The Low Density Lipoprotein Receptor-Related Protein/alpha 2-Macroglobulin

Receptor is a Receptor for Connective Tissue Growth Factor (CTGF)

Patricia R. Segarini +,%, James E. Nesbitt#,+ , Dongxia Li+, Lara G. Hayes*,@, John R. Yates

III *,$ and David F. Carmichael+

#To whom correspondence should be addressed.(jnesbitt@fibrogen.com)

+FibroGen, Inc.225 Gateway Blvd.

South San Francisco, CA 94080phone: 650-866-7200FAX: 650-866-7205

%current address :Aspira Biosystems571 Eccles Ave.

South San Francisco, CA 94080

*University of WashingtonDepartment of Molecular Biotechnology

Box 357730Seattle, Washington 98195

@current address: Zymogenetics

1201 Eastlake Ave East Seattle, WA 98102

$current address:The Scripps Research Institute

Department of Cell Biology, SR1110550 North Torrey Pines Road

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Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on August 22, 2001 as Manuscript M105180200 by guest on M

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La Jolla, CA 92037

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RUNNING TITLE

LRP is a CTGF Receptor

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SUMMARY

Connective tissue growth factor (CTGF) expression is regulated by transforming growth factor-ß

(TGF-ß) and strong upregulation occurs during wound healing; in situ hybridization data

indicate that there are high levels of CTGF expression in fibrotic lesions. Recently the binding

parameters of CTGF to both high and lower affinity cell surface binding components have been

characterized1. Affinity crosslinking and SDS-PAGE analysis demonstrated the binding of

CTGF to a cell surface protein with a mass of ~620 kDa. We report here the purification of this

protein by affinity chromatography on CTGF coupled to Sepharose and sequence information

obtained by mass spectroscopy. The binding protein was identified as the multi-ligand receptor,

low density lipoprotein receptor-related protein / alpha-2-macroglobulin receptor (LRP). The

identification of LRP as a receptor for CTGF was validated by several studies: (1) binding

competition with many ligands that bind to LRP, including Receptor-Associated Protein (RAP);

(2) immunoprecipitation of CTGF-receptor complex with LRP antibodies; and (3) cells that are

genetically deficient for LRP were unable to bind CTGF. Lastly, CTGF is rapidly internalized

and degraded and this process is LRP-dependent. In summary, our data indicate that LRP is a

receptor for CTGF, and may play an important role in mediating CTGF biology.

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INTRODUCTION

Connective tissue growth factor2 (CTGF; Mr approximately 38,000 Da), is a member of

the CCN family of growth factors, which is characterized by the presence and conserved spacing

of some 38 cysteine residues. CTGF was initially identified in and subsequently purified from

HUVEC conditioned media (1). Early studies demonstrated the strong induction of CTGF

expression by transforming growth factor beta (TGF-ß), and the promoter region of the CTGF

gene contains a unique TGF-ß response element not shared by other members of the CCN

family (2). In an anchorage-independent growth assay of TGF-ß, neutralization of CTGF

activity with antibodies or inhibition of CTGF expression with antisense oligonucleotides

reduced the ability of the cells to form colonies (3), suggesting that CTGF is a necessary part of

the cascade for induction of anchorage-independent growth. More recently, CTGF was shown

to be positively regulated by vascular endothelial growth factor (VEGF) (4,5), epidermal growth

factor, fibroblast growth factor (4), plasma clotting factor VIIa (6,7), thrombin (7,8), and by

lysophosphatidic acid and serotinin activation of heptahelical receptors (9), but negatively

regulated by tumor necrosis factor alpha (10) and the Wilms tumor suppressor WT1 (11).

The initial discovery of CTGF in vascular endothelium (1), and the subsequent

demonstration that CTGF is involved in the proliferation and migration of vascular endothelial

cells (12), suggests that CTGF is also an angiogenic factor. The isolation of CTGF from uterine

fluid and localization in embryonic and placental tissues suggests of role for CTGF in embryo

implantation (13,14).

CTGF is expressed at high levels during granulation tissue deposition in normal healing

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wounds (15,16). Expression of the extracellular matrix proteins fibronectin, α5 integrin, and

type I collagen is regulated by CTGF (16-18). Co-administration of CTGF with TGF-ß

produced a persistent fibrotic reaction that lasted for 14 days, but had resolved and was absent by

7 days in animals treated with TGF-ß-alone (19). The overexpression of TGF-ß in fibrotic

lesions is well documented (reviewed in (20,21)), and now many reports indicate that CTGF, too,

is overexpressed in many fibrotic lesions ((22-28) and reviewed in (29)). The emerging

understanding that CTGF is actively involved in the induction and/or maintenance of persistent

fibrosis has provided a target for the modulation of matrix overproduction in fibrotic disease.

The low density lipoprotein receptor-related protein (LRP)/alpha-2-macroglobulin

receptor (α2mR) (hereto referred to as LRP), is a member of the family of low density

lipoprotein (LDL) receptors (30). The LDL receptor family includes two subfamilies: one

containing “small” receptor members of approximately 120 kDa, including the LDL receptor

(LDLR), apo E receptor-2 (apoER2), and very low density lipoprotein receptor (VLDLR); and

one containing “large” receptor members of approximately 600 kDa, including LRP, epithelial

glycoprotein 330/megalin, and a related protein, sorLA. LDL family receptors bind multiple

ligands. At least two of these protein ligands, apolipoprotein E (apoE) and a 39 kDa Receptor-

Associated Protein (RAP), bind to all LDL receptor family members (30). A number of

functionally and structurally distinct ligands bind LRP and the diversity of these ligands suggests

that it may function in a variety of distinct physiological processes, such as lipoprotein

metabolism, protease regulation, tissue repair and remodeling, and embryonic development

(reviewed in (30)).

Here we have further characterized the major CTGF binding protein1. This protein was

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affinity purified and identified as LRP. Competitive inhibition of CTGF binding using other

LRP ligands and immunoprecipitation of CTGF receptor complexes with LRP antibodies has

confirmed that LRP is a receptor for CTGF. Additionally, this report shows that CTGF is rapidly

internalized and degraded by cells through an LRP-dependent pathway. This is the first

identification of direct interaction of a growth factor with LRP. Our findings suggest that LRP

has a regulatory role in the biology of CTGF.

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EXPERIMENTAL PROCEDURES

Materials

A baculovirus expression system was used for the preparation of recombinant human

CTGF (rhCTGF) and the protein was purified as described1. Apolipoprotein E (apoE), low

density lipoprotein (LDL), lactoferrin (LF), alpha 2-macroglobulin (α2m), and chloroquine were

purchased from Sigma Chemical Co. (St. Louis, MO). Membrane grade Triton® X-100 was

purchased from Boehringer Mannheim Corp. (Indianapolis, IN). Heparin was purchased from

Gibco/BRL (Bethesda, MD). A panel of receptor grade detergents, including Nonidet® P-40

(NP40), Triton® X-100, Tween® 20, digitonin, dodecyl-ß-D-maltoside, octylglucoside,

deoxycholic acid, and CHAPS, was purchased from Boehringer Mannheim Corp.

Cell lines

The cell lines MG63, MEF, PEA10, and PEA13 were purchased from the American Type

Culture Collection (ATCC). Cells were maintained as recommended by the ATCC. The murine

bone marrow stromal cell line BMS-2 was graciously provided by Dr. Jeff Gimble (formerly of

the Oklahoma Medical Center, currently at Artecel Sciences, Inc., Durham, NC). BMS-2 cells

were maintained in DMEM, containing pyruvate, 55 µM ß-mercaptoethanol, and 10% fetal

bovine serum.

Binding of CTGF to monolayer cultures

rhCTGF was iodinated with chloramine-T (Sigma Chemical Co.) by the procedures

described1. For binding analysis, cells were plated at 2 5 x 104 cells/cm2 in 24 well dishes, 16 –

24 hours prior to a binding experiment. The cells were washed twice with binding buffer

(Dulbecco’s phosphate buffered saline (PBS) (Life Technologies, Inc.) containing 0.2% bovine

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serum albumin and 0.2% sodium azide) at 4oC. Binding experiments were performed by

incubating monolayers of cells with various concentrations of 125I-rhCTGF in binding buffer

for 4 hours at 4oC with gentle rocking. Duplicate wells were incubated with at least 100-fold

excess of unlabeled rhCTGF for the determination of nonspecific binding.

We determined the kinetics of binding of 125I-rhCTGF to cells as follows. Supernatants

were collected from the cells and directly counted on a Beckman Gamma 5500B counter.

Radioactivity measured from this was called the “free” fraction. The cells were then washed

four times with cold binding buffer, and lysed with 1% Triton® X-100. The lysis fraction was

counted as above, and contained the “total bound” fraction. The nonspecific bound fraction was

also determined and subtracted from each point to yield specific bound. The specific bound and

free fractions were analyzed for one or two site binding using non-linear regression (GraphPad

Prism, version 3.0, GraphPad Software, Inc., San Diego, CA). The same protocol and analysis

was used for competitive binding experiments.

Affinity labeling and crosslinking of monolayer cultures

Cells to be affinity labeled were washed twice with cold binding buffer, then incubated

with 50 - 100 pM 125I-rhCTGF in binding buffer at 4oC for 3 – 4 hours. The binding medium

was removed and replaced with 0.5 mM Bis-[succinimidyl suberate] (BS3, Pierce Chemical Co.,

Rockville, IL). Alternatively, 0.1 mM ethylene glycobis [sulfo-succinimidyl succinate] (Sulfo-

EGS, Pierce Chemical Co.) was also used for crosslinking. Crosslinking proceeded at room

temperature for 15 minutes (BS3) or at 4oC for 30 min (Sulfo-EGS) and was terminated by

removing the medium and washing the cells several times with buffer containing 250 mM

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sucrose, 10 mM Tris pH 7.4, and 10 mM EDTA at 4oC. The complexes were collected in the

soluble fraction following cell lysis with 1% Triton® X-100 (unless indicated otherwise) in PBS

with a cocktail of protease inhibitors (Calbiochem®, San Diego, CA). In some experiments,

0.5% NP40 was used for cell lysis.

Preparation of crude cell membranes

Membranes were prepared from BMS2 cells by a modification of the procedure described

by Atkinson (31). The cells were grown to confluence in roller bottles, and then dissociated with

5 mM EDTA in Dulbecco’s PBS lacking calcium and magnesium. Cell pellets were then

collected by centrifugation, and washed. The cells were then suspended in hypotonic phosphate

buffer (7.5 mM NaPO4, pH 7.2) and incubated 10 min on ice. The membranes were disrupted

by sonication, and the nuclei were stabilized in buffer containing10 mM NaPO4, pH 7.2,

10 mM NaCl, and 3 mM MgCl2. The nuclei and whole cells were removed by centrifugation for

5 min at 800 x g, and the supernatant was collected. The supernatant was centrifuged over a

cushion of 45% sucrose in Dulbecco’s PBS, pH 7.2, for one hour at 24,000 x g. The membrane

fraction located at the sucrose/PBS interface was carefully collected, diluted, and concentrated

by centrifugation at 100,000 x g for 15 min. The membrane pellet was resuspended in

Dulbecco’s PBS, and protein content estimated with the Pierce BCA reagent against an albumin

standard (Pierce Chemical Co.). From a preparation of an estimated 109 cells, 10 – 20 mg of

crude membrane protein was frequently obtained.

Affinity chromatography

One milligram of rhCTGF prepared from baculovirus was immobilized on Reacti-Gel®

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GF-2000 (Pierce Chemical Co.) according to the manufacturer’s specifications. The membranes

(10 – 15 mg) were solubilized in 0.2% Triton® X-100, 20% glycerol, in Dulbecco’s PBS

(Buffer A) containing a cocktail of protease inhibitors, centrifuged (14,000 x g) for 10 min at

4oC to remove insoluble material, and applied to the CTGF affinity matrix. The sample was re-

circulated over the column 5 – 10 times. The flowthrough was collected and the column was

washed with 20 column volumes of Buffer A. The bound sample was eluted with a gradient of

0.135 – 2M NaCl in buffer containing 0.2% Triton X-100, 20% glycerol, and PBS. The CTGF

receptor-containing fractions were identified using a solution binding assay (described below).

Further purification was achieved by electrophoresis of the fractions in 5% SDS-PAGE under

non-reducing conditions.

Solution binding assay

Preparations of membrane proteins (5 µg each) were solubilized in 0.2% Triton® X-100,

20% glycerol in Dulbeccos PBS (Buffer A) and the insoluble material was removed by

centrifugation (14,000 x g) for 10 min at 4oC. Fractions from the affinity column were used

directly in the solution binding assay (10 µl/fraction). The samples were incubated with 0.2 nM

125I-rhCTGF for 3 4 hours. The crosslinker, BS3, was added to a final concentration of

0.5 mM, and the reaction proceeded at room temperature for 15 min. Gel sample buffer was

added to each sample, the samples were then heated for 2 min at 100oC, and applied to 5%

SDS-PAGE. Following electrophoresis, the gels were dried and analyzed by autoradiography.

Mass spectroscopy

The gel band of interest (migrating above the 220 kDa marker) was excised with a fresh

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razor band, destained, and subjected to trypsin digestion (32). The recovered peptide fragments

were analyzed by LC/MS/MS. Microelectrospray columns of 360 µm o.d. x 100 µm i.d. fused

silica capillary were packed with 10 12 cm POROS 10R2, a reversed phase packing material

(PerSeptive Biosystems, Framingham, MA) (33). The flow from the HPLC pump (typically

150 µl/min) was split pre-column to achieve a flow rate of 500 nl/min. The mobile phase for the

gradient elution consisted of (A) 0.5% acetic acid and (B) acetonitrile/water 80:20 (v/v)

containing 0.5% acetic acid. The gradient was linear from 0 – 60% B in 30 min. Mass spectra

were recorded on an LCQ ion trap mass spectrometer (Finnigan MAT, San Jose, CA) equipped

with a microelectrospray ionization source (33). Tandem mass spectra were acquired during the

entire gradient automatically as previously described (34). Protein sequence databases were

searched with the tandem mass spectra using the computer program SEQUEST (35). SEQUEST

correlates tandem mass spectra of peptides with amino acid sequences from protein and

nucleotide databases (15 public databases available). The FBSC Non-Redundant Protein

Database (NRP) database was obtained as an ASCII file in the FASTA format from Frederick

Biomedical Supercomputing Center (ncbi.nlm.nih.gov in /pub/nrdb) by anonymous ftp. Each

sequence produced by SEQUEST was verified by manually inspecting the fit of the amino acid

sequence to the corresponding tandem mass spectrum.

Purification of receptor-associated protein (RAP)

Plasmid DNA containing the human RAP protein sequence was purchased from the

ATCC as the I.M.A.G.E. Consortium CloneID 511113 (36). The RAP cDNA sequence was

excised from the vector with the restriction enzymes BamHI and XhoI, and the 1 kb fragment

was gel purified. The pGEX-4T-1 vector (Pharmacia) was cut with BamHI and XhoI, into

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which the RAP cDNA was ligated. Expression of RAP from this expression construct results in

the formation a fusion protein of RAP and glutathione S-transferase. Correct RAP cDNA

insertion was verified by sequence analysis. The RAP-GST fusion protein and the GST protein

alone (control vector) were expressed in E. coli and purified as described (37).

Antibodies and immunoprecipitation

A mouse monoclonal antibody reactive to the C-terminus of LRP was prepared from

IgG-11H4 hybridoma supernatant (ATCC) and purified by Protein G-Sepharose (Pharmacia).

Monoclonal antibodies reactive with the extracellular domains of the α chain and ß chain of LRP

were purchased from American Diagnostica (catalog #3402 and #3501 respectively).

Affinity labeled cells were extracted with 1% Triton® X-100 or 0.5% NP40 in PBS

containing a cocktail of protease inhibitors. The extracts were incubated for 2.5 hours at 4oC

with 1 µg of antibody to LRP. Protein G-Sepharose was added to the sample and the incubation

continued at 4oC for 1 hour. The sample that bound to the suspension was collected by

centrifugation, washed four times with PBS, then eluted by boiling with Laemmli gel buffer.

The eluted proteins were applied to 5% SDS-PAGE, and analyzed by autoradiography.

Internalization experiments

To determine the kinetics of internalization of CTGF following binding to the cell

surface, MG63 cells grown in 6-well dishes were affinity labeled at 4oC with 0.2 nM 125I-

rhCTGF for 3 hours in binding buffer prepared with Minimal Essential Medium containing

0.2% bovine serum albumin (MEM/BSA). Nonspecific binding, internalization, and degradation

were determined by the addition of 50 nM unlabeled CTGF and subtracted from experimental

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values. In some samples, 3 µg/ml heparin or 250 nM lactoferrin was added to the cells during

binding. After the binding period, the binding medium was removed and the cells were carefully

rinsed twice with fresh, cold MEM/BSA. Pre-warmed MEM/BSA was then added to the cells

and the cells were incubated at 37oC in a CO2 humidified incubator. At 0, 15, 30, 60, 120, and

180 min after transfer to 37oC, the cells were harvested. The medium was collected, treated with

10% trichloroacetic acid (TCA), and the precipitable fraction was separated by centrifugation. In

these experiments, the supernatant resulting from TCA precipitation contained the degradation

products of 125I-rhCTGF that had been internalized, degraded, and returned to the extracellular

space (i.e., to the conditioned media). The internalized fraction was determined following

trypsinization of the cell layer for 10 minutes. The resulting cell pellet was collected by

centrifugation. The radioactivity associated with the trypsin-resistant pellet represented the

internalized fraction of 125I-rhCTGF. To demonstrate LRP-mediated endocytosis of CTGF,

MG63 cells were affinity labeled with 0.2 nM 125I-rhCTGF in MEM/BSA in the presence or

absence of 50 nM RAP-GST. Nonspecific binding, internalization, and degradation of 125I-

rhCTGF were determined by the addition of 50 nM unlabeled CTGF and subtracted from

experimental values. To inhibit lysosomal activity, 200 µM of chloroquine was added to some

of the cultures. After a 2 hour incubation period at 37oC, the degraded and internalized fractions

were determined as described above.

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RESULTS

Solubilized Receptor Assay.

The bone marrow stromal cell (BMS2) line was chosen as a source for purification of the

receptor due to their high level of CTGF-binding to a low affinity receptor1. The ease of growth

and subculture combined with the high level of CTGF binding were factors involved in choosing

these cells for receptor purification. Prior to affinity purification, we optimized the binding and

solubilization conditions that would allow for solubilization of cell membrane proteins as well as

retain the capacity to bind CTGF. Crude membranes were prepared from BMS2 cells by

homogenization and differential centrifugation. A panel of receptor-grade detergents was

examined individually in order to solubilize the membrane proteins. Solubilized membranes

were incubated with 0.2 nM 125I-rhCTGF in the presence or absence of 200-fold excess

unlabeled CTGF. The samples were crosslinked with BS3 and separated by 5% SDS-PAGE.

Competitive crosslinking and binding was achieved using 1% Triton® X-100 containing

glycerol to help stabilize the membrane proteins (Figure 1A). The left panel demonstrates CTGF

binding and crosslinking to intact membrane fragments; the addition of 10% and 20% glycerol to

the solubilized membranes gave similar binding results, with the inclusion of 20% glycerol

providing more favorable results (middle and right panels, Figure 1A). These conditions were

used for the affinity purification of the receptor.

Affinity Purification.

Membranes prepared from BMS2 cells were solubilized and applied to a column of

CTGF coupled to Sepharose, washed with the same solvent, and bound proteins were eluted with

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a salt gradient as described above. Fractions were collected and incubated with 125I-rhCTGF to

assay for CTGF binding activity (Figure 1B). The peak CTGF binding fractions eluted between

50 – 75% buffer B (1.15M – 1.65M NaCl). Peak fractions were pooled and separated by SDS-

PAGE (Figure 1C). A band migrating above the highest Mr standard (220 kDa) and estimated as

>400 kDa was observed in the Coomassie stained gel. We occasionally observed additional

proteins migrating at Mr = 200 kDa, and Mr = 150 kDa (as shown in Figure 1C). These bands

were excised and analyzed by mass spectroscopy. The >400 kDa band contained the peptides

listed in Table I and database analysis identified the protein as LRP. Repeat purification and

mass spectroscopy analysis confirmed the result. Identification of the Mr 200 kDa and 150 kDa

proteins was not achieved.

LRP Ligands Compete for CTGF binding.

That LRP was a binding protein for CTGF was further confirmed. A number of

commercially available LRP ligands were tested for their ability to compete with CTGF for

binding to cells (Figure 2). All ligands tested inhibited 125I-rhCTGF binding to cells (Figure

2A) albeit with 5 – 10 fold lower affinity than that of CTGF. Additionally, receptor associated

protein, RAP, which is able to displace all LRP ligands, prevented CTGF binding (Figure 2B).

Crosslinking analyses confirmed that the inhibition was due to lack of binding to the high Mr

receptor protein (Figure 2C). These data support the identity of LRP as a CTGF binding protein.

Binding of CTGF to LRP Deficient Cells.

The availability of cells isolated from LRP gene deletion mouse embryos provided an

opportunity to study CTGF binding on cells genetically lacking LRP. The cell lines examined

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include a homozygous LRP-deficient mouse embryo fibroblast cell line, PEA 13 (-/-), a

heterozygous LRP-deficient cell line, PEA 10 (+/-), and a wildtype mouse embryo fibroblast,

MEF1, (+/+).

Each of these cell lines was examined for CTGF binding in a CTGF-binding assay. The

binding parameters obtained (determined by nonlinear regression analysis) are summarized in

Table II. MEF1 (+/+) and PEA10 (+/-) cells bound 125I-rhCTGF with single site binding

kinetics, while the LRP-deficient cells, PEA13, did not bind 125I-rhCTGF in this assay. The

heterozygous cell line, PEA10, appeared to have approximately one-fifth the number of CTGF

binding sites as observed in wild-type MEF1 cells, yet had the same Kd for binding. These data

are suggestive of a gene dosage affect on LRP protein expression levels, but not on the kinetics

of ligand association/dissociation.

Crosslinking analysis was performed using these LRP-deficient cells to confirm that the

observed binding of CTGF to these cells involved the high Mr protein (Figure 3). Both MEF

(+/+) and PEA10 (+/-) cells, but not PEA 13 (-/-) cells, bound and could be cross-linked to

CTGF to the high Mr protein. The binding of CTGF to the LRP-expressing cells, but not to the

LRP-deficient cells, supports the identification that LRP is a CTGF binding protein.

Immunoprecipitation of CTGF/High Mr Complexes.

Further confirmation that LRP is the high Mr CTGF binding protein was demonstrated

using LRP antibodies. Detergent lysates were prepared from 2 cell types, BMS2 and MG63, that

had been affinity labeled with 125I-rhCTGF, cross-linked, and immunoprecipitated with

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antibodies directed to LRP (Figure 4). Antibodies specific to the extracellular epitopes in both

the α and β subunit chains of LRP, as well as an antibody that recognizes a cytoplasmic domain

of LRP, immunoprecipitated the CTGF-containing complex, while control normal murine IgG

did not. CTGF, migrating at the front of the gel, was poorly immunoprecipitated with these LRP

antibodies. Notably, the antibody recognizing the LRP α chain was most active for MG63

(human origin) cells while the antibody recognizing the LRP β chain was most active for BMS2

(murine origin) cells. The LRP α chain antibody is immunoreactive with the human protein

only. While the β chain antibody is reactive with both human and rodent protein, the difference

in recognition could be due to different detergent extraction conditions.

Internalization of CTGF.

The internalization kinetics of CTGF following binding was examined. For these

experiments, the MG63 cell line was utilized because the conditions often promoted lifting of

BMS2 cells from monolayers whereas the MG63 cultures remained intact. Monolayer cultures

of MG63 cells were incubated at 4oC with 0.2 nM 125I-rhCTGF in medium. The cells were

then transferred to 37oC and internalization and degradation of 125I-rhCTGF were followed at

specific time points (Figure 5). In one third of the cultures, an LRP ligand, lactoferrin, was

included to compete for LRP binding. To the other third of the cultures, heparin was added to

inhibit binding of CTGF to the cell surface. The results demonstrated that CTGF is rapidly

internalized by cells, occurring within 30 minutes following the temperature shift to 37°C.

Degradation of CTGF, determined by the release of label into the medium, was detected after 30

minutes, and continued throughout the time course of the experiment. Lactoferrin moderately

reduced internalization and degradation, suggesting LRP-mediated internalization.

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An LRP internalization pathway transports ligands through an endosomal compartment,

wherein the acidic lumen of the endosome promotes ligand dissociation, and the ligand is

subsequently degraded (38). This process is sensitive to the drug, chloroquine, which raises the

pH of the endosome and thus inhibiting the pH-dependant ligand dissociation. We examined

whether CTGF is internalized by LRP and degraded through the same pathway (Figure 6). In

these experiments, cultures of MG63 cells were incubated at 37oC with 125I-rhCTGF in the

presence or absence of the LRP antagonist, RAP, or with chloroquine. MG63 cells treated with

chloroquine showed in an increased level of intracellular 125I-rhCTGF. Degraded 125I-

rhCTGF was barely detectable in these chloroquine-treated cell cultures, whereas the control

cultures had high levels of degraded CTGF. These results support an endosomal-dependent

pathway for the LRP-mediated internalization of CTGF, with ligand dissociation required for its

degradation. RAP addition to the cultures eliminated CTGF internalization and degradation.

These results provide evidence that CTGF uptake and degradation by cells are LRP dependent.

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DISCUSSION

In this study, we have presented evidence that the major cell membrane protein to which

CTGF binds is a high Mr protein, identified previously as LRP. This study provides the first

report of a growth factor that binds directly to LRP, although recently the Wnt family of secreted

molecules has been demonstrated to bind to other members of the LRP family (39). Previous

suggestion of growth factor interaction with LRP has been through complexes with α2m as a

clearance molecule (40).

We were unable to obtain sequence information for two lower Mr proteins that were

occasionally affinity co-purified with LRP (estimated Mr of 150 and 200 kDa). These proteins

were not detected in the crosslinking/binding assay. CTGF, and the structurally related protein,

Cyr61, bind to a number of integrins, including αvß3, αIIbß3 and α6ß1 (41-44). Recently it has

been demonstrated that CTGF-dependent cell adhesion induces cell signaling through integrin-

mediated pathways (42). While the identity of the co-purifying bands is unclear, the Mr

estimates are suggestive for some of the integrin subunits. The possibility exists that these bands

represented breakdown products of the large LRP protein. Further purification and analysis will

be necessary for unambiguous sequence identity of these proteins.

Our data indicate that at least part of the CTGF-binding site on LRP is similar or

common to the binding site utilized for many of the LRP ligands, as these ligands competed for

CTGF binding, although with lower affinity (Figure 2). LRP contains multiple copies of

cysteine-rich repeats known as LDLR class A repeats (complement-type repeats) arranged in 4

clusters, forming ligand binding sites (45-49). The binding site for many LRP ligands has been

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sub-localized (50), and NMR solution structure of the complement-like repeat CR3 from LRP

has recently been determined (51). It will be of interest to sub-localize the binding site for

CTGF within the extracellular domain of LRP.

With the exception of RAP, which binds to LRP with very high affinity (Kd = 3 nM

(52)), most LRP ligands bind LRP with a moderate affinity. For example, hepatic lyase binds

LRP with a Kd of 52 nM (53); PEA binds LRP with a Kd of 14 nM (54); and, coagulation factor

VIII binds LRP with a Kd of approximately 50 nM to LRP (55). Urokinase-type plasminogen

activator (uPA) is synthesized as a single chain zymogen, pro-urokinase, and binds LRP with a

Kd of 45 nM; the active two-chain enzyme (tc-uPA) binds LRP with a Kd of 60 nM. Complex

formation with plasminogen activator inhibitor type I (PAI-1) increases the affinity of uPA to

LRP, such that the uPA/PAI-1 complex binds LRP with a Kd of 1-3 nM (56,57).

Many LRP ligands are polyanionic proteins that bind heparin, suggesting that their

binding to cell surface HSPG may be required for interaction with LRP (58). HSPG may serve

to concentrate the ligands at the cell surface, promoting binding with the relatively low affinity

interaction with LRP. The observed Kd for CTGF binding to LRP is 0.5 -1 nM (the present

study, for MEF1 and PEA10 cells; and in Nesbitt et al.1), which is a much higher binding

affinity than reported for other LRP ligands. In Nesbitt et al.1, we showed that, while CTGF is a

heparin binding protein, CTGF is capable of binding to the surface of cells depleted of GAGs.

Perhaps the higher affinity of CTGF for LRP obviates concentration of CTGF on the cell surface

by HSPGs. Nevertheless, the significance of the high affinity binding of CTGF to LRP is

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unclear at this time, but suggests an important role for LRP in CTGF biology.

Experiments aimed at creating mice lacking LRP by genetic deletion of the LRP allele

failed to produce viable embryos (59), suggesting an important role for LRP during early

development. CTGF is highly expressed in uterine epithelium and in decidualizing endometrial

stromal cells during early pregnancy, as well as in embryonic ectoderm, endoderm, and at the

ectoplacental cone after implantation (60). LRP is localized to invading trophoblastic cells, in

decidualizing tissue, and at the ectoplacental cone after implantation (61,62). The similar

developmental localization patterns support possible interaction between CTGF and LRP during

embryonic development.

We have demonstrated that CTGF is rapidly internalized and degraded by cells, and that

this represents an LRP-mediated process. We have experienced a rapid disappearance of CTGF

from culture medium after addition of CTGF to cell cultures. A rapid internalization and

degradation pathway would account for the inability to detect CTGF in conditioned medium.

We suggest that one function of LRP in CTGF biology is to modulate the concentration of free

CTGF in the extracellular space or at the cell surface.

Whether or not LRP serves as a signaling receptor for CTGF bioactivity remains to be

determined. LRP binds and internalizes numerous ligands that would necessitate separate

ligand-specific signaling mechanisms; yet, signaling involving LRP has been suggested in at

least some systems. Most recently, signaling by the Wnt family of proteins requires the LRP-5

or LRP-6 members of the LRP family to function as co-receptors with the Frizzled family of

receptors (39,63,64). In a different system, LRP mediates long-term potentiation in

hippocampal neurons (65) and activation with activated α2M promotes calcium influx in

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neuronal cells (66). Other members of the LDLR family have recently been demonstrated to

transduce extracellular signals. For instance, VLDLR and apoER2 function as receptors in the

Reelin/disabled-mediated neuronal migration pathway (67-70). The short cytoplasmic domain

of LRP contains multiple potential endocytosis motifs including 2 NPXY motifs; but, a recent

study demonstrated that a YXXL motif serves as the dominant signal for LRP endocytosis (71),

leaving the NPXY motifs available for interaction with other signaling or adaptor proteins.

Disabled-1 (dab-1) interacts with the NPXY motifs of LRP in neuronal cells (69,70,72,73).

When tyrosine phosphorylated, mDab1 binds nonreceptor tyrosine kinases, such as src, fyn and

abl (74). Another member of this family, mDab2/p96/DOC-2, has approximately 50% sequence

conservation with the amino terminal sequence of mDab1, but is expressed in a wider variety of

cells (75). Dab2/p96/DOC-2 has been shown to compete with SOS to bind Grb2, suggesting a

negative regulatory role in the Ras signaling cascade (76) and may negatively regulate

mitogenesis (77). Whether Dab2/p96/DOC-2 or an unidentified member of the Dab family

binds to LRP remains unknown and is of much interest to investigate.

We have been unable to detect tyrosine phosphorylation of LRP in cells treated with

CTGF (data not shown). In addition, we have examined LRP purified by CTGF affinity

chromatography in kinase assays. Although an associated kinase activity towards casein co-

purified with LRP, this kinase activity was found not from LRP itself, but rather from an

associating protein. Most importantly, the kinase activity did not respond to CTGF stimulation3.

Therefore it remains unclear whether LRP phosphorylation plays any role in mediating

biological functions of CTGF.

LRP and many of its ligands have been localized to the senile plaques of Alzheimer’s

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disease (78-80), in plaque-associated activated astrocytes, but not in resting astrocytes (80).

Recent biochemical data support a role for LRP in the pathogenesis of Alzheimer’s disease

(81,82). There is an increase in LRP expression in monocytes from patients with coronary heart

disease (83), and LRP is abundantly expressed by smooth muscles cells and macrophages in

human atherosclerotic lesions (84). In the renal ablation model of experimental kidney fibrosis,

LRP expression is significantly increased in the glomeruli and interstitium with preferential

localization to fibrotic lesions (85). While to date CTGF has not been documented in

Alzheimer’s lesions, it is highly expressed in fibrotic kidney disease (22) and atherosclerotic

plaques (24). With co-localization of CTGF and LRP in these diseases, it will be important to

understand the functional role of the high affinity interaction of these proteins.

It will be of interest to examine whether altered LRP expression is a general marker of

disease characterized by impaired wound healing or in fibroproliferative disorders. Future work

examining the role of LRP in CTGF signaling by itself or as a co-receptor to a yet unidentified

CTGF receptor, and the effect of the modulation of LRP on CTGF activity will be important

studies to probe the biology of CTGF and for the design of therapeutics for intervention in

fibrotic disease.

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ACKNOWLEDGMENTS

We acknowledge and thank Miss Cathyrn Baker for preparation of cell membranes and Dr. Leah

Allen for subcloning the RAP gene into the GST fusion vector. We thank Dr. George R. Martin

for valuable advice and critical review of this work.

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FOOTNOTES

1. Abbreviations: LRP, Low density lipoprotein- receptor related protein/α2-

macroglobulin receptor; CTGF, Connective Tissue Growth Factor; rhCTGF, recombinant

human CTGF; TGF-ß, Transforming Growth Factor Beta; PEA Pseudomonas Exotoxin

A; IgG, Immunoglobulin gamma; RAP,Receptor associated protein; RAP-GST,RAP

fusion protein wit2. h glutathione S-transferase; α2m, alpha 2-

macroglobulin; α2m-Me, methylamine activated α2M; α2mR, α2m receptor; LF,

lactoferrin; ApoE; apolipoprotein E; LDL, low density lipoprotein; VLDLR, very low

density lipoprotein receptor; TCA, trichloroacetic acid; MEM/BSA, minimal essential

medium with 0.2% bovine serum albumin

3. Nesbitt, J. E., Segarini, P. R., Carmichael, D. F. Manuscript submitted.

4. Li, D. Unpublished observations.

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FIGURE LEGENDS

Table I. The gel band of interest (migrating above the highest Mr standard of 220 kDa) was

excised with a clean razor blade, destained and subjected to trypsinization. The recovered

peptide fragments were analyzed by LC/MS/MS and mass measurements were analyzed using

the SEQUEST analysis program.

Table II. Binding parameters of CTGF with LRP wildtype and deficient cells. Equilibrium

binding was performed on monolayer cultures of cells and analyzed as described in Materials

and Methods.

Figure 1. Purification of a CTGF receptor.

A. The solubilized receptor binds CTGF. Crude membranes were prepared from BMS2 cells

and solubilized in 1% Triton in PBS with 10% glycerol, or 20% glycerol, and a cocktail of

protease inhibitors. Intact membranes were used as a binding control (far left panel). The

membranes were affinity labeled with 0.2 nM 125I-rhCTGF and crosslinked. Half the

samples also contained a 200 fold excess of unlabeled CTGF. The crosslinked samples were

separated on 5% SDS-PAGE under non-reducing conditions. The gels were dried and

visualized by autoradiography. Molecular size markers are indicated at the left in kDa, and an

arrow indicates the CTGF-receptor complex. The far right panel demonstrates 125I-

rhCTGF crosslinked to BMS2 cells in monolayer culture.

B. Binding assay of solubilized membrane proteins separated by CTGF affinity

chromatography. Crude membrane preparations were solubilized in 1% Triton® X-100 in

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PBS with 20% glycerol and protease inhibitors. The sample was applied to a CTGF affinity

matrix and washed through with Buffer A. The bound material was eluted with a NaCl

gradient. An aliquot of the loaded sample, the flowthrough, and each fraction collected

during wash and elution were analyzed by affinity labeling, crosslinking, and separation on

non-reducing 5% SDS-PAGE. Peak fractions are indicated with a bracket. An arrowhead

indicates the CTGF-receptor complex.

C. Coomassie staining of affinity purified receptor. The peak fractions were pooled and

separated on non-reducing 5% SDS-PAGE. The upper band (long arrow) migrates at the

expected position for the previously identified complex. Short arrows indicate 2 other

proteins co-purifying on the column. The three indicated bands were excised for further

analysis.

Figure 2. Competition of LRP/α2mR ligands with CTGF.

A. Monolayers of BMS2 cells were incubated with 100 pM 125I-rhCTGF in the presence of

indicated concentrations of unlabeled CTGF, apo E4, LpL or LDL. The bound material was

collected and determined by counting. Bound is represented as a fraction of total (non-

competing) CTGF bound. Each point is the average of duplicate samples and error bars

indicate the standard deviation.

B. Monolayers of BMS2 cells were incubated with 50 pM 125I-rhCTGF in the presence of

indicated concentrations of unlabeled CTGF, RAP or RAP-GST. The bound material was

collected and determined by counting. Bound is represented as a fraction of total (non-

competing) CTGF bound. Each point is the average of duplicate samples and error bars

indicate the standard deviation.

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C. Monolayers of BMS2 cells were incubated with 50 pM 125I-rhCTGF in the presence of

unlabeled competitor: 21.5 nM CTGF, 250 nM LF, 574 nM apo E4, 400 nM LpL or 100 nM

α2M-Me. The bound material was crosslinked to the cells. Cell lysates were separated by

SDS-PAGE, dried and visualized by autoradiography. An arrow indicates the position of the

CTGF-receptor complex.

Figure 3. Affinity labeling and crosslinking of 125I-rhCTGF to LRP/α2mR -deficient cells.

MEF1 (+/+), PEA 10 (+/-) and PEA 13 (-/-) cells were grown in monolayer culture; affinity

labeling with 125I-rhCTGF and crosslinking were performed. The Triton® X-100 soluble

fraction was run on SDS-PAGE; the gel was dried and visualized by autoradiography. An arrow

indicates the position of the CTGF-receptor complex.

Figure 4. Immunoprecipitation of CTGF complexes with anti- LRP/α2mR antibodies.

A. Immunoprecipitation from MG63 cells. MG63 cells were affinity labeled and crosslinked

with 125I-rhCTGF. The complexes were extracted with Triton® X-100 and

immunoprecipitated with anti LRP-α, antiLRP-β or nonimmune murine IgG. A portion of

the extract was saved and applied to the gel without immunoprecipitation. An arrow

indicates the CTGF-receptor complex.

B. Immunoprecipitation from BMS2 cells. BMS2 cells were affinity labeled and crosslinked

with 125I-rhCTGF. The complexes were extracted with NP40 and immunoprecipitated with

anti LRP-α, antiLRP-β or mAb11H4. An arrow indicates the CTGF-receptor complex.

Figure 5. Kinetics of the internalization and degradation of CTGF. MG63 cells were affinity-

labeled with 125I-rhCTGF alone (solid circles), with 3 µg/ml heparin (open squares), or with

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250 nM lactoferrin (open circles), at 4oC. After 3 hours, the cells were shifted to 37oC and at

the indicated time points, the internalized (A) or degraded (B) fractions were determined.

Internalized CTGF was measure as the trypsin-resistant cell pellet. Degraded CTGF was

measure in the supernatant of TCA-treated cell medium. Each point is the average of

triplicate samples and error bars indicate the standard deviation.

Figure 6. Internalization and degradation of CTGF is LRP/α2mR dependent. MG63 cells were

incubated with 125I-rhCTGF alone, with RAP-GST, or with chloroquine for 2 hours at

37oC. After 2 hours, the internalized (A) and degraded (B) fractions were measured as described

in the legend to Figure 5 and as detailed in the methods section. Each point is the average of

triplicate samples and error bars indicate the standard deviation.

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TABLE I

First round of mass spectroscopy yielded following peptides:(R)AALSGANVLTLIEKDIR(K)NAVVQGLEQPHGLVVHPLR(R)SERPPIFEIR(K)TVLWPNGLSLDIPAGR(R)TTLLAGDIEHPR(R)YVVISQGLDKPR

Second round of mass spectroscopy yielded following peptides:(R)DGILFWTDWDASLPR(R)GWDTLYWTSYTTSTITR(R)IFFSDIHFGNIQQINDDGSGR(R)ILWIDAR(K)ITWPNGLTVDYVTER(K)NAVVQGLEQPHGLVVHPLR(R)SERPPIFEIR(R)TTLLAGDIEHPR(K)TVLWPNGLSLDIPAGR

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TABLE II.

Cell line Phenotype Kd (pM) Bmax (fmol/106cells) #receptors/cell

MEF1 +/+ 489 40 24,008PEA10 +/- 456 9 5227PEA13 -/- -- <0 --

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David F. CarmichaelPatricia R. Segarini, James E. Nesbitt, Dongxia Li, Lara G. Hayes, John R. Yates III and

is a receptor for connective tissue growth factor (CTGF)The low density lipoprotein receptor-related protein/alpha 2-Macroglobulin receptor

published online August 22, 2001J. Biol. Chem. 

  10.1074/jbc.M105180200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

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