The transport o f macromolecules into cells by receptor-m ediated endocytosis first emerged as a distinct mechanism following studies carried out on human fibroblasts by Brow n and Goldstein (Brow n and Goldstein, 1975). These studies w ere designed to investigate the normal function o f LD L the major carrier o f cholesterol in human plasma. On exposure o f fibroblast cell cultures to LDL, the cellular endogenous cholesterol synthesis was decreased while the intracellular content o f cholesterol remained largely unchanged. Following further biochemical studies it em erged that in mammals, the delivery o f LD L-derived cholesterol into hepatic and extra-hepatic cells w as mediated by a specific cell surface receptor know n as the apo B/E receptor. The sequential process o f receptor-m ediated endocytosis o f LDL and the subsequent regulation o f cellular synthesis o f cholesterol has been term ed the LD L receptor pathway.
4.(A).1. The LD L receptor pathw ay
This im portant receptor pathway is represented schematically in figure 4.1. It depicts the events that are involved in the receptor-m ediated endocytosis o f LD L which allows cells to control their intracellular cholesterol content.
LD L receptors are found in specialised regions o f cell plasma membranes know n as “coated pits” into which they move continuously and spontaneously even in the absence extracellular ligand (Goldstein et al. 1979a). These pits are o f a diam eter o f ~ 100-500 nm and have a lining o f fuzzy appearance on their
cytoplasm side when viewed under an electron microscope (Goldstein et al. 1979a). They may contain a variety o f receptors in addition to LDL receptors but LDL only binds its receptor due to its high affinity and specificity. W hen LDL binds its receptor the coated pit invaginates and pinches o ff enclosing its contents to form an endocytic “coated vesicle” in the interior o f the cell. This process occurs constitutively even when LDL or other ligands in the pit are not bound to their respective receptors (Brow n et al. 1983a).
The next step in this process is the movement o f the endocytic vesicle coated on its cytoplasmic side with netw ork o f fibrous protein mainly consisting o f clathrin into the interior o f the cell (Schneider, 1989). Thereafter there is a rapid loss o f coat and fusion with un-coated endocytic vesicles followed by acidification, a process involving ATP-pum ps (Xie et al. 1983; Stone et al. 1983). Subsequently LDL detaches from its receptor due to the low pH conditions and is delivered to lysosomes w here it is degraded. The LDL receptor how ever is not degraded but recycles back to the cell surface (Goldstein et al. 1979a). Lysosomal degradation o f LD L results in the liberation o f cholesterol from LD L cholesterol esters. This cholesterol or cholesterol derived oxysterols precipitate a series o f events that lead to the regulation o f intracellular cholesterol. Firstly, it inhibits the rate limiting enzyme o f cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-CoA reductase (H M G CoA reductase) thus inhibiting the de-novo synthesis o f cholesterol (Brown et al. 1974). Secondly, the cholesterol esterifying enzyme, cholesterol acyl-CoA transferase (ACAT) is activated by the cholesterol allowing any excess to be stored
as droplets o f choiesteryi ester (G oldstein et al. 1974). Finally, new LD L receptor protein synthesis is inhibited to ensure that recep to r m ediated delivery o f cholesterol into the cell is decreased. These events prevent the over-accum ulation o f cholesterol in cells (B row n and G oldstein, 1975).
1. # H M G C o A r c d u c l a s ^ LDL re cep to rs choiesteryi leate 2. # A C A l lolesterol C hoiesteryi oleate J . # L D L r e c e p to r s an nno acids
4
apoprotein1.1)1. bim lino Inli'niiiliziition L ysosom al hyd to lysis R ogulatory
actiuiis
Figure 4.1 The LDL receptor pathway. See le.xt for details o f the pathway.
4.(A).2. The LDL receptor protein
The structure o f the LD L receptor from four species (human, rabbit, bovine and ham ster) has been well characterised using protein chemistry, m olecular and cell biology techniques (Schneider et al. 1982). It is a highly conserved integral m em brane glycoprotein with five main dom ains (Schneider et al. 1982). These dom ains listed in order o f their appearance from the amino term inus o f the protein are: 1) The LD L binding dom ain, 2) a dom ain which has a strong hom ology to the epidermal grow th factor (E G F) precursor, 3) a dom ain in
which there is a cluster o f 0-linked carbohydrate chains, 4) a transmembrane
domain and 5) a short region that extends into the cytoplasm (Schneider, 1989).
4.(A).3. The human LDL receptor gene
The LDL receptor gene locus is located on the distal short arm o f
chromosome 19 and spans about 45 kb o f DNA. The gene consists o f 18 exons
which are separated by 17 introns (Sudhof et al. 1985). There is a strong
correlation between the proposed structural arrangement o f the receptor protein
and the sequence o f exons on its gene. The first exon encodes the non-translated
signal sequence and exons 2-6 the 7 cysteine-rich repeats o f the LDL binding
domain (Yamamoto et al. 1984; Schneider, 1989). The next 8 exons code for the
EGF precursor homology domain and the third receptor domain (0-linked sugar
cluster) is translated from a single exon found between introns 14 and 15. The
membrane spanning and cytoplasmic domains are encoded by 2 exons and the 18 ^
and final exon is translated into the carboxyl end amino acids o f the receptor
protein and also contains a 2.5 kb non-translated stretch o f mRNA (Yamamoto et
al. 1984; Schmid and Jelinek, 1982).
4.(A).4. Familial hypercholesterolaemia
The understanding of lipoprotein metabolism and has been greatly
advanced by functional and molecular studies using fibroblast cell lines isolated
was recognised in 1974, to arise due to mutations that result in a defectively
functioning LDL receptor (Brown et al. 1974). It is an example o f a single-gene
mutation that leads to obligatory atherosclerosis. This disease is characterised by:
a) a raised level o f plasma cholesterol localised in LDL b) cholesterol deposition in
tendons (xanthomata) and arteries (atheromata). c) disease inheritance in an
autosomal dominant fashion with severity o f the disease depending on the number
o f genes affected. Homozygotes, individuals who have inherited two mutant
alleles, have an accumulation o f LDL derived cholesterol deposition in the intimai
region o f major arteries. Sudden death may occur due to myocardial infarction
often before the age o f 15. Heterozygotes, individuals who have inherited one
mutant allele have less severe and more variable clinical manifestations of
atherosclerosis than homozygotes (Goldstein and Brown, 1978). The probability of
FH men having a myocardial infarction before the age o f 60 being 75%. This risk
in normal men is 15% (Stone et al. 1974). Although female FH heterozygotes
display the same genetic abnormalities and raised plasma LDL levels as male
patients the risk o f them experiencing a myocardial infarction before the age o f 60
is 45% compared with 10% for normal females (Stone et al. 1974). About 11
different mutant alleles o f the LDL receptor protein have thus far been identified
4.(A).5. Multiligand lipoprotein receptors
Receptors for LDL, described above and most other mammalian cell-
surface receptors that mediate endocytosis, adhesion or signalling have two
essential properties; A high affinity for its ligand and a very narrow specificity.
More recently, receptors for lipoproteins that have a high affinity for their ligands
but also exhibit a broad specificity have been identified. These include the
macrophage scavenger receptors Type Al and All, the B type scavenger receptors
and the LDL receptor-related protein (LRP). The binding o f ligands to the
receptors above is o f a high affinity and broad specificity. This enables binding o f
both lipoprotein and non-lipoprotein ligands to these receptors.
4.(A).6. Macrophage scavenger receptors
These receptors were first described by Brown and Goldstein during their
studies examining the link between elevated plasma LDL levels and the
development o f atherosclerosis (Goldstein et al. 1979b). An early feature o f
atherosclerotic lesions is the presence o f cholesterol laden macrophages. These
investigators found that cholesterol uptake by the LDL receptor pathway described
in the section above did not lead to the massive accumulation o f cholesterol in cells
because uptake was tightly coupled to the concentration o f intracellular
cholesterol. They noticed however, when LDL was chemically modified,
macrophages in culture were able to accumulate large amounts o f lipid and convert
similarity to lipid laden macrophage foam cells found in atherosclerotic plaques.
Chemical modification that enable scavenger receptors to bind LDL include
acétylation or oxidation (Brown and Goldstein, 1990; Brown and Goldstein, 1983;
Steinberg et al. 1989). These receptors were first termed acetyl LDL (AcLDL)
receptors but are now known as macrophage scavenger receptors (SCR) because
o f their multiligand binding capacity.
4.(A).7. Scavenger receptor ligands
Initial studies indicated that SCRs found on mouse peritoneal macrophages
were capable o f high affinity binding, internalisation and degradation o f
labelled AcLDL (Goldstein et al. 1979b). A wide variety o f compounds were
found to able to competitively inhibit this binding (Krieger et al. 1993). Using
direct binding assay and competitive inhibition studies a variety o f compounds that
can bind SCRs with a high affinity have been identified (table 4.1). Ligands that
bind macrophage SCRs identified so far are either polyanionic molecules or
macromolecular complexes. However many polyanions are not ligands for SCRs.
This suggests exact determinants that make compounds SCR ligands have not been
elucidated. The structures needed for one class o f ligands that include
oligodeoxynucleotides and polyribonucleotides to bind SCRs have been identified.
These nucleic acids need to form base-quartet stabilised four-stranded helices,
known as quadraplexes before they can bind scavenger receptors with a high
thought to form a charged surface complementary with that o f the SCR binding
domain (Krieger and Herz, 1994).
Table 4.1 Macrophage scavenger receptor ligands adapted from Krieger and
Herz (1994)
Effective competitors Ineffective competitors
M od ified p rotein s
AcLDL, OxLDL, M -LDL, M-HDL, M -album in
N ative an d m odified p rotein s Poly (D-glutam ate), Phosvitin, thyroglobulin,
orosomucoidin, fetuin, asialoorosom ucoidin, lysozym e, acetylated proteins including albumin,
y-glubulin, a - 1-antitrypsin, transferrin, ovalbumin, histones, ovomucoid, a - 1-acid
glycoprotein, HDL and methylated LDL Four stran ded n ucleic acids
polyinosinic acid (poly 1), poly G, poly 0:1, polyxanthinylic acid, telomere models [dCG^TOsl
N on-four stran ded n ucleic acids poly A, poly C, poly U, single and double
stranded D N A Polysaccharides
Dextran sulphate, Fucoidin, carragheenan
Polysaccharides
Heparin, chondroitin sulphate A and C, colom inic acid (polysialic acid), yeast mannan P hosph olip ids
Phosphatidylserine
P hosph olip ids Phosphatidylcholine O thers
B ovine sulfatides, polyvinyl sulphate, endotoxin, lipoteichoic acid, crocidolite asbestos
O th ers Polyphosphates
4.(A).8. Cloning and predicted quaternary structure o f SCR
Scavenger receptors have been detergent isolated and purified from bovine
lung membranes using ligand affinity and immuno-affinity chromatographic
techniques (Kodama et al. 1988). These purified bovine receptors have been used
to identify corresponding cDNAs. Homologues o f the bovine SCR cDNA have
been cloned in the mouse (Ashkenas et al. 1993), rabbit (Bickel and Freeman,
murine receptor genomic DNA has been isolated and the organisation o f the
intron-exon arrangement has been determined (Krieger and Herz, 1994).
The two isoforms o f SCR, type Al and type A ll are produced by
alternative splicing o f a message encoded by a single gene located on chromosome
8 in mice (Freeman et al. 1990) and humans (Matsumoto et al. 1990). Both type
IA and type HA SCR are expressed in macrophages in vitro (Naito et al. 1991)
and in vivo (Matsumoto et al. 1990).
The quaternary structure o f type A SCRs have been determined by the
cDNA sequences o f the receptor together with other biochemical (Kodama et al.
1988) and biophysical methods (Krieger and Herz, 1994). These studies predict
the type I receptors to be monomers made up o f 451-454 amino acids (aa) with an
elongated homotrimeric integral membrane protein structure. The SCR protein has
been determined to be arranged into 6 distinct domains (figure 4.2) (Krieger et al.
1992; Ashkenas et al. 1993). I: The N-terminal cytoplasmic domain [aa residues 1-
50]. II; A single transmembrane domain per chain (aa 51-76). Ill: A spacer region
(aa 77-150). IV: An a-helical coiled coil domain composed o f three helices, each
consisting o f a series o f up to 16, seven amino acid repeats known as heptads (aa
151-271). V: A second coiled-coil domain composed o f a right handed,
collagenous triple helix containing 23 or 24 uninterrupted Gly-X-Y triplet repeats
(aa 272-343). VI: A C-terminal cysteine-rich domain (SRCR) which is thought to
Type II receptors have dom ains I -V found in type I receptors but lack the cysteine-rich dom ain VI which is replaced by a truncated C -term inus consisting o f 6-17 aa. A lthough type II receptors lack SR CR they still have a broad ligand- specificity suggesting that this region is not essential to ensure binding o f m ultiple ligands (R ohrer et al 1990).
Figure 4.2 M odels of the predicted quaternary structure of m acrophage type A
scaven ger receptors.
Type I and type II receptors consist o f 6 dom ains (see text). Type II receptors share dom ains I-V but the C -term inal dom ain VI (the SRCR region) is replaced by a short oligopeptide (6-17 aa
residues depending on the
species). T he aa residues o f the
bovine type I receptor are
indicated in parenthesis.
A dapted from K rieger and Herz, (1994)
VI. SR C R (H O )
V. C ollagen-like (72)
G ly-X -Y repeat
IV. a -H e lic a l coiled coil (121) Meptad repeat III S pacer (74) II.TM (26) I. C ytoplasm ic (50)
N N N
T ype I
N N N
Type II
4.(A).9. Ligand binding properties of SCR
Binding o f ligands to type I and type II scavenger receptors is a process that exhibits a num ber o f com plex features.
1) B road specificity (K rieger and H erz, 1994).