The very low density lipoprotein receptor

The Very Low Density Lipoprotein Receptor A Second Lipoprotein Receptor That May Mediate Uptake of Fatty Acids into Muscle and Fat Cells Tokuo Yamamoto, Sadao Takahashi, Juro Sakai, and Yutaka Kawarabayasi The isolation of a cDNA highly homologous to that of the low-density lipoprotein (LDL) receptor revealed the presence of a lipoprotein receptor that specifically binds apolipoprotein-E-containing lipoproteins, including very low density lipoprotein (VLDL), intermediate-density lipoprotein, and pmigrating VLDL. This new receptor, designated VLDL receptor, consists of five domains that resemble those of the LDL receptor. The VLDL receptor mRNA is abundant in tissues petforming active fatty acid metabolism, suggesting that the receptor may be responsible for the uptake of fatty acids in triglyceride-rich lipoproteins into muscle and fat cells. (Trends Cardiovasc Med 1993;3: 144-l 48)

Receptor-mediated uptake of plasma lipoproteins into cells plays a key role both in the intracellular metabolism of cholesterol and fats and in the clearance of lipoproteins from plasma. The bestcharacterized lipoprotein receptor is the receptor for low-density lipoprotein (LDL), a major cholesterol carrying lipoprotein in plasma (Brown and Goldstein 1986). The LDL receptor binds with high affinity to apolipoprotein (apo) BlOOcontaining LDL, and to apoE-containing lipoproteins, including very low density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and p-migrating VLDL @-VLDL). A genetic defect of the LDL receptor causes familial hypercholesterolemia @I-I), one of the most common genetic diseases in humans (Brown and Goldstein 1974). It leads to a reduced clearance of LDL from plasma, and thereby elevates the levels of plasma cholesterol. The resulting hypexcholesterolemia is a major risk factor for the development of premature atheroscleroTokuo Yamamoto, Sadao Takahashi, Juro Sakai, and Yutaka Kawarabayasi are at the Tohoku University Gene Research Center, Sendai 981, Japan.

144

sis and increased frequency of myocardial infarction (Brown and Goldstein 1976). The discovery of the LDL receptor by Goldstein and Brown has led to dramatic progress toward elucidating the mechanisms that mediate cholesterol homeostasis. In contrast, little is known of the mechanisms for the uptake of other lipoproteins that carry triglycerides. For many years, it was believed that the uptake of triglyceride fatty acids in chylomicron and VLDL into muscle and fat cells was mediated by lipoprotein lipase (LPL) (Nilson-Ehle et al. 1980). However, the cloning of a lipoprotein receptor that is abundant in muscle and fat cells has led to the discovery of a new mechanism of transport of fatty acids into these cells. In this review, we describe the structure of the newly discovered receptor (Takahashi et al. 1992) and discuss its physiologic function.

.

LDL Receptor-Related Protein/ Activated a,-Macroglobulin Receptor: A CandidateReceptor for ApoE-Containing Lipoproteins

Although the LDL receptor binds both apoE- and apoB 100-containing lipopro-

teins, the presence of specific receptors for apoE-containing lipoproteins (including chylomicrons, chylomicron remnants, VLDL, and IDL) was suggested for many years. One of the most convincing pieces of evidence for this was derived from clearance studies of chylomicrons in the Watanabe heritable hyperlipidemic (WHHL) rabbit, which has a defect in the LDL receptor. Kita et al. (1982) demonstrated that apoEcontaining chylomicron remnants were cleared normally in WHHL rabbits, despite the defect in the LDL receptor, suggesting the presence of a distinct receptor for apoE-containing lipoproteins. A candidate receptor for apoE-containing lipoproteins arose from the isolation by Herz et al. (1988) of a cDNA encoding the LDL receptor-related protein (LRP). Both the LRP and the LDL receptors contain two types of cysteinerich repeats, one related to complement proteins C8 and C9 and the other to the epidermal growth factor (EGF) precursors. LRP contains one transmembrane domain and two copies of an NPXY sequence in the cytoplasmic domain that is necessary for the internalization of the LDL receptor. Beisiegel et al. (1989) showed that LRP in HepG2 cells is chemically cross-linked to apoE. Kowal et al. (1989) demonstrated that apoEenriched &VLDL causes marked increases in intracellular cholesterol esterification in LDL receptor-defective FH homozygote fibroblasts and that this interaction is blocked by antibodies against LRP. Although numerous studies implied that LRP plays a role in the metabolism of apoE-containing lipoproteins (Brown et al. 1991). it turned out to be a receptor for activated a,-macroglobulin (Strickland et al. 1990, Kristensen et al. 1990). Recently, mice lacking the LRP/a,macroglobulin receptor gene were produced by means of homologous recombination in embryonic stem cells (Herz et al. 1992). Disruption of this gene results in failure of embryogenesis, indicating that the protein is required for the early development of the mouse embryo. Animals heterozygous for the LRP/cx,macroglobulin receptor gene developed normally and their plasma total cholesterol and triglyceride levels were normal, so the role of the LRP/a,-macroglobulin receptor in lipoprotein metabolism is at present unclear.

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To obtain a cDNA encoding a receptor for apoE-containing lipoproteins, we employed a novel method (Figure 1). The rabbit LDL receptor cDNA contains a unique Sal I restriction site -400 bp upstream of the termination codon (Yamamoto et al. 1986). This restriction enzyme enabled us to exclude LDL receptor cDNAs from a cDNA library, because the Okayama-Berg vector used for the construction of the cDNA library also contains a unique Sal I site (Okayama and Berg 1983). Digesting a whole cDNA library with Sal I and recircularizing with T, DNA ligase, we successfully excluded the LDL receptor cDNA from the library. We conjecture that a putative receptor for apoE-containing lipoproteins should have the same binding motif as the LDL receptor, so we used a cDNA fragment that corresponds to the binding domain of the rabbit LDL receptor as a probe for screening. This exclusion method enabled us to obtain cDNAs that crosshybridized with a cDNA fragment coding for the binding domain of the LDL receptor. The positive cDNAS were then introduced into LDL receptor-deficient cells and the ligand-binding activities were analyzed. By this procedure, we obtained a cDNA that expresses a receptor for apoE-containing lipoproteins. Since the newly identified lipoprotein receptor is specific for apoE-containing lipoproteins, the receptor was designated VLDL receptor (Takahashi et al. 1992).

l

Structure of the VLDL Receptor

As expected from cross-hybridization with the LDL receptor cDNA fragment that encodes the ligand-binding domain, the new receptor has significant sequence homology to the ligand-binding repeats of the LDL receptor. However, an unexpected finding was that the entire region of the VLDL receptor is also strikingly homologous to the LDL receptor (Figure 2). The LDL receptor is composed of five domains (Yamamoto et al. 1984 and 1986, Lee et al. 1989, Mehta et al. 1991): (a) an amino-terminal ligandbinding domain composed of seven cysteine-rich repeats (Yamamoto et al. 1984 and 1986, Lee et al. 1989, Mehta et al. 1991); (b) an EGF precursor homology domain that mediates the acid-depend-

TCM Vol. 3, No. 4, 1993

VLDL cDNA

Other cDNA

LDL cDNA

Novel Strategy for Isolating a cDNA for ApoE-Containing Lipoproteins

salI @-‘I

wIl@

%llG

Sal I digestion Self-ligation

Sal1 digestion Self-ligation

xm

Sal I digestion Self-ligation

+ Transformation

1

LDL Receptor-Subtracted

cDNA Library

Figure 1. cDNA cloning strategy for the rabbit vety low density lipoprotein (VLDL) receptor. A cDNA library was constructed in the Okayama-Berg vector with use of poly(A) + RNA from

rabbit heart. To exclude low-density lipoprotein (LDL) receptor cDNAs from the library, the plasmid DNA isolated from the library was digested with Sal I and recircularized with T,DNA ligase. The resulting LDL receptor-subtracted cDNA library was screened with the rabbit LDL receptor cDNA fragment corresponding to the ligand-binding domain of the LDL receptor.

Figure 2. Functional domains in the low-density lipoprotein (LDL) receptor and amino acid

sequence similarity of rabbit very low density lipoprotein (VLDL) receptor with that of the rabbit LDL receptor. The percentage homology between the two proteins in a given domain is indicated. The cysteine-rich repeats in the ligand-binding domains are numbered. The cysteine-rich repeats in the epidermai growth factor (EGF) precursor homology domains are lettered A to C.

VLDL Receptor NH,Homology(%)

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Function in the

apoBand apoE-conthing lipoproteins

LDL Receotor

01993, Elsevier Science Publishing

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Figure 3. Ligand specificity of the rabbit very low density lipoprotein (VLDL) receptor and the human low-density lipoprotein (LDL) receptor. (a) Binding and internalization of ‘251-labeled VLDL, f3-VLDL, intermediatedensity lipoprotein (IDI), and LDL in Chinese hamster ovary (CHO) cells expressing rabbit VLDL receptor or human LDL receptor. CHO cells transfected with a plasmid encoding the human LDL receptor (pLDLR2), or the rabbit VLDL receptor (pVLDLRl), were incubated with the indicated concentration of 1251VLDL (753 cpm/ng), 1251-B-VLDL(614 cpm/ ng), 12sI-IDL (378 cprn!ng), or l*sI-LDL (1006 cpm/ng). After incubation for 4 h at 37”C, the values for bound and internalized 12sI- labeled VLDL, fl-VLDL, IDL, and LDL were determined. (b) Competition of unlabeled ligands for the binding and internalization of 1251-fi-VLDLin CHO cells expressing rabbit VLDL receptor. CHO cells transfected with a plasmid encoding the rabbit VLDL receptor (pVLDLR1) were incubated with 2.5 pg protein/ mL usI- fl-VLDL (614 cpm/ng) in the absence (w) or presence of the indicated concentrations of unlabeled ligands. After incubation for 4 h at 37”C, the values for bound and internalized t2SI-13-VLDL were determined. The 100% control value, in the absence of unlabeled lipoprotein, was 54.5 ng/mgprotein.

146

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200

ent dissociation of ligands (Davis et al. 1987); (c) an O-linked sugar domain of unknown function; (d) a transmembrane domain; and (e) a cytoplasmic domain that mediates the clustering of the receptor into coated pits (Chen et al. 1990). These domains are also present in the VLDL receptor. The major structural difference is the number of cysteine-rich repeat sequences in the ligand-binding domain: the VLDL receptor contains an eightfold repeat, whereas that of the LDL receptor is only sevenfold. Except for the extra binding repeat in the VLDL receptor, the hydropathy profiles of the two receptors are virtually superimposable. The extensive similarity between tbe VLDL and LDL receptors suggests that they have similar secondary and tertiary structures and are probably derived from a common ancestral gene. The features of each domain are highly similar between the two receptors. The NH,-terminal 328-amino-acid sequence of the rabbit VLDL receptor is composed of a single 40-amino-acid unit repeated

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eight times (Figure 2). This unit is similar to that in the ligand-binding domain of the LDL receptor (Yamamoto et al. 1984 and 1986, Lee et al. 1989, Mehta et al. 1991). The spacing of the cysteines is almost completely conserved among the repeats (Takahashi et al. 1992). The sequence SDE, which forms part of the ligand-binding site of the LDL receptor (Russell et al. 1989), is also completely conserved in each of the eight repeats of the VLDL receptor. The novel structural difference is the presence of the extra repeat sequence at the NH, terminus of the VLDL receptor. Excluding this first repeat, 55% of the amino acids within the ligand-binding domain of the rabbit VLDL receptor are identical with those of the rabbit LDL receptor. This value is -52% for the EGF precursor homology domain, 32% for the transmembrane domain, and 46% for the cytoplasmic domain. Within the cytoplasmic domain of the VLDL receptor, the sequence FDNPVY, which is required for coated pit-mediated internalization of the LDL receptor, is also completely conserved. The O-linked sugar domain of the rabbit VLDL receptor contains 28 amino acids, including I2 serine or threonine residues. In the rabbit LDL receptor, this domain of 48 amino acids contains 16 serine or threonine residues, most of which are modified with O-linked sugars (Yamamoto et al. 1986). The O-linked sugar domain of the LDL receptor has little conservation among species (Mehta et al. 199 l), so it is not surprising that the overall amino acid sequence identity between O-linked sugar domains of the LDL and VLDL receptor is only 19%.

l

Ligand Specificity and Tissue Distribution of the VLDL Receptor

The rabbit VLDL receptor cDNA was introduced into ldlA-7 cells, a mutant Chinese hamster ovary (CHO) cell line lacking an LDL receptor (Kingsley and Kt-ieger 1984), and the ligand specificity of the VLDL receptor expressed in the cells was analyzed by using t251-labeled rabbit VL.DL, B-VLDL, IDL, and LDL. Figure 3a shows that the rabbit VLDL receptor binds with high affinity to apoE-containing lipoproteins, including VLDL, fl-VLDL, and IDL, but not LDL, whereas the human LDL receptor binds both apoB- and apoE-containing lipo-

TCA4 Vol. 3, No. 4, 1993

fatty acids into muscle and fat cells in collaboration with capillary endothelial

LDL . . 0

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Receptor

Extrahepatic

t HDL

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.::* .*. . ..

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Figure 4. Diagram summarizing present views of the metabolism of plasma lipoproteins. The details of this model are described in the text. FFA, free fatty acid; HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein; LCAT, lecithin-cholesterol acyltransferase; LP lipase, lipoprotein lipase; LDL, low-density lipoprotein; and VLDL, very low density lipoprotein. proteins

with

high

affinity.

‘251-fi-VLDL

binding to the transfected VLDL receptor was inhibited by unlabeled apoE liposomes

(Figure

3b),

the receptor recognizes VLDL receptor dant in heart,

indicating

and adipose

sue, whereas the LDL receptor expressed

in most tissues,

tis-

mRNA is

including

ad-

renal gland, liver, and fibroblasts (Yamamoto et al. 1986). In vivo hepatic LDL receptor

is most important

in con-

trolling plasma levels of LDL. Administration of a high-cholesterol diet to African

green

monkeys

decreases

levels of LDL receptor consequent

decrease

hepatic

mRNA, and the

in hepatic

LDL re-

ceptors causes LDL to accumulate in the plasma (Sorci-Thomas et al. 1989). Despite the importance of the liver in cholesterol homeostasis in vivo, very little VLDL receptor mRNA is expressed there, which

TCM Vol. 3, No. 4, 1993

l

Acknowledgment

We thank

Dr.

comments

on the manuscript.

Ian

Gleadall

that

for

helpful

References

apoE.

mRNA is most abun-

muscle,

may suggest that the VLDL receptor does not play a critical role in the metabolism of cholesterol-carrying lipoproteins.

LPL (Figure 4). After partial hydrolysis of VLDL and chylomicron particles by LPL, these particles become smaller and more enriched with apoE (Manley and Hussain 1991). These intermediate particles may then have access to the VLDL receptor on the surface of muscle and fat cells, and are likely to be subsequently taken up into the cells by the receptor and metabolized within the cells. The bases of this hypothesis are (a) the receptor-mediated endocytosis of lipoproteins is one of the most active transport systems for macromolecules; (b) the VLDL receptor is abundant in muscle and fat cells, and this pattern of expression is very similar to that of the LPL mRNA (Wion et al. 1987, Kirchgessner et al. 1987, Senda et al. 1987); (c) the VLDL receptor recognizes apoE that is high in triglyceride-rich lipoproteins, VLDL, and chylomicrons; and (d) patients deficient in LPL can still store triglycerides in the adipose tissue (Eckel 1989). Although isolation of the cDNA has demonstrated the presence of a new lipoprotein receptor, its exact nature and function remain to be elucidated, so we are continuing our characterization experiments. One of the best approaches is to produce mice lacking the VLDL receptor gene. Efforts to produce such animals are under way.

l

Possible Physiologic Role of the VLDL Receptor in the Metabolism of Triglyceride-carrying Lipoproteins

Structural LDL

similarity

receptors

of the VLDL

suggests

that

they

and have

similar functions. Based on the ligand specificity and tissue expression of the VLDL receptor, we propose that its basic function is to provide muscle and fat cells with fatty acids. The uptake of fatty acids into these cells has previously been believed to be mediated mainly via LPL on the surface of capillary endothelial cells. However, the finding of a specific receptor that binds apoE-containing lipoproteins may alter this view. We believe that VLDL receptor somehow mediates the uptake of

01993,Elsevier Science Publishing Co., 10%1738/93/$6.00

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14%

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