to apply the techniques of cell culture. Our studies
led to the discovery of a cell surface receptor for LDL and to the
elucidation of the mechanism by which this receptor carries LDL
particles into cells through coated vesicles. We soon found that FH is
caused by genetic defects in the LDL receptor. The LDL receptor studies
provided the first clear evidence for selective vesicular transport,
giving rise to the concept of receptor-mediated endocytosis.
|
|
..
|
|
Background
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
|
|
..
|
|
In 1938 Carl Müller, a Norwegian
clinician, described FH as an "inborn error of metabolism" that
produces high blood cholesterol and myocardial infarctions (heart
attacks) in young people (2140). Müller concluded that FH is
transmitted as an autosomal dominant trait. In 1964 Khachadurian, at
the American University in Beirut, showed that FH exists in two forms:
the less severe heterozygous form and the more severe homozygous form (2141).
|
|
..
|
|
FH heterozygotes, who are now known to
carry a single copy of a mutant LDL receptor gene, are quite common,
accounting for 1 out of every 500 persons among most ethnic groups
throughout the world (2142). These individuals have a 2-fold
increase in the number of LDL particles in blood from birth and begin
to have heart attacks at 30 years of age. Among people under age 60 who
suffer myocardial infarctions, about 5% have the heterozygous form of
FH.
|
|
..
|
Figure 1
A 10-year-old girl with homozygous FH. Note the elevated
orange-yellow xanthomas lying superficially over the knees, the wrists,
and interdigital webs. These xanthomas arise from the deposition of
plasma LDL-derived cholesterol into macrophages of the skin. The rate
of deposition is proportional to the severity and duration of the
elevation in plasma LDL. A similar deposition of LDL-derived
cholesterol occurred in the coronary arteries of this girl, producing
atheromas of artery wall, which led to her first myocardial infarction
at age 8.
|
|
..
|
|
The attractiveness of FH as an experimental model stems from the
existence of homozygotes (Figure 1).
These individuals, who number about 1 in 1 million, are now known to
inherit two mutant LDL receptor genes, one from each parent. Their
disease is severe. They have 6- to 10-fold elevations in plasma LDL
from birth, and they often have heart attacks in childhood (2142).
The early atherosclerosis in FH patients without any other risk factors
is formal proof that elevated LDL can produce atherosclerosis in
humans. The availability of FH homozygotes permits study of the
manifestations of the mutant allele without any confounding effects
from the normal allele.
|
|
..
|
|
At the time we began our studies in 1972,
it was felt that all of the important events in cholesterol metabolism
take place in the liver or intestine. It was impossible to perform
meaningful studies in livers of humans with FH. Our only chance
depended on the mutant phenotype being faithfully manifest in cells,
such as skin fibroblasts, which can be readily obtained from patients
and cultured in vitro. Techniques for growing such cells had
been established over the preceding two decades. Moreover, a handful of
inherited enzyme defects were known to be detectable in cultured
fibroblasts from patients with rare recessive diseases, such as
galactosemia and the Lesch-Nyhan syndrome.
|
|
..
|
|
There was some reason to believe that the
FH derangement might be manifest in cultured fibroblasts. Studies in
the 1960s by Bailey and Rothblat had demonstrated that cultured mouse
lymphoblasts and L cells synthesize cholesterol and that this synthesis
is subject to regulation (2143; 2135). They showed
that when whole serum, containing lipoproteins, is present in the
medium, cultured cells produce little cholesterol from radioactive
acetate. When serum lipoproteins are removed from the culture medium,
cholesterol synthesis increases. Incubation of cells with radiolabeled
acetate is a convenient way to monitor the overall action of the 30
enzymes that are needed to convert the simple 2-carbon precursor
(acetate) to the complex 27-carbon, four-ring structure of cholesterol.
This biosynthetic pathway was delineated in the 1950s by Konrad Bloch
and Feodor Lynen, who received the 1964 Nobel Prize in Physiology or
Medicine for this tour de force of biochemistry.
|
|
..
|
|
The discovery of the LDL receptor
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
|
|
..
|
Figure 2
Regulation of HMG CoA reductase activity in fibroblasts
from a normal subject and from an FH homozygote. Left panel: Monolayers
of cells were grown in dishes containing 10% fetal calf serum. On day 6
of cell growth (zero time), the medium was replaced with fresh medium
containing 5% human serum from which the lipoproteins had been removed.
At the indicated time, extracts were prepared, and HMG CoA reductase
activity was measured. Right panel: 24 hours after addition of 5% human
lipoprotein-deficient serum, human LDL was added to give the indicated
cholesterol concentration. HMG CoA reductase activity was measured in
cell free extracts at the indicated time. ( Modified from (2110)).
|
|
..
|
|
We began in 1972 by setting up a
microassay for measuring the activity of 3-hydroxy-3-methylglutaryl
coenzyme A reductase (HMG CoA reductase), the rate-controlling enzyme
of cholesterol biosynthesis, in extracts of cultured fibroblasts (2136).
Earlier studies in rat livers had shown that this activity is reduced
when rats ingest cholesterol and that this reduction limits cholesterol
synthesis (2144). We soon found that HMG CoA reductase is also subject to
negative regulation in fibroblasts (2136). When normal human fibroblasts
are grown in the presence of serum, HMG CoA reductase activity is low.
As shown in Figure 2, when the lipoproteins are removed from the culture medium, the activity
of HMG CoA reductase rises by at least 50-fold over a 24 hr period. The
induced enzyme is rapidly suppressed when LDL is added back to the
medium.
|
|
..
|
|
Not all lipoproteins could suppress HMG
CoA reductase activity. Of the two major cholesterol-carrying
lipoproteins in human plasma, LDL and HDL, only LDL is effective (2136).
This specificity was our first clue that a receptor might be involved.
Our second clue was the concentration of LDL that is required. The
lipoprotein is active at concentrations as low as 5 μg of protein per
ml, which is ~10
–
9 molar. A high affinity receptor mechanism seemed likely.
|
|
..
|
|
The key to this mechanism emerged in 1973 from studies of cells from
patients with homozygous FH (2110).
When grown in serum containing lipoproteins, the homozygous FH cells
have HMG CoA reductase activities that are 50 to 100-fold above normal
(Figure 2). This activity does not increase significantly when the lipoproteins are
removed from the serum, and there is no suppression when LDL is added
back.
|
|
..
|
|
The simplest interpretation of these
results was that FH homozygotes have a defect in the gene encoding HMG
CoA reductase that renders the enzyme resistant to feedback regulation
by LDL-derived cholesterol. This working hypothesis was immediately
disproved by our next experiment in which cholesterol was delivered in
a way that would bypass the putative receptor-mediated uptake.
Cholesterol, dissolved in ethanol, was added to normal and FH
homozygote cells. When mixed with albumin-containing solutions,
cholesterol forms a quasi-soluble emulsion that enters cells by
adsorption to the plasma membrane. When cholesterol is added in this
form, the HMG CoA reductase activities of normal and FH homozygote
fibroblasts are equally suppressed (2111).
|
|
..
|
|
Clearly, the defect in the FH homozygote
cells must reside in their ability to extract cholesterol from the
lipoprotein, and not in the ability of the cholesterol, once extracted
by the cells, to act. But how do normal cells extract the cholesterol
of LDL? The high affinity suggested that a cell surface receptor is
involved. The existence of cell surface receptors for hormones (such as
epinephrine and glucagon) had been known for many years. It was
generally thought that these receptors act by binding the ligand at the
surface and then generating a "second messenger" on the intracellular
side of the plasma membrane. The classic "second messenger" in 1973 was
cyclic AMP. Perhaps LDL was binding to a receptor and generating some
second messenger that suppresses HMG CoA reductase.
|
|
..
|
Figure 3
Internalization and degradation at 37°C of 125I-LDL
by fibroblasts from a normal subject (Top panel) and from J.D., a
patient with the internalization-defective form of FH ( Bottom panel).
Each cell monolayer was allowed to bind 125I-LDL at 4° C,
after which the cells were washed extensively. In one set of dishes,
the amount of 125I-LDL bound was determined by measuring the amount of 125I-LDL
that could be released from the surface by treatment with heparin.
Replicate dishes then received warm medium and were incubated at 37°C.
After the indicated interval, the amounts of surface-bound
(heparin-releasable) 125I-LDL, internalized (heparin-resistant)
125I-LDL, and degraded (trichloroacetic acid-soluble)
125I-LDL were measured. (Modified from (2123)).
|
|
..
|
|
The existence of the postulated LDL receptor was formally
demonstrated in 1974 when we radiolabeled LDL with 125
Iodine and showed that normal cells have high affinity binding sites for 125I-LDL,
whereas FH homozygote cells lack these sites (2137).
This seemed to explain the genetic defect in FH, but it did not reveal
how LDL releases its cholesterol so as to suppress HMG CoA reductase.
The answer came from studies of the fate of the surface-bound 125I-LDL.
Techniques were developed to distinguish surface-bound from intracellular 125
I-LDL (2112), and these revealed that the receptor-bound LDL remains on the surface
of normal cells for less than 10 min on average (Figure 3).
Within this time most of the surface-bound LDL particles has entered
the cell; within another 60 min the protein component of 125
I-LDL is digested completely to amino acids and the 125I,
which had been attached to tyrosine residues on LDL, is released into the culture medium as 125
I-monoiodotyrosine (Figure 3) (2137; 2112).
Meanwhile, the cholesteryl esters of LDL are hydrolyzed, generating unesterified cholesterol
that remains within the cell (2113).
|
|
..
|
|
Our laboratory purified the LDL receptor from bovine adrenal glands in 1982 (2114),
cloned its human cDNA shortly thereafter (2115), and isolated the gene in 1985
(2116). These advances laid the groundwork for the molecular analysis of the mutations
underlying FH (2142). As of October, 2001, more than 700 allelic mutations in the LDL
receptor gene have been identified in FH patients.
|
|
..
|
|
Feedback regulation of cholesterol synthesis
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
|
|
..
|
|
Having demonstrated the
receptor-mediated internalization of LDL, we next sought to determine
where in the cell the LDL was degraded and how this degradation
suppressed HMG CoA reductase activity and cholesterol synthesis. The
only cellular organelle in which LDL could have been degraded so
completely and rapidly is the lysosome, which contains acid hydrolases
that could easily digest all of the components of LDL (Figure 4).
We confirmed the lysosomal digestion of LDL through the use of chloroquine (2117),
which raises the pH of lysosomes and inhibits lysosomal enzymes, and
through studies of cultured fibroblasts from patients with a genetic
deficiency of lysosomal acid lipase (2118). Cells from these
patients bind and internalize LDL but fail to hydrolyze its cholesteryl
esters, even though they did degrade the protein component.
|
|
..
|
|
The cholesterol that is generated from
LDL within the lysosome proved to be the "second messenger" responsible
for suppressing HMG CoA reductase activity (Figure 4 and Figure 5).
We now know that LDL-derived cholesterol (or an hydroxylated derivative
formed within the cell) acts at several levels, including suppression
of transcription of the HMG CoA reductase gene through the sterol
regulatory element-binding protein (SREBP) pathway (discussed below)
(2119) and acceleration of the degradation of the enzyme protein (2120).
The LDL-derived cholesterol also regulates other processes in a
coordinated action that stabilizes the cell's cholesterol content. It
activates a cholesterol-esterifying enzyme, acyl CoA: cholesterol
acyltransferase (ACAT), so that excess cholesterol can be stored as
cholesteryl ester droplets in the cytoplasm (2121).
It also suppresses transcription of the LDL receptor gene through the SREBP pathway (2121).
This latter action allows cells to adjust the number of LDL receptors
to provide sufficient cholesterol for metabolic needs without causing
cholesterol overaccumulation (2138). Through these regulatory
mechanisms, cells keep their level of unesterified cholesterol
remarkably constant despite wide fluctuations in cholesterol
requirements and exogenous supply.
|
|
..
|
Figure 4
Sequential steps in the LDL receptor pathway of mammalian cells. (Modified from (2148)).
|
Figure 5
Actions attributable to the LDL receptor in fibroblasts
from a normal subject and from a homozygote with the receptor-negative
form of FH. Cells were incubated with varying concentrations of 125
I-LDL or unlabeled LDL at 37°C for 5 hr and assayed as described (2148).
All data are normalized to 1 μg of total cell protein. The units for each assay
are as follows: Binding, mg of 125I-LDL bound to cell surface; Internalization,
mg of 125I-LDL contained within the cell; Hydrolysis of apoprotein B-100, mg of 125
I-LDL degraded to 125I-monoiodotyrosine per hr; Hydrolysis of cholesteryl esters, nmol of [3H]
cholesterol formed per hr from the hydrolysis of LDL labeled with [3H]cholesteryl linoleate;
Cholesterol synthesis, nmol of [14C]acetate incorporated into [14C]cholesterol per hr by intact cells;
Cholesterol esterification, nmol of [14C]oleate incorporated into cholesteryl [14C]oleate
per hr by intact cells. (Modified from 2148).
|
|
..
|
|
Figure 4 summarizes the sequential steps in the LDL receptor pathway as deduced
from the biochemical and genetic studies performed between 1972 and
1977 (2163). Figure 5 shows the striking "all-or-none" biochemical differences in the
metabolism of LDL and its regulatory actions in fibroblasts derived
from a normal subject and from an FH homozygote with a complete
deficiency of LDL receptors.
|
|
..
|
|
The most recent development in the LDL
receptor story relates to the discovery of the molecular basis of the
feedback regulation of LDL receptors and the cholesterol biosynthetic
pathway. The key was our discovery of sterol-regulated, membrane-bound
transcription factors called SREBPs (2119). Unlike other
transcription factors, the SREBPs are synthesized as membrane-bound
proteins attached to the endoplasmic reticulum. In cholesterol-depleted
cells, the active portions of the SREBPs are released from the membrane
by proteolysis. They enter the nucleus where they bind to regulatory
sequences in the promoters of numerous genes involved in lipid
metabolism. The SREBPs enhance transcription of the genes encoding HMG
CoA reductase and other enzymes of cholesterol biosynthesis as well as
the LDL receptor. When LDL-derived cholesterol enters cells, it blocks
the proteolytic release of SREBPs from membranes. Transcription of the
target genes declines, and the cells produce less cholesterol, thus
preventing cholesterol overload.
|
|
..
|
|
Receptor-mediated endocytosis: Origin of the concept
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
|
|
..
|
|
The rapidity of internalization and degradation of receptor-bound LDL (Figure 3)
implied that cells possess a special mechanism for transport of
receptor-bound LDL from the cell surface to the lysosome. The likely
mechanism was endocytosis, the process by which surface membranes pouch
inward and pinch off to form vesicles. Endocytosis was first
demonstrated by cinematography of phagocytic cells in the 1930s, and
its universal occurrence was established in the 1950s by the electron
microscopic studies of Palade. Prior to 1975, endocytosis was felt to
be a nonspecific process that transported bulk fluid and its contents
into cells. There was no precedent for selective entry of specific
receptors into cells by this route.
|
|
..
|
|
To determine whether endocytosis was
involved in LDL uptake, we began in 1975 a collaboration with Richard
G. W. Anderson, a cell biologist at our medical school in Dallas.
Through the use of LDL coupled to electron-dense ferritin, we found
that receptor-bound LDL is internalized by endocytosis. But more
importantly, these morphological studies explained the efficiency of
internalization: efficiency is contingent upon the clustering of the
LDL receptors in coated pits (2122; 3138). Coated
pits had been described in 1964 by Roth and Porter during electron
microscopic studies of the uptake of yolk proteins by mosquito oocytes (2145).
These investigators showed that coated pits pinch off from the surface
to form coated endocytic vesicles that carry extracellular fluid and
its contents into the cell. In 1976, Barbara Pearse purified coated
vesicles and discovered that the cytoplasmic coat was composed
predominantly of one protein, clathrin (2132). The finding
that LDL receptors are clustered in clathrin-coated pits raised the
general possibility that these structures serve as gathering places for
cell surface receptors that are destined for endocytosis (3138).
Other cell surface proteins, being excluded from coated pits, could not rapidly enter the cell.
|
|
..
|
|
The interpretation of coated pit
function was strengthened by study of fibroblasts from a unique FH
homozygote. Cells from most FH homozygotes simply fail to bind LDL (Figure 5).
But cells from one FH patient, whose initials are J.D., bind LDL, but fail to internalize it
(Figure 3) (2123; 2139). In collaboration with Anderson,
we showed that the receptors in these mutant cells are excluded from coated pits (2124).
This was an important finding, for it established the essential role of
coated pits in the high efficiency uptake of receptor-bound molecules (2125).
|
|
..
|
|
Later molecular studies showed that the
J.D. mutation results from a single base pair change that substitutes a
cysteine for a tyrosine in the receptor's cytoplasmic domain (2126; 2127).
This observation stimulated a series of in vitro
mutagenesis experiments, which revealed that this tyrosine is part of a
tetrameric sequence NPVY (Asn-Pro-Val-Tyr) that directs LDL receptors
to clathrin-coated pits for rapid internalization (2128).
A variant of this sequence, NPxY (where x
can be any amino acid), is present in one or more copies in the
cytoplasmic tails of other members of the LDL receptor gene family (2142). An NPxY
sequence is also present in the cytoplasmic domains of other
cell-surface receptors, including the amyloid precursor protein and
several receptors with tyrosine kinase domains (EGF, c-erb-B/neu, insulin, IGF-1).
In many of these receptors, the NPxY also serves as a binding site for adaptor proteins
that are involved in signal transduction.
|
|
..
|
|
The LDL receptor studies also exposed another feature of receptor-mediated endocytosis
—
namely, that receptors can be recycled (2129; 2130).
After internalization the receptors dissociate from their ligands when
they are exposed to a drop in pH in endosomes. After dissociation, the
receptors find their way back to the cell surface. The LDL receptor
makes one round trip into and out of the cell every 10 min for a total
of several hundred trips in its 20-hr lifespan (2130).
Inasmuch as each LDL particle contains 1600 molecules of cholesterol,
this rapid recycling of LDL receptors provides an efficient mechanism
for delivery of cholesterol to cells.
|
|
..
|
|
The Legacy
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
|
|
..
|
|
The early work on the LDL receptor, summarized above, introduced three concepts to biology and medicine:
|
-
The concept of lateral sorting of proteins within membranes, which
forms the basis for receptor clustering in coated pits and is a
prerequisite for receptor-mediated endocytosis.
-
The concepts of receptor-mediated endocytosis and receptor recycling,
which provide a mechanism by which cells selectively and efficiently
internalize macromolecules, including transport proteins, hormones,
growth factors, lysosomal enzymes, and certain viruses.
-
The concept of receptor regulation (2109),
which explains the cholesterol-lowering effects of the statins, a class
of drugs that inhibit cholesterol synthesis in liver. The consequent
depletion of hepatic cholesterol activates the SREBP regulatory
pathway, producing an increase in hepatic LDL receptors that take up
plasma LDL, a fall in plasma LDL levels, a reduction in heart attacks,
and a prolongation of life (2146).
|
|
The authors
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Michael S. Brown (right) and Joseph L. Goldstein (left) are currently Regental
Professors in the Department of Molecular Genetics at the University of
Texas Southwestern Medical Center in Dallas where they have worked
together since 1972. Brown received his M.D. from University of
Pennsylvania in 1966. Goldstein received his M.D. from University of
Texas Southwestern Medical School in 1966. From 1966-68, both Brown and
Goldstein were interns and residents in Medicine at the Massachusetts
General Hospital in Boston. Brown and Goldstein then did postdoctoral
research at the NIH, Brown working in the laboratory of Earl Stadtman
and Goldstein in the laboratory of Marshall Nirenberg.
Brown and Goldstein are members of the U.S. National Academy of
Sciences, American Philosophical Society, and Institute of Medicine.
They are also Foreign Members of the Royal Society. Among their awards
are the Lasker Award in Basic Medical Research (1985), Nobel Prize in
Physiology or Medicine (1985), and U.S. Medal of Science (1988). Both
have received honorary degrees from numerous institutions, including
the University of Chicago, University of Paris, and Rockefeller
University.
Michael S. Brown, M.D.
Department of Molecular Genetics
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd., Room L5.238
Dallas, TX 75390-9046
Phone: 214 648 2179
Fax: 214 648 8804
E-mail: mbrow1@mednet.swmed.edu
Joseph L. Goldstein, M.D.
Department of Molecular Genetics
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd., Room L5.238
Dallas, TX 75390-9046
Phone: 214 648 2141
Fax: 214 648 8804
E-mail: jgolds@mednet.swmed.edu
|
|
..
|
-
2109 Goldstein, J. L. and Brown, M. S.
(2001).
The cholesterol quartet.
Science 292, 1310-1312.
PubMed Journal
-
2125 Goldstein, J. L., Anderson, R. G., and Brown, M. S.
(1979).
Coated pits, coated vesicles, and receptor-mediated endocytosis.
Nature 279, 679-685.
PubMed
-
2130 Brown, M. S., Anderson, R. G., and Goldstein, J. L.
(1983).
Recycling receptors: the round-trip itinerary of migrant membrane proteins.
Cell 32, 663-667.
PubMed Journal
-
2135 Rothblat, G. H.
(1969).
Lipid metabolism in tissue culture cells.
Adv. Lipid. Res. 7, 135-163.
PubMed
-
2142 Goldstein, J.L., Hobbs, H.H., and Brown, M.S.
(2001).
Familial hypercholesterolemia. In The Metabolic and Molecular Bases of Inherited Disease Scriver, R, Beaudet, A L, Sly, W S, and Valle, D, eds. (New York: McGraw-Hill), pp. 2863-2913.
-
2143 Bailey, J.M.
(1973).
Regulation of cell cholesterol content. In Atherogenesis: Initiating Factors Porter, R, and Knight, J, eds. (Amsterdam: Ciba Foundation Symposium), pp. 63-92.
-
2144 Siperstein, M.D.
(1970).
Regulation of cholesterol biosynthesis in normal and malignant tissues.
Curr. Topics Cell. Reg. 2, 65-100.
PubMed
-
2146 Brown, M. S. and Goldstein, J. L.
(1996).
Heart attacks: gone with the century?
Science 272, 629-692.
PubMed
-
2148 Brown, M. S. and Goldstein, J. L.
(1979).
Receptor-mediated endocytosis: insights from the lipoprotein receptor system.
Proc. Natl. Acad. Sci. USA 76, 3330-3337.
PubMed
-
2163 Brown, M. S. and Goldstein, J. L.
(1986).
A receptor-mediated pathway for cholesterol homeostasis.
Science 232, 34-37.
PubMed
|
-
2110 Goldstein, J. L. and Brown, M. S. (1973).
Familial hypercholesterolemia: identification of a defect in the
regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity
associated with overproduction of cholesterol. Proc. Natl. Acad.
Sci. USA 70, 2804-2808. PubMed
-
2111 Brown, M. S., Dana, S. E., and Goldstein, J.
L. (1974). Regulation of 3-hydroxy-3-methylglutaryl coenzyme A
reductase activity in cultured human fibroblasts. Comparison of cells
from a normal subject and from a patient with homozygous familial
hypercholesterolemia. J. Biol. Chem. 249, 789-796. PubMed Journal
-
2112 Goldstein, J. L., Basu, S. K., Brunschede, G. Y., and Brown, M. S.
(1976).
Release of low density lipoprotein from its cell surface receptor by sulfated glycosaminoglycans.
Cell 7, 85-95.
PubMed Journal
-
2113 Brown, M. S., Dana, S. E., and Goldstein, J. L.
(1975).
Receptor-dependent hydrolysis of cholesteryl esters contained in plasma low density lipoprotein.
Proc. Natl. Acad. Sci. USA 72, 2925-2929.
PubMed
-
2114 Schneider, W. J., Beisiegel, U., Goldstein, J. L., and Brown, M. S.
(1982).
Purification of the low density lipoprotein receptor, an acidic glycoprotein of 164,000 molecular weight.
J. Biol. Chem. 257, 2664-2673.
PubMed Journal
-
2115 Yamamoto, T., Davis, C. G., Brown, M. S., Schneider, W. J., Casey, M. L., Goldstein, J. L., and Russell, D. W.
(1984).
The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA.
Cell 39, 27-38.
PubMed Journal
-
2116 Sudhof, T. C., Goldstein, J. L., Brown, M. S., and Russell, D. W.
(1985).
The LDL receptor gene: a mosaic of exons shared with different proteins.
Science 228, 815-822.
PubMed
-
2117 Goldstein, J. L., Brunschede, G. Y., and
Brown, M. S. (1975). Inhibition of proteolytic degradation of low
density lipoprotein in human fibroblasts by chloroquine, concanavalin
A, and Triton WR 1339. J. Biol. Chem. 250, 7854-7862. PubMed Journal
-
2118 Goldstein, J. L., Dana, S. E., Faust, J. R.,
Beaudet, A. L., and Brown, M. S. (1975). Role of lysosomal acid
lipase in the metabolism of plasma low density lipoprotein.
Observations in cultured fibroblasts from a patient with cholesteryl
ester storage disease. J. Biol. Chem. 250, 8487-8495. PubMed Journal
-
2119 Brown, M. S. and Goldstein, J. L.
(1999).
A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood.
Proc. Natl. Acad. Sci. USA 96, 11041-11048.
PubMed Journal
-
2120 Gil, G., Faust, J. R., Chin, D. J., Goldstein, J. L., and Brown, M. S.
(1985).
Membrane-bound domain of HMG CoA reductase is required for sterol-enhanced degradation of the enzyme.
Cell 41, 249-258.
PubMed Journal
-
2121 Brown, M. S., Dana, S. E., and Goldstein, J. L.
(1975).
Cholesterol ester formation in cultured human fibroblasts. Stimulation by oxygenated sterols.
J. Biol. Chem. 250, 4025-4027.
PubMed Journal
-
2122 Anderson, R. G., Goldstein, J. L., and
Brown, M. S. (1976). Localization of low density lipoprotein
receptors on plasma membrane of normal human fibroblasts and their
absence in cells from a familial hypercholesterolemia homozygote.
Proc. Natl. Acad. Sci. USA 73, 2434-2438. PubMed
-
2123 Brown, M. S. and Goldstein, J. L. (1976).
Analysis of a mutant strain of human fibroblasts with a defect in
the internalization of receptor-bound low density lipoprotein.
Cell 9, 663-674. PubMed Journal
-
2124 Anderson, R. G., Goldstein, J. L., and
Brown, M. S. (1977). A mutation that impairs the ability of
lipoprotein receptors to localize in coated pits on the cell surface of
human fibroblasts. Nature 270, 695-699. PubMed
-
2126 Lehrman, M. A., Goldstein, J. L., Brown, M.
S., Russell, D. W., and Schneider, W. J. (1985).
Internalization-defective LDL receptors produced by genes with
nonsense and frameshift mutations that truncate the cytoplasmic domain.
Cell 41, 735-743. PubMed Journal
-
2127 Davis, C. G., Lehrman, M. A., Russell, D.
W., Anderson, R. G., Brown, M. S., and Goldstein, J. L. (1986).
The J.D. mutation in familial hypercholesterolemia: amino acid
substitution in cytoplasmic domain impedes internalization of LDL
receptors. Cell 45, 15-24. PubMed Journal
-
2128 Chen, W.-J., Goldstein, J. L., and Brown, M.
S. (1990). NPXY, a sequence often found in cytoplasmic tails, is
required for coated pit-mediated internalization of the low density
lipoprotein receptor. J. Biol. Chem. 265, 3116-3123. PubMed Journal
-
2129 Basu, S. K., Goldstein, J. L., Anderson, R. G., and Brown, M. S.
(1981).
Monensin interrupts the recycling of low density lipoprotein receptors in human fibroblasts.
Cell 24, 493-502.
PubMed Journal
-
2132 Pearse, B. M.
(1976).
Clathrin: a unique protein associated with intracellular transfer of membrane by coated vesicles.
Proc. Natl. Acad. Sci. USA 73, 1255-1259.
PubMed
-
2136 Brown, M. S., Dana, S. E., and Goldstein, J. L.
(1973).
Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human fibroblasts by lipoproteins.
Proc. Natl. Acad. Sci. USA 70, 2162-2166.
PubMed
-
2137 Goldstein, J. L. and Brown, M. S. (1974).
Binding and degradation of low density lipoproteins by cultured
human fibroblasts. Comparison of cells from a normal subject and from a
patient with homozygous familial hypercholesterolemia. J. Biol.
Chem. 249, 5153-5162. PubMed Journal
-
2138 Brown, M. S. and Goldstein, J. L.
(1975).
Regulation of the activity of the low density lipoprotein receptor in human fibroblasts.
Cell 6, 307-316.
PubMed Journal
-
2139 Goldstein, J. L., Brown, M. S., and Stone, N. J.
(1977).
Genetics of the LDL receptor: evidence that the mutations affecting binding and internalization are allelic.
Cell 12, 629-641.
PubMed Journal
-
2140 Muller, C.
(1938).
Xanthomata, hypercholesterolemia, angina pectoris.
Acta. Med. Scand. 89, 75-84.
-
2141 Khachadurian, A.K.
(1964).
The inheritance of essential familial hypercholesterolemia.
Am. J. Med. 37, 402-407.
PubMed
-
2145 Roth, T.F. and Porter, K.R.
(1964).
Yolk protein uptake in the oocyte of the mosquito Aedes aegypti.
J. Cell Biol. 20, 313-332.
PubMed
-
3138 Anderson, R. G., Brown, M. S., and Goldstein, J. L.
(1977).
Role of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts.
Cell 10, 351-364.
PubMed Journal
|
|
..
|
|
©Jones and Bartlett Publishers (2007)
|
|