exogenous receptor-associated protein binds to two distinct sites

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 21, Issue of May 27, pp. 15172-15178, 1994 Printed in U.S.A.

Exogenous Receptor-associated Protein Binds to Two Distinct Sites on Human Fibroblasts but Does Not Bind to the Glycosaminoglycan Residues of Heparan Sulfate Proteoglycans*

(Received for publication, January 6, 1994, and in revised form, March 11, 1994)

Gerard VassiliouS and Keith K. Stanley From the Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, New South Wales 2050, Australia

We have investigated the proposal that the receptor- associated protein (RAP) of the low density lipoprotein receptor-related protein/az-macroglobulin receptor binds to heparan sulfate proteoglycans (HSP). ‘2sI-RAP binds to two sites on the surface of fibroblasts as follows: a high affinity site with a Kd of 1.4 MI and a low affinity site (K, = 188 MI) with a capacity of more than 1000-fold the maximum amount of lipoprotein receptor-related protein/au,-macroglobulin receptor on the cell surface. ‘2gI-RAP binding to the low affinity site was abolished by heparin or Suramin. However, maximal digestion of the glycosaminoglycan chains of HSP with heparinase or culturing the cells in chlorate, an inhibitor of proteoglycan sulfation, did not affect the binding of ‘261-RAP or of 12SI-labeled, methylamine-activated a,-macroglobulin. Comparison of ‘2BI-RAp degradation at two different concentrations suggests that the low affinity, high capacity site on the surface of human fibroblasts partici- pates in the endocytosis of 1251-RAP. The nature of the low affinity site remains to be elucidated, but we can exclude the glycosaminoglycan chains of HSP.

~~~~~~~~~~~~

The low density lipoprotein receptor-related protein/a,-macroglobulin receptor (LRP/a,MR)’ is a membrane glycoprotein of about 600 kDa that shares a striking homology with the LDL receptor (1). The LRP/a,MR contains four clusters of 2, 8, 10, and 11 cysteine-rich, class A repeats, similar to the seven repeats in the LDL receptor that are responsible for binding of LDL and apolipoprotein E containing lipoproteins (2). The LRP/a,MR binds a number of ligands, including apolipoprotein E-enriched lipoprotein remnants (3-5), lipoprotein lipase (LpL) (6), complexes between plasminogen activators and their inhibitors (7-9) and a,M-protease complexes or aZM that has been treated with methylamine (10, 11).

In addition to these ligands, a 39-kDa protein termed receptor-associated protein (RAP) co-purifies with the LRP/a,MR (12). RAP is not secreted by cells (8, 13) but associates with LRP/a,MR soon after synthesis (13) and remains with the LRP/ a,MR during receptor recycling (13, 14). RAP may therefore be considered a subunit of the LRP/a,MR. In contrast, exog-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisenent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of a National Heart Foundation ofAustralia postgraduate scholarship.

The abbreviations used are: LRP/a,MR, lipoprotein receptor-related proteidol,-macroglobulin receptor; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; HBSS, Hanks’ balanced salt solution; HSP, heparan sulfate proteoglycans; LpL, lipoprotein lipase; LDL, low density lipoprotein; a,M, a 2-macroglobulin; a,M*, activated or “fast-form” a 2-macroglobulin; RAP, receptor-associated protein; p-VLDL, p-migrating very low density lipoprotein.

enously added RAP behaves as a ligand; it binds to the LRP/ a,MR on cells (7, 9, 15-17) and inhibits the binding of all ligands to the LRP/a,MR (8,9,15,17-20). Despite this evidence that exogenous RAP may modulate ligand binding to the LRP/ a,MR, its physiological function is yet to be demonstrated.

Recently, it has been shown that lipoproteins enriched with apolipoprotein E or LpL bind to heparan sulfate proteoglycans (HSP) on the cell surface and are delivered to the LRP/a,MR (or, in some cases, the LDL receptor) with subsequent endocytosis (21-24). Chappell et al. (16) have proposed that RAP also binds to HSP based on the observation that RAP inhibits the cell-surface binding of LpL (HSP contribute the majority of this binding) and on the evidence that RAP, like LpL, is a heparin- binding protein (25).

We report here that RAP binds to a low affinity, high capacity site on human fibroblasts in addition to binding to the LRP/ a,MR. RAP binding to this site is inhibited by heparin and Suramin as would be expected for a HSP-binding protein. How- ever, treatment of the cells with heparinase or inhibition of glycosaminoglycan sulfation does not alter RAP binding. We conclude that RAP does not bind to the glycosaminoglycan chains of HSP.

MATERIALS AND METHODS Ligands-a,-Macroglobulin was purified from human plasma by

zinc-chelate chromatography according to the method of Porath et al. (26). RAP-glutathione S-transferase was prepared as described by Herz et al. (X), from a RAP-glutathione S-transferase-pGEX construct gen- erously supplied by J. Herz. The RAP-glutathione S-transferase was cleaved by incubating 1 mg of RAP-glutathione 5’-transferase, dissolved in 20 mM Tris-HCI, 30 mM NaCI, pH 7.6, with 30 NIH units of human a-thrombin (American Diagnostica Inc., New York, N Y ) for 1 h at 37 “C, and the RAP was purified from the glutathione S-transferase portion by heparin-agarose chromatography as follows: the RAP and glutathione S-transferase mixture was loaded onto a 5-ml (bed-volume) heparin- agarose column (KEM EN TEC, Resourceful Science, Copenhagen, Den- mark), equilibrated with 20 mM sodium citrate, 30 mM NaCl, pH 6.0, and the column was washed with this buffer. The glutathione S-transferase elutes in this wash. The RAP was then eluted from the column with a linear salt gradient from 30 to 1500 mM in 20 mM citrate, pH 6.0. a,M (1 mg) was activated overnight at room temperature in 250 p1 of 10 m~ citrate, 20 mM sodium phosphate, 50% (v/v) glycerol, 400 mM methylamine, pH 7.4. The a,M* was shown to be in the activated or ”fast- form” (27) by nondenaturing polyacrylamide gel electrophoresis. The a,M* was radiolabeled for 30 s with 1 mCi of NalZ5I (Amersham Corp.) using a borosilicate glass tube coated with 25 pg of Iodogen reagent (Pierce Chemical Co.). The unincorporated radiolabel was removed by Sephadex G-25 chromatography followed by extensive dialysis (6 changes of 1 liter each) of the Iz51-a,M* at 4 “C against nitrogen-sparged phosphate-buffered saline containing 0.1% Chelex (Bio-Rad). The specific activity of the Iz5I-azM* was normally between 500 and 1000 cpm/ng of protein, and more than 98% of the radioactivity was tricarboxylic acid-precipitable. RAP was radiolabeled with IODO-BEADS (Pierce) as described (16) to retain normal binding, and the unincorporated radiolabel was removed by chromatography and dialysis, exactly as described for 1251-a,M*. The specific activity of ’251-RAP was normally about 1500-2000 cpdng, and more than 98% of the radioactivity was

15172

W Binding to Human Fibroblasts 15173

tricarboxylic acid-precipitable. F'rior to use, both '=I-RAP and 1z51-aL;M* were filtered through 0.2 pm (low protein binding) Acrodisc filters (Gelman Sciences).

Human Fibroblast Culture-Primary human skin fibroblasts were obtained from the forearm of normal subjects and used before the 10th passage. The fibroblasts were seeded into 24-well plates (16-mm diam- eter dishes) and grown in DMEM (Flow Laboratories, Irvine, Scotland) with 8 mM NaHCO,, 20 mM Hepes pH 7.45 and containing 10% bovine fetal calf serum (Flow) and 50 IUfml penicillin, 50 pgiml streptomycin (Sigma). The cells were grown in a 37 "C incubator with 2% CO, until they were about 90% confluent. The cells were fed with fresh DMEM media 2 days before they were used.

Cell-surface Binding of 1251-a$f* and 1251-RAl"Prior to the binding assay, the cells were washed 1 time at 37 "C in 1 ml of HBSS, 25 mM Hepes, 2 mglml of BSA, pH 7.45 and then incubated in 1 ml of the same medium at 37 "C for 10 min. The cells were placed on ice for 15 min. The radioiodinated ligands, 1251-a2M* or '251-RAP, were added to the cells at the concentrations indicated in the relevant figures, dissolved in 250 pi of HBSS, 25 m~ Hepes, 2 mglml BSA, pH 7.45. lzsI-ol,M* and 1251-RAP were incubated with the cells on ice for 2 and 3 h, respectively. Non- specific binding was determined in the presence of a 30-50-fold excess of unlabeled ligand. The cells were washed 6 times (with 1 ml each time) with ice-cold HBSS, 25 m~ Hepes pH 7.45. The last 2 washes were for 5 min each. The cells a t this stage have been shown to he greater than 99% viable according to the trypan blue exclusion assay. After the final wash, all the medium was removed from the wells, and the cells were solubilized in 200 pl of 100 rn NaOH and agitated on a rotary platform for 2 h. Following this, 1 ml of the Bicinchoninic acid protein reagent (Sigma) was added to the wells, and the plates were agitated on a rotary platform for 10 min and then incubated at 50 "C for 45 min to develop the color. For a standard comparison, 0-50 pg of BSA was simulta- neously measured by the same method. The radioactivity of 1 ml of the solution in each well was measured (to determine the amount of ligand bound), and the absorbance of 150 p1 of this solution was measured at 550 nm in a 96-well titertek plate to determine the cellular protein content, which was generally 20-30 pg/well. Specific binding (total minus nonspecific) was used for the Scatchard plots, and the binding parameters were determined by the nonlinear, least squares curve- fitting program LIGAND (28) using a molecular weight of 720,000 and 40,000 for a,M and RAP, respectively. For studies of the inhibition of ligand binding by polyanions, either porcine intestinal heparin (163 unitdmg) from Calbiochem or Suramin (Bayer) were dissolved in the ligand-containing medium at the concentrations indicated in the relevant figures, and ligand binding was assayed as described.

Degradation of'2sI-a$f* or 1251-RAP by Human Fibroblasts-Prior to the degradation assay, the cells were washed at 37 "C with 1 ml of HBSS, 25 mM Hepes, 10 mg/ml BSA at pH 7.45. The cells were incubated in this medium for 10 min at 37 "C. The cells were then incubated at 37 "C for up to 8 h in 500 p1 of HBSS, 25 mM Hepes pH 7.45,lO mgiml BSA containing the indicated concentrations of rz51-a2M* or 1251-RAl? At the beginning of the incubation period and at each time point, 450 pl of the medium was transferred to a microcentrifuge tube containing 150 pl of ice-cold, 45% tricarboxylic acid. This material was immediately mixed by inversion and kept on ice for 1 h. The rest of the medium was removed from the wells, the cells were washed in ice-cold buffer, and the amount of cell-associated radioactivity was determined exactly as described for the cell-surface binding assay. The tricarboxylic acid-precipitated proteins were centrifuged at 16,000 x g for 4 min, and the radioactivity of 500 pl of the supernatant (tricarboxylic acid-soluble material) was measured.

Deatment of Cells with Heparinuse-Human fibroblasts were cultured as described above and then grown for 48 h in Basal medium EagleMam's F-12 (Sigma) buffered to pH 7.45 with 15 m x Hepes, 6 mM NaHCO, at 37 "C, 2% CO,. The cells were treated with heparinase as described (22). Briefly, the cells were washed 4 times at 37 "C in HBSS, 25 mM Hepes pH 7.45 containing 10 mglml BSA, then incubated for 45 min at 37 "C with up t o 2 unitdml of heparinase (E.C. 4.2.2.7) from Sigma, dissolved in DMEM, 25 mM Hepes pH 7.45, 10 mg/ml BSA. The cells were again washed 4 times at 37 "C in HBSS, 25 mM Hepes pH 7.45, containing 2 mgiml BSA and then cooled to 3 "C and 1251-a2M* f 1 pgiml) or '%RAP (0.26 pgiml) binding was assessed as described above. The efficiency of heparinase treatment was determined as follows: the cells were labeled with 20 pCi/ml of [36S]sulfate (Amersham) 36 h before the binding experiment. Upon heparinase treatment, the %-labeled material, which was released into the media, was expressed as a per- cent of the total incorporated radiolabel, After washing the cells twice in phosphate-buffered saline, pH 7.5, the remaining cell-surface ,%-labeled material was released by digestion with 0.25% trypsin for 15 min

0 0 1 2 3 4 5

t h e 0 FIG. 1. Time dependent binding of radioiodinated a&* and

bated at 3 "C with 1 pg/ml 1251-a&* or lSI-RAP in HBSS, 25 mM Hepes, RAP to human fibroblasts at 3 "C. Human fibroblasts were incu-

2 mgfd BSA, pH 7.45. After the time indicated, the cells were washed 6 times in HBSS, 25 mM Hepes pH 7.45 to remove unbound ligand, and the bound radioactivity and cellular protein were measured. The total binding (0) of lasI-a,M* (A) or '251-RAP ( B ) is the mean of three measurements, and the error bars indicate the S.D., which lie within the symbols in some instances. Nonspecific binding (0) was a single meas- urement a t each time point in the presence of a 30-fold excess of unlabeled a,M* or in the presence of 1 mgimt Suramin (Fig. lB).

at 37 "C. This treatment releases the cells from the tissue culture dishes, so the cells were transferred to a microcentrifuge tube and centrifuged at 500 x g for 5 min, and then the supernatant (trypsin- released) and pellet (cell-associated) radioactivity were measured sepa- rately.

Inhibit~on of Sulfation of Cellular ProteogZycans-The sulfation of cellular proteoglycans was inhibited for 48 h with up to 20 mglml sodium chlorate, as described (29). 1251-a2M* (1 pg/ml) or '251-RAP (0.26 pg/mV binding was assessed as described above. The efficiency of chlorate treatment was assessed by labeling with [35Slsulfate for the last 36 h of chlorate treatment. The amount of labeled material released with 2 unitdml heparinase was then determined.

Metabolic Labeling and Immune Precipitation-Human fibroblasts were metabolic~ly labeled using 100 pCL'm1 35S-Trans label (DuPont NEN) as described (30). L ~ P / f f ~ ~ was immune-precipitated with 3 ~1 of polyclonal anti-COOH terminal LRP/a,MR antiserum (1) or 3 1-11 of anti" (13) and with 30 pl of a 1:l slurry of protein A-Sepharose (Bio-Rad).

RESULTS 1251-RAP Binds to lzoo Distinct Sites on Human Fibro-

blasts-In order to compare the binding of RAP and a,M*, we first established that each ligand attained near-equilibrium binding. The amount of '251-a,M* bound to human fibroblasts reached a plateau after 2 h (Fig. lA), while lpsI-RAP binding was slightly slower (Fig. 1B). Therefore, in the following experiments, 1251-(u,M* was bound to cells for 2 h, while lZ5I-R,AP was bound for 3 h. The proportion of nonspecific binding is similar for both ligands.

In 24 separate experiments, using primary skin fibroblasts from four different subjects, we found that the maximum binding capacity for 1z51-~,M* varied from 22 to 66 fmol/mg of cell protein, with a mean of 37 fmol/mg of cell protein and a S.D. of 12 fmol/mg of cell protein. Therefore, to compare 1251-~zM* and lzsI-RAP binding, these ligands were bound to identical cul-

15174 RAP Binding to Human Fibroblasts

0.12

0.1

0.08

0.06

0.04

0.02

0

~~

0.14

0.12 0.1

300[1 0.08

2m 0.06

0.04 1000 0.02

0 0 10 20 30 0 2wo 4m 6Mo

0

'25I-Ligmd (pg/ml) BI-Ligand bound

(fmol/mg cell protein)

FIG. 2. Binding of radioiodinated a,M* and RAP to human fibroblasts at 3 "C. Human fibroblasts were incubated at 3 "C with the

HBSS, 25 mM Hepes, 2 mg/ml BSA, pH 7.45. After the incubation indicated concentrations of lzsI-a,M* (for 2 h) or lZsI-RAP (for 3 h), in

period, the cells were washed as described in Fig. 1, and the bound radioactivity and cellular protein were measured. The total binding (0) of 'z51-a,M* (A) is the mean of three measurements, and the total binding of lEI-RAP (C) is the mean of six measurements from two experiments. Nonspecific binding (0) was determined at each ligand Concentration in the presence of a 30-fold excess of unlabeled a2M* (A) or a 50-fold excess of unlabeled RAP (C). The error burs indicate the S.D., which lie within the symbols in some instances. The Scatchard plots are drawn for the specific binding (total binding minus nonspecific binding) of 12sI-a,M* ( B ) or lz'I-RAl' (D) , and the parameters of binding have been determined by the LIGAND computer program. 12sI-a,M* binds to a single site with a Kd of 0.32 nM and a B,,, of 43 fmoVmg of cell protein. The curve drawn through the lZ5I-RAP Scatchard plot (D) is the sum of 12sI-RAP binding to two populations of sites (D), which are indicated by dotted lines on the graph.

tures of human fibroblasts in the same experiment (Fig. 2). The binding isotherm for lZ5I-a,M* to the cells (Fig. 2 4 ) approached saturation, and the straight-line Scatchard transfo~ation of this data (Fig. 2B ) indicated a single class of binding sites with a Kd of 0.32 nM and a B,,, of 43 fmolimg of cell protein. This value corresponds to 15,200 binding siteskelf, as we have determined that there are 1.7 x lo6 human fibroblastsimg of cell protein. Similar dissociation constants (0.2-0.54 nM) and maximum binding capacities (10,500-15,000 siteskell) have been reported for a2M* binding to human fibroblasts in previous studies (32, 33). The specific binding of lZ5I-RAP attained saturation above 20 yg/ml (Fig. 2C). Scatchard transformation of the "%RAP binding data resulted in a curvilinear plot that was analyzed by the computer program LIGAND. The best-fit of this data necessitated the assumption of two distinct binding sites, including a high affinity, low capacity binding site that bound lZ5I-RAP with a Kd of 1.4 nM and a B,, of 226 fmoL'mg of cell protein. Asimilar Kd of 3.2 ( 2 0.9) nra has been reported for lZ5I-RAP binding to the LRP/a,MR on rat MH,C, hepatoma cells (34), suggesting that the high affinity site is the LRP/ a,MR. However, the B,,, of this site was over &fold greater than the B,, for a2M* binding. '251-RAP also bound to a low affinity, high capacity binding site with a Kd of 188 nM and B,,, of 6100 fmolimg of cell protein. This value is more than 1000- fold higher than the maximum amount of LRP/a2MR on the cell surface (based on the binding of lZ5I-a2M*). Thus the low affinity, high capacity binding site for lZ5I-RAP cannot be the LRP/ a2MR.

The Low Affinity, High Capacity Site Internalizes Iz5I- W - W e next addressed the question of whether the low af-

i g 0 2 4 6 8 0 2 4 6 8

C RAP (ceil associated)

.*

0 2 4 6 8 0 2 4 6 8

Time (h)

a#* and RAP by human fibroblasts at 37 "C. Human fibroblasts FIG. 3. CelI association and degradation of radioiodinated

were washed at 37 "C to remove any residual fetal calf serum. The cells were incubated at 37 "C for the indicated times in HBSS, 25 m Hepes pH 7.45, 10 mgiml BSAcontaining 1.4 nM 1251-a&* or 1.4 t l ~ '2sI-R.AI? At the beginning of the incubation period and at each time point, the medium was precipitated with ice-cold tricarboxylic acid, and the tricarboxylic acid-soluble material was used as a measure of the degraded protein ( B and D, 0). The cells were then washed, and the cell-associated radioactivity was measured as described for the cell-surface binding assay (A and C, *). Background cell association and degradation (0) were measured either in the presence of 2 mgiml Suramin or in the absence of any cells, and both measures were essentially the same. Note that about 10-fold more lzsI-RA.P is degraded compared with lzsI-a,M*.

finity, high capacity site for RAP binding a t 3 "C was able to endocytose 1251-RAP leading to its degradation. Cells were incubated over 8 h with equimolar (1.4 nM) lZ5I-RAJ? or 1251-a@*. Both ligands gave an approximately linear release of tricarboxylic acid-soluble radioactivity from the cells after an initial lag phase of 1 h (Fig. 3 , B and D). The rate of 1251-ftAp degradation was 9-fold that of 1z51-a2M* degradation, but only 4-fold more 1251-RAP bound to the cells at this concentration compared with '"I-a,M* as calculated from the measured binding parameters. The slower degradation of 1251-a2M* cannot be explained by its resistance to proteolysis (31), since the cell-associated radioactivity attains steady state; if the lysosomal degradation of 12'I-a,M* was the rate-limiting step, it would continue to accumulate in the cells, We next compared the steady-state rates of lZ5I-RAl' degradation by cells at 1.4 and 25 n~ concentration of this ligand (Table I). The higher concentration was well below the Kd €or the low affinity site (188 nM) but suffl- ciently above the Kd for the high affinity site (1.4 nMf that some degree of saturation and, hence, attenuation in the rate of degradation, would be expected. At 25 nM, 6-fold more 12'f-RAP

bound to the cell surface at 3 "C compared with the amount bound at 1.4 nM (by extrapolation of Fig. 2C), although this represents an 18-fold difference in ligand concentration (Table I). Surprisingly, however, the increase in the rate of degradation (29-fold) exceeded the increase in ligand concentration (18- fold). Thus, an increase in the rate of degradation of 1251-RAP was observed when proportionally more 12sI-RAP bound to the low affinity site. This data demonstrates that the low afflnity site not only internalizes RAP but may do so more efficiently than the high afflnity (LRP/a~MR) site.

RAP Binding to Human Fibroblasts 15175

TABLE I Cell surface binding and degradation of1251-RAP

The values of 1251-RAp binding were measured from interpolation of Fig. 2C. The degradation rates of '251-RAp were determined as described in the legend to Fig. 3. The values for cell-surface binding and degradation are the mean of six and three measurements, respectively, *1 S.D.

Concentration Cell-surface binding Degradation

n.w fmollmg cell fmollmg cell

1.4 protein protean Ih

25 160 f 40 930 f 172

272 f 4.8 7819 2 119

Ratio 18 6 29

Influence of Heparin and Suramin on '"I--.&* and lZ5I-RAP Binding to Human Fibroblasts-It has been proposed that in addition to the LRP/a,MR, RAP may bind to HSP (16). Most ligands that bind to HSP do so via the anionic clusters of sulfate groups of the glycosaminoglycan residues (for review, see Ref. 35), and binding is therefore inhibited by polyanions such as heparin or Suramin. 1251-a2M*, which does not bind to the glycosaminoglycan residues (22, 36) is not inhibited by up to 10 mg/ml(l630 unitdml) of heparin (Fig. 4.4 ). In addition, up to 20 mg/ml of dextran sulfate did not affect lZ5I-a,M* binding (data not shown). In contrast, 1251-a2M* binding was reduced to background levels with 250 pg/ml Suramin (Fig. 4B). This inhibition was not due to denaturation of the LRP/a,MR, since pretreatment of the cells with up to 1 mg/ml Suramin in HBSS, 25 mM Hepes pH 7.45, for 1 h a t 3 "C, followed by removal of the Suramin by washing the cells twice in HBSS, 25 mM Hepes pH 7.45, at 3 "C, did not alter 1251-~,M* binding (data not shown). lZ5I-RAP binding was inhibited by both heparin and Suramin (Fig.4, C and D ) , and binding of 1251-RAP was reduced to background levels with 125 pg/ml Suramin. Binding of 1251-RAP in the absence of the polyanions was about 350 fmoVmg of cell protein of which at least 50% must be to the low affinity site, as calculated from the measured binding parameters. Therefore, inhibition of lZ5I-RAP binding to the LRP/a,MR (15, 18) cannot account for the extent of inhibition we observed, indicating that '251-RAP binding to the low affinity, high capacity site must also be inhibited by heparin and suramin.

1251-RAP Does Not Bind to the Glycosaminoglycan Chains of HSP-If RAP binds to HSP, then it should be possible to abolish most of this binding by digestion of the HSP with heparinase. Conditions were chosen that abolish binding of apolipoprotein E-enriched p-VLDL to cells (22). As previously reported (22, 361, heparinase treatment of the human fibroblasts had no effect on 1251-a,M* binding (Fig. 5 A ) , indicating that the LRP/ a,MR was not adversely affected by this treatment. When tested on the same cells, lZ5I-RAP binding was also found to be unaffected by heparinase treatment (Fig. 5B).

To verify that heparinase treatment resulted in the digestion of surface HSP, one flask of cells was labeled with [35S]sulfate, and heparinase activity assayed by release of surface incorporated radioactivity. Approximately 40% of the total cell-associated 35S-labeled material was released into the medium (Fig. 5C), comparable with the earlier studies (22). The plateau in release of 35S-labeled material also indicates that the majority of surface HSP had been removed.

A further 40% of the cell-associated ?S-labeled radioactivity could be released by protease digestion (Fig. 6). This was pre- sumably present in dermatan sulfate and chondroitin sulfate, which constitutes about 30% of the total sulfated proteoglycans in fibroblasts (37) and would not be digested by heparinase. To test whether dermatan sulfate or chondroitin sulfate could be responsible for RAP binding, we grew cells in chlorate, which inhibits the enzyme sulfate adenylyltransferase that is neces-

100

75 t

0 0 2 4 6 8

Heparin (mg/ml)

B a2M + Suramin I

D RAP + Suramin

I I

0 0.2 0.4 0.6 0.8 1 Suramin (mg/ml)

FIG. 4. Influence of heparin and Suramin on lzaI-a&"* and lmI- RAP binding. Human fibroblasts were incubated at 3 "C with 1 pg/ml 1251-a2M* (A and B ) or 0.26 pg/ml lZ5I-RAp (C and D) in HBSS, 25 mM Hepes, 2 mg/ml BSA, pH 7.45, in the presence of the indicated concentrations of heparin or Suramin. After 2 h, the cells were washed 6 times, and the cell-associated radioactivity was measured as described in Fig. 1. The means of three measurements are plotted, and the S.D. are shown. Nonspecific binding has not been subtracted, but this was usu- ally about 15% of total binding. Control values, in the absence of heparin or Suramin, were 352 (f15) fmol/mg of cell protein for 1251-RAp binding and 33 (f8.1) fmol/mg of cell protein for 1251-a2M* binding.

sary for sulfation of all proteoglycans (29). Blocking sulfation of proteoglycans by chlorate treatment does not affect 1251-a,M* binding (Fig. 7A) nor does it affect lZ5I-RAP binding (Fig. 7B) even when HSP sulfation (measured by heparinase release) was reduced by 60% (Fig. 7C).

Most of the Endogenous RAP Is Associated with LRPI a&R-Having shown that exogenously added RAP can bind to a site other than the LRP/u,MR, we investigated whether endogenous RAP was associated with other proteins. Human fibroblasts were metabolically labeled, and the LRP/a,MR and RAP were immune-precipitated. The anti-COOH-terminal LRP/a,MR antibody immune-precipitates the 515- and 85-kDa subunits of the LRP/a,MR as expected (Fig. 8, lane 1 ) . In addition, a faint band of RAP was visible. RAP was poorly labeled, because it contains a single methionine and no cysteines. When RAP was immune-precipitated using anti" serum, the LRP/a,MR was found to co-precipitate (Fig. 8, lane 2 ) . The similar intensity of the LRP/a,MR and RAP bands immune- precipitated with both antibodies confirms that the majority of LRP/a,MR molecules have RAP bound and that most of the cellular RAP is associated with LRP/a,MR.

DISCUSSION Previous studies have demonstrated that exogenously added

RAP binds to the cell-surface LRP/a,MR in a variety of tissue culture cell lines (7, 9, 15-17). Our results using primary cultures of human skin fibroblasts show that RAP binds to two distinct sites on these cells. This includes a high affinity, low capacity site that bound RAP with a Kd = 1.4 I", similar to the Kd of 3.3 nM previously reported for human RAP binding to rat MH,C, cells (34) but lower than 4-20 I", which has been reported (14) for RAP binding to purified LRP/a,MR. It is likely


0 0.5 1.0 1.5 500 '

I

0 0.5 1.0 1.5 2

n 1

R 0

0 0.5 1.0 1.5 2 Heparinase (U/ml)

FIG. 5. Heparinase treatment of human fibroblasts does not alter the binding of 1251-~,M* or '251-RAp to these cells. Heparinase was dissolved in DMEM, 25 mM Hepes pH 7.45, 10 mg/ml BSA and immediately incubated with the fibroblasts for 45 min at 37 "C. The cells were then washed to remove residual digested proteoglycans and heparinase and were then cooled to 3 "C. The cell-surface binding of 1 pg/ml 1251-o(2M* (A) or 0.26 pg/ml 1251-RAp ( B ) was measured as described in Fig. l. Each value for ligand binding (A and €3, .), is the mean of three measurements. Background binding (0) is a single measure- ment at each concentration in the presence of 1 mg/ml Suramin. C, the release of 35S-labeled radioactivity following treatment with heparinase. Cells were labeled with [35Slsulfate and incubated with heparinase as for panels A and B . At higher concentrations of heparinase, more %-labeled radioactivity is released into the media upon digestion with heparinase (A), indicative of the digestion of sulfated glycosaminoglycan residues of heparan sulfate proteoglycans.

that the purified receptor binds RAP with lower affinity, as it binds a,M* with lower affinity (12). The Kd for RAP binding to the high affinity site and the evidence that RAP binds to the LRP/a,MR on human skin fibroblasts suggest that this site is the LRP/a,MR. However, the B,,, of the high affinity site was 226 fmol/mg of cell protein, which was just over &fold the maximal binding of a,M*. This agrees with Iadonato et al. (34) who reported 5-7 times more RAP binding to MH,C, cells compared with the binding of tissue plasminogen activator, which also binds to the LRP/a,MR. Although two molecules of RAP bind to the purified LRP/a,MR (14), one RAP molecule is al- ready bound to this cell-surface receptor (14, E ) , so a stoicho- metric binding of RAP and a2M* should be expected. Our results imply that up to five RAP molecules can bind each molecule of LRP/a,MR, as Iadonato et al. (34) have also proposed. Alternatively, a,M* may bind only to a fraction of the LRP/a,MR molecules on the cell surface or RAP may bind to other receptors that cannot be distinguished from the LRP/ a,MR by cell-surface binding experiments.

RAP has been proposed as a modulator of a,M* binding to LRP/a,MR, as exogenously added RAP inhibits binding of all ligands. Removal of endogenously bound RAP would therefore

40

30

20

10

0

I T

T

I I

FIG. 6. Release of 36S-labeled radioactivity following treatment of human fibroblasts with heparinase and trypsin. Cells were labeled with [35Slsulfate and incubated with the indicated amounts of heparinase for 45 min at 37 "C as in Fig. 5 or with 0.25% trypsin for 15 min at 37 "C. About 20% of the radioactivity remains cell-associated after heparinase and trypsin treatment (Hep/Tryp-resistant). Each value plotted is the mean of four measurements. The error bars show the S.D.

be predicted to increase a,M* binding. We find that while heparin or Suramin treatment of cells effectively removes the exogenously bound RAP, and these polyanions also remove endogenous RAP, no increase in a,M* binding occurs upon treatment of the cells with heparin or pretreatment with Sur- amin. This implies that endogenous RAP, which is associated with LRP/a,MR on the cell surface, does not modulate a,M* binding.

RAP also bound to a low affinity, high capacity site (K, = 188 nM, Bmax = 6100 fmol/mg of cell protein). In contrast to our results, a single class of high affinity binding sites for RAP were observed on rat MH,C, cells (341, but this could be due to a species or cellular difference. The curvilinear Scatchard plot of RAP binding to human skin fibroblasts could not be explained by negative cooperativity, as the total amount of RAP bound is in excess of 1000-fold the amount of LRP/a,MR on the cell surface.

What is the molecular identity of the second binding site for RAP on the surface of primary skin fibroblasts? Gp330, another member of the LDL receptor family that is similar in structure to the LRP/a,MR (39), is known to bind RAP (20) but not a,M* (40). It might, therefore, account for an excess of RAP binding with respect to a,M*. Fibroblasts, however, do not express gp330 (17). The LDL receptor can also be eliminated as a can- didate for the low affinity, high capacity site, as it does not bind RAP (15). A further possibility is that RAP could bind directly to the plasma membrane, since RAP contains two regions with potential for forming amphipathic helices (13), which have been proposed to bind to the plasma membrane in the mouse homologue of RAP, HBP-44 (25) . However, if RAP was bound directly to the lipid bilayer of the plasma membrane, it is un- likely to be degraded at the high rate we have observed or to be inhibited by heparin or Suramin.

LpL and apolipoprotein E-enriched P-VLDL bind to cells with a vast molar excess compared with ",MY, since these

RAP Binding to Human Fibroblasts 15177

80 70

a2M A

30 20

lo 0 1 0 5 10 15 20

300

0' I 0 5 10 15 20

Y I

0 5 10 15 20 Chlorate (mM)

FIG. 7. Inhibition of proteoglycan sulfation does not alter the binding of 'zsI-(u&M* or 12sI-RAF' to human fibroblasts. Cells were cultured for 48 h in DMEMMam's F-12 containing up to 20 mM sodium chlorate. The cells were washed to remove residual chlorate and fetal calf serum and were then cooled to 3 "C. The binding of 1 pg/ml "'I- a,M* (A) or 0.26 pg/ml 12'I-RAP ( B ) was assessed as described in Fig. 1. To assess the efficiency of chlorate treatment, the cells were labeled with ["Slsulfate and then the labeled material was released with 2 unitdml heparinase. At higher concentrations of chlorate, less [3sSlsul- fate is incorporated into HSP and, therefore, there is less released upon heparinase treatment. Each value for ligand binding (A and B, O), is the mean of three measurements. Background binding (0) is a single meas- urement of binding in the presence of 1 mg/ml Suramin. The amount of "S-labeled radioactivity released upon heparinase treatment (C , 0) is the mean of three measurements. The error bars show S.D.

ligands bind to HSP (21-24). This explains the stimulation of /3-VLDL binding and endocytosis when the lipoprotein is enriched with apolipoprotein E or with LpL (4,6), since the high capacity binding to HSP would increase the concentration of the ligand in the vicinity of the receptor and therefore enhance the rate of endocytosis. We have tested the proposal (16) that RAP also binds to HSP. RAP binds to a low affinity, high capacity site with a Kd of 188 nM, which is around the range of Kd values reported for other proteins that bind to HSP (41-43). Furthermore, the B,,, of the low affinity site (2.2 x lo6 sites/ cell) is similar to the binding capacities that have been reported for HSP-binding proteins (41, 43).

RAP binding was sensitive to heparin or Suramin, indicating that the binding could be via the heparin binding domain of RAP as would be expected if RAP was binding to the glycosaminoglycan chains of HSP (35). However, RAP binding was not affected by heparinase treatment of the cells even at an extent of glycosaminoglycan digestion (40%) that abolished the binding of apolipoprotein E-enriched /3-VLDL to the cell surface (22). Similarly, RAP binding was not affected by chlorate treatment of the cells, which reduced HSP sulfation by 60%. Al- though 40% of the HSP sulfate groups are still present, one

200 - 92.5 -

69 - 46 - 30 -

I 4

4

L J

and LRP/a,MR Human fibroblasts at 90% confluence, in 60-mm Petri FIG. 8. Metabolic labeling and immune precipitation of RAP

dishes were metabolically labeled with %Trans label for 4 h at 37 "C. The cells were then washed and cooled on ice and solubilized with Nonidet P-40 in the presence of protease inhibitors. The nuclei and cytoskeleton were removed by low speed centrifugation. The solubilized membrane proteins in the supernatant were immune-precipitated using protein A-Sepharose as the immunoadsorbent and with polyclonal anti-COOH-terminal LRP/a,MR antiserum (lane 1 ), polyclonal anti RAP (lane 2), or with the immunoadsorbent in the absence of any antibody (lane C) . After washing the immunoadsorbent, the adsorbed proteins were eluted in Laemmli sample buffer a t 95 "C for 5 min, and the proteins were separated by 4 1 2 % SDS-polyacrylamide gel electro-

arrowheads identify the 515- and 85-kDa subunits of the LRP/a,MR phoresis with 35S-labeled molecular mass markers (Amersham). The

and the 39-kDa RAP. The labeled proteins were identified by autora- diography. RAP is only faintly visible, because it has only one methionine and no cysteines.

would expect that the spacial distribution of the anionic sites along the glycosaminoglycan chains would be significantly dis- rupted with a concomitant disruption of ligand binding (35). Furthermore, since dermatan sulfate and chondroitin sulfate are more susceptible than HSP to inhibition of sulfation by chlorate (371, we conclude that the glycosaminoglycan chains of these proteoglycans, in addition to HSP, do not bind RAP.

This result is consistent with the observation of Nykjaer et al. (8 ) that RAP does not inhibit the cell association of LpL (which is known to bind to HSP) on human monocytes. Never- theless, RAP may inhibit LpL binding to human fibroblasts (consistent with Chappell et al. (16)) by binding to a core protein of a HSP and altering the conformation of the glycosaminoglycan chains or sterically interfering with LpL binding. A number of proteoglycan core proteins have been shown to me- diate the binding and endocytosis of ligands; a core protein of a fibroblast HSP was found to have properties similar to the transferrin receptor (44), and an iduronic acid-rich proteoglycan from fibroblasts binds fibronectin (45). It is noteworthy, in this example, that the binding was via the heparin binding domains of fibronectin. In addition, an approximately 400-kDa core protein of an HSP, termed Perlecan (46) is expressed by a variety of cell types including fibroblasts (47,481 and contains four cysteine-rich, class A motifs, similar to the repeats in the LRP/a,MR, which are reported to bind RAP (38).

By metabolic labeling and immune precipitation, we find that most of the endogenous RAP is associated with the LRPI a,MR and not with a second site. In contrast, exogenous RAP binds to a site in addition to the LRP/a,MR, but this site is not the glycosaminoglycan residues of HSP as previously proposed. The physiological relevance, if any, of the low affinity RAP binding site remains to be determined; it is possible that endogenous RAP remains attached to the LRP/a,MR but interacts


transciently with the low affinity site. In any case, given that we know very little of the function of RAP, and RAP is used extensively as a tool to study the LRP/a,MR, the nature of the low affinity, high capacity RAP binding site deserves further investigation.

Acknowledgments-We thank Dudley Strickland for providing a,M, which was used during the early stages of this investigation, and for providing RAP antisera; Joachim Herz for providing the W-glutathione S-transferase-pGEX construct; Nicholas Dudman for the human fibroblast cultures; and Adrian Zammit for helpful discussions regard- ing the chlorate experiments.

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exogenous receptor-associated protein binds to two distinct sites

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