Proc. Natl. Acad. Sci. USA

Vol. 76, No. 5, pp. 2311-2315, May 1979 Cell Biology

Rate and equilibrium constants for binding of apo-E HDLC (a cholesterol-induced lipoprotein) and low density lipoproteins to human fibroblasts: Evidence for .multiple receptor binding of apo-E HDLC (cholesterol metabolism/low density lipoprotein receptors/atherosclerosis)

R. E. PITAS*, T. L. INNERARITY*, K. S. ARNOLD*, AND R. W. MAHLEY11 *Meloy Laboratories, Inc., 6715 Electronic Drive, Springfield, Virginia 22151; and tLaboratory of Experimental Atherosclerosis, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20205

Communicated by Donald S. Fredrickson, February 12, 1979

ABSTRACT Competitive binding assays have demonstrated that a cholesterol-induced canine lipoprotein containing only the E apoprotein (apo-E HDLc) binds to the same cell surface receptors of human fibroblasts as human low density lipoproteins (LDL). However, the apo-E HDLC have a much greater binding activity than LDL. Equilibrium and kinetic binding studies were conducted at 40C to determine the mechanism for this enhanced receptor binding activity. Based on the data, the binding of both LDL and apo-E HDLC appears to be a simple bimolecular receptor interaction, and no heterogeneity of binding sites or cooperative effects among the receptor sites were observed. Equilibrium dissociation constants determined by Scatchard analysis of the equilibrium binding data for apo-E HDLC (Kd = 0.12 X 10-9 M) and LDL (Kd = 2.8 X 10-9 M) revealed a 23-fold greater affinity of HDLC for the receptors. Association and dissociation rate constants for the lipoproteinreceptor complex were determined from the time course of binding at various lipoprotein concentrations. The equilibrium dissociation constants calculated from these kinetic data confirmed that apo-E HDLC had a much higher affinity for the receptor than LDL. Furthermore, the kinetic studies indicated that apo-E HDLC bound more rapidly than LDL with rates of association of 18.0 X 104 and 5.5 X 104 M-1 sect, respectively. The rate of dissociation of the apo-E HDL,-receptor complex (1.7 X 10-5 sec') was slower than that of the LDL receptor complex (6.3 X 10-5 sec'). An additional important difference between the binding of apo-E HDLC and LDL was that 4 times (3.6 i 0.4) as many LDL particles as HDLC particles were required for saturation of the receptors at maximal binding. These Eaa indicate that each HDLC particle binds to multiple cell surface receptors at a ratio of 4:1 for LDL receptor binding. Cell surface receptors-that bind specific plasma lipoproteins have been identified on the plasma membrane of cultured human fibroblasts and extensively studied (for review see ref. 1). Two distinct classes of plasma lipoproteins have been shown to interact with these high-affinity cell surface receptors. These are the apo-B-containing low density lipoproteins (LDL) and certain high density lipoproteins (HDL1 and HDLC) that contain the arginine-rich apoprotein (apo-E) (2-4). Previous studies have indicated that both classes of lipoproteins (the B- and E-containing lipoproteins) bind to the same cell surface receptors (4). Furthermore, we have shown that selective modification of a limited number of the lysyl or arginyl residues of the apo-B of LDL and the apo-E of HDLC results in loss of the binding activity of these lipoproteins (5, 6). These data indicated that the protein moieties of LDL (apo-B) and HDLC (apo-E) are the specific binding determinants and suggested that apo-B and apo-E share a common structural sequence responsible for the lipoprotein-receptor interaction. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 2311

There is, however, a significant difference between the binding of LDL and HDLC. The apo-E HDLC§ exhibit a 100fold greater ability than LDL to displace '25I-labeled LDL (125I-LDL) from the cell surface receptors in competitive binding studies at 40C. It has been postulated (8) that the enhanced binding of apo-E HDLc could be due to a greater number of recognition sites on the HDLC molecule as compared to LDL, a greater affinity of apo-E HDLc for the cell surface receptors, or binding of the HDLc particle to more cell surface receptors than the LDL particle. In this communication, we report results of direct binding experiments which indicate that the enhanced binding of apo-E HDLc is a result of all three of these factors. MATERIALS AND METHODS Materials. Dulbecco's modified Eagle's medium (catalogue no. 430-2100) and the trypsin/EDTA solution were purchased from GIBCO. Sodium [125I]iodide (carrier free) in NaOH and Bolton and Hunter reagent [N-succinimidyl 3-(4-hydroxy 5[125I]iodophenyl)propionate] were purchased from Amersham/Searle. Heparin (porcine, grade II) and Hepes buffer were obtained from Sigma. Lipoproteins: Isolation, Characterization, and Iodination. Human LDL (p = 1.02-1.05 g/ml) were isolated from plasma by centrifugation for 18 hr at 59,000 rpm in a 60 Ti rotor. The LDL were washed by recentrifugation (p = 1.05 g/ml) for 16 hr at 59,000 rpm. Canine HDLc (p = 1.02-1.063 g/ml) and apo-E HDLc (p = 1.006-1.02 g/ml) were isolated from the plasma of foxhounds fed a semisynthetic hypercholesterolemic diet, as described (7, 9). Lipoprotein purity was determined by paper and polyacrylamide gel electrophoresis (10, 11). Canine apo-E HDLc were iodinated (125I) as described by Bolton and Hunter (12) with slight modification as described (13). Human 12I-LDL were prepared as described (14). Cells. Human fibroblasts derived from a preputial specimen from a normal infant were maintained and harvested as described (5, 8). Primary cultures were established with 9 X 104 cells in 60-mm petri dishes. The cells were maintained for 5 days in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and then allowed to grow for 2 days in medium containing 10% lipoprotein-deficient human plasma. ExperiAbbreviations: LDL, low density lipoproteins; HDLC, cholesterolinduced plasma lipoproteins; apo-E, arginine-rich apoprotein. t To whom reprint requests should be addressed. § Apo-4 HDLc, as characterized in cholesterol-fed dogs (4, 7), contain the E apoprotein as the only protein constituent, are cholesteryl ester rich, and float at much lower plasma density than do the typical high density lipoproteins. Typical HDL lack the E apoprotein and do not bind to the cell surface receptors of fibroblasts (3, 4, 8).

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Cell Biology: Pitas et al.

ments were conducted 7 days after plating. The petri dishes were placed on crushed ice in a 4VC cold room 0.5 hr before the experiment. The media were then removed and the indicated amounts of 125I-LDL or 125I-apo-E HDLC were added in Dulbecco's modified Eagle's medium (25 mM Hepes, pH 7.4) containing 10% lipoprotein-deficient human plasma. The solution containing the iodinated lipoprotein was cooled to 40C prior to use. After incubation, the plates, which remained on ice in a 4VC cold room, were rapidly washed three times with phosphate-buffered saline containing 2 mg of bovine serum albumin per ml, twice for 10 min each with phosphate-buffered saline/bovine serum albumin (2 mg/ml), and once with phosphate-buffered saline alone. This washing procedure (15) separates the cellular-bound lipoprotein from the free lipoprotein in the media. The procedure for determining the amount of specific receptor-bound lipoprotein as compared with nonspecifically bound lipoproteins is given below. Cells were transferred from the plates for protein determination and gamma counting in three successive 0.5-ml aliquots of 0.1 M NaOH. A hemacytometer was used to count cells on four to six plates from each set of plates used in the binding studies. Competitive binding studies were performed at 40C as described (8) except that the cells were incubated for 3 hr with the lipoproteins. At 40C very little internalization or degradation of 125I-LDL occurs; therefore, the 4VC assay measures only

surface-bound lipoproteins (15). Determination of Equilibrium Constants. The affinity constants for specific binding of LDL and apo-E HDLC to the high-affinity cell surface receptors of human fibroblasts were determined from data obtained in direct binding studies with the iodinated lipoproteins. Todinated LDL at concentrations of 0.3-12 ,tg/ml and '25I-apo-E HDLC at concentrations of 0.01-0.8 jig/ml were incubated with the fibroblasts at 40C. Receptor-bound lipoprotein, the saturable or specific component of binding, was determined at each lipoprotein concentration by subtracting the quantity of nonspecifically bound lipoprotein from the total cell-bound lipoprotein. As previously described (16), nonspecific binding was measured by the addition of human LDL (500 ,ug/ml) or canine HDLC (p = 1.02-1.063 g/ml; 50 ,ug/ml) at levels that saturate the highaffinity receptors. Under these conditions the quantity of lipoprotein bound to the cell represents the nonspecifically bound lipoprotein. Receptor-bound lipoprotein was also determined by measuring the quantity of lipoprotein released after the cells were incubated with heparin. Goldstein et al. (15) have shown that only receptor-bound LDL are released from the cells under these conditions. Both methods for the determination of specific LDL binding gave identical results. However, the heparin release method was not capable of releasing the receptor-bound HDLC, so the saturation method for the determination of HDLC receptor (specific) binding was used in all HDLC studies (see Discussion). Equilibrium binding of lipoproteins to the cell surface receptors can be described by analogy with hormone-receptor interactions (for review see ref. 17). kl

[Lp] + [R] 4'k-1±[Lp-R],

[1] where [Rl, [Lp], and [Lp-R] represent the concentrations of receptor, lipoprotein, and receptor-bound lipoprotein, respectively. The association and dissociation rate constants are given by ki and k-1. The equilibrium dissociation constant (Kd; Eq. 2) was determined by plotting the ratio of receptor-bound to free lipoprotein against receptor-bound lipoprotein, as described by Scatchard (18). The slope of the resulting straight line is equal

Proc. Natl. Acad. Sci. USA 76 (1979)

to -1/Kd and the x intercept is the maximum lipoprotein bound (ng of lipoprotein protein per dish). To convert from ng of lipoprotein protein to moles, molecular weights of 3 X 106 (of which 20% is protein) for LDL (19) and 3.6 X 106 (of which 15% is protein) for apo-E HDLC (8) were used. Determination of Rate Constants. As expressed in Eq. 2, Kd is the ratio of k-1 to kj, and it is possible to obtain an independent estimation of Kd by determining the association and dissociation rate constants. Kd = k-_ =

[Lp][R]

[2] ki [Lp-R] Rate constants k, and k-1 for the interaction of LDL and

apo-E HDLC with the cell surface receptors were determined from the time course of binding at five lipoprotein concentrations. If the simple bimolecular model expressed in Eq. 1 is correct, the rate of formation of the lipoprotein-receptor complex [Lp-R] is given by = ki[Lp][R] - k-i[Lp-R]. d[ [3] dt Solution of this equation as described by Kahn (17) for hormone-receptor interactions yields ln 2 ki[Lp] + k-1, [4] tl/2(assoc.) -

where tl/2(assoc.) is the time required for [Lp-R] to reach half of its equilibrium value. Time courses for binding of 125I-LDL and 125I-apo-E HDLC were ascertained at five lipoprotein concentrations (see figure captions). The tl/2(assoc.) at each concentration of lipoprotein was obtained from a double-reciprocal plot of nanograms bound (1/B, ordinate) against time (lIt, abscissa). The maximum bound lipoprotein at equilibrium was determined from the y intercept. Half of this value gave the half maximum bound, and tl/2(assoc.) was obtained from the graph. A plot of ln 2/tl/2(assoc.) against the lipoprotein concentration gave a straight line with slope ki and intercept k- (see Eq. 4). LDL Dissociation. During all procedures, petri dishes were kept on ice in a 4°C cold room and all solutions were at 4°C. After the cell monolayers had been incubated with 5 ,tg of 125I-LDL per ml for 1 hr, they were washed with phosphatebuffered saline/bovine serum albumin (2 mg/ml), first three times rapidly and then twice for 2 min. The plates were then incubated with 2 ml of Dulbecco's modified Eagle's medium containing 7.5% lipoprotein-deficient human plasma and 5 ,ug of native LDL per ml for different intervals of time. At the times indicated, the media were removed and the plates were washed once with phosphate-buffered saline. The heparinreleasable (receptor-bound) and -nonreleasable 125I-LDL were determined by incubation of the cells with 10 mg of heparin per ml in phosphate-buffered saline (15). RESULTS Studies. Human LDL, which contain Competitive Binding the B apoprotein, and canine HDLC, which contain only the E apoprotein, displaced essentially all of the iodinated apo-E HDLC§ from the cell surface receptors on cultured human fibroblasts in competitive binding studies conducted at 4°C (Fig. 1). Likewise, we have shown previously that iodinated LDL were displaced by LDL and HDLC (8, 20). These data demonstrate that LDL and HDLC are bound to the same cell receptor sites. Furthermore, as shown in Fig. 1, the requirement for much higher concentrations of LDL than of apo-E HDLC to displace the 125I-apo-E HDLC from the receptors supports the contention that apo-E HDLC have enhanced binding ac-

Natl. Acad. Sci. USA 76 (1979) al. Cell Biology: Pitas eteProc. I

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FIG. 1. Competitive binding of canine 125I-apo-E HDLC (204 cpm/ng of protein) to the cell surface receptors plotted against unlabeled human LDL (0) and canine apo-E HDLC (0). The 1251-apo-E HDLC (0.05 ,ug/ml) were added to the plates in media containing 25 mM Hepes, 10% human lipoprotein-deficient serum, and the concentrations of unlabeled lipoproteins indicated. Incubation was for 3 hr at 4VC. The mean cellular protein was 0.35 mg/dish.

tivity, an activity 80- to 100-fold greater than that of LDL (8). The equilibrium and kinetic studies reported below were performed to determine the mechanism of this enhanced binding. Equilibrium Studies. The direct binding of canine 1251. apo-E HDLC and human 125I-LDL as a function of lipoprotein concentration in the media is shown in Fig. 2. Apo-E HDLC saturated the cell surface receptors at a lower lipoprotein concentration than did LDL and, at saturation, the cells bound fewer apo-E HDLC than LDL. To obtain quantitative values for the affinity of the lipoproteins for the receptors and to determine the maximum amount of lipoprotein bound to the receptors, we linearized the binding curves of LDL and apo-E HDLC (Fig. 2) by the method of Scatchard (18). As shown in Fig. 3, both LDL and HDLC gave linear Scatchard plots. Linearity of the Scatchard plots indicates that only one class of receptor exists (homogeneity of the receptor sites) and that there is-no cooperativity among receptor sites (for review see ref. 17). Furthermore, the slope of the apo-E HDLC plot was greater than that of the LDL plot, indicating that HDLC have a higher affinity for the receptor (Fig. 3). The Kd for apo-E HDLC and

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FIG. 3. Scatchard plot for binding of LDL (-) and apo-E HDLC (0) to human fibroblasts. "Bound/free" is the lipoprotein bound (ng of protein/dish)/Iipoprotein free in the media (ng of protein/2 ml). The data are those shown in Fig. 2.

LDL, derived from the slopes of the Scatchard plots, were 0.13 X 10-9 M and 2.53 X 10-9 M, respectively. The Scatchard plots also confirmed that saturation of the receptors occurred at much lower levels of HDLC than LDL. The values, as determined from the x intercept in Fig. 3, were 30.8 and 106 ng of lipoprotein protein per plate for HDLC and LDL, respectively. The equilibrium affinity constant for LDL was the same whether

specific receptor binding was determined as heparin-releasable lipoprotein or by subtraction of the nonspecific binding (see Materials and Methods). Specific binding of HDLC was determined by subtraction of the nonspecific binding because receptor-bound HDLC were only minimally released by heparin treatment of the cells. Resistance to heparin release is probably explained by the higher affinity of the HDLC for the receptors and by the weaker interaction of HDLC with heparin. HDLC are less firmly bound to heparin affinity columns than LDL (unpublished observations). The results of several independent studies are summarized in Table 1. In six experiments, Kd was determined to be 2.8 X 10-9 M for LDL and 0.12 X 10-9 M for apo-E HDLC, a 23-fold higher affinity of HDLC for the receptors. In addition, it was possible not only to determine the amount of LDL and HDLC bound to the cell receptors at saturation, but also to convert from nanograms to total lipoprotein particles bound per cell (see Materials and Methods and Table 1). In five studies the average numbers of particles bound per cell were 99,600 and 27,700 for LDL and HDLC, respectively. These data showed that approximately 4 times (mean ± SD, 3.6 + 0.4) more LDL particles were required to saturate the receptors than HDLC and indicated that an HDLC particle binds to multiple receptors at a ratio of 4 (3.6):1 for LDL (see Table 1 and Discussion). The ratio of 4:1 remained constant regardless of the total number of receptors expressed by the cells and was even maintained by fibroblasts from patients with heterozygous type II hyperlipoproteinemia which have only about half as many receptors as normal human fibroblasts (unpublished observations). Rate Constants. The Scatchard method of analysis involves several important assumptions (see ref. 17). One of these assumptions is that the lipoprotein-receptor interaction is a simple bimolecular reaction as expressed in Eq. 1. To evaluate further the validity of this model and to obtain an independent estimate for Kd, we undertook kinetic analyses. The rate constants for LDL and apo-E HDLC association and

Proc. Natl. Acad. Sci. USA 76 (1979)

Cell Biology: Pitas et al.

2314

Table 1. Constants for interaction of LDL and apo-E HDLC with cell surface receptors HDLC LDL Constant 0.12 X 10-9 M 2.8 X 10-9 M Apparent dissociation (+0.4 X 10-9 M) (:0.006 X 10-9 M) constant, Kd (4SD)* 1.7 X 10-5 6.3 X 10-5 Dissociation rate sec-1 t sec'1 t constant, k-1t 18.0 X 104 5.5 X 104 Association rate M-1 sec' M-1 sec'1 constant, k1t LDL/HDLC particles 3.6 + 0.4 per cell§ * Data derived from Scatchard plots of equilibrium binding data. Binding was for 2, 3, or 4 hr at 4VC. Data corrected for nonspecific binding. t Derived from the time course of binding at several lipoprotein concentrations. Data for LDL and HDLC are shown in Figs. 4 and 5. The dissociation constants for LDL and apo-E HDLC are equivalent to half-lives of dissociation (t1i2 = 0.693/k-1) of 3.1 and 11.3 hr, respectively. § Mean + SD for five studies. Ratio calculated from the number of particles bound per cell, determined as follows: (g protein/dish) mol/dish. (M, lipoprotein X % lipoprotein protein) (mol/dish)(Avogadro's number) = molecules (particles)/dish,

particles/dish particles bound/cell. cells/dish

dissociation were determined from the time course of binding of the lipoproteins at different concentrations (Figs. 4 and 5). Plotting the data as described in Eq. 4 yielded a straight line for both apo-E HDLC and LDL (Figs. 4 and 5). The k1 and k-, for binding derived from these data were 5.5 X 104 M-1 secand 6.3 x 10-5 sec-1, respectively, for LDL and 18.0 X 104 M-1 sec-1 (kj) and 1.7 X 10-5 sec-I (k-1) for apo-E HDLC. The Kd calculated from the rate constants (Kd = k-1/kj) was 1.14 X 10-9 M for LDL and 0.094 X 10-9 M for apo-E HDLC. These results are in agreement with data obtained in the equilibrium binding studies (Table 1) and clearly indicate that apo-E HDLC has a higher affinity for the receptors than LDL.

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FIG. 5. Determination of the rates of association (k 1) and dissociation (k-1) of the 1251-apo-E HDLc-receptor complex. (Left) Time course of binding of 1251-apo-E HDLC (2869 cpm/ng) to cultured human fibroblasts as a function of concentration. Cells were incubated at 4VC for the indicated times with: 1, 0.01; 2,0.02; 3,0.04; 4,0.08; or 5, 0.2 ,ug of 1251-apo-E HDLC per ml. The data were corrected for nonspecific binding. Each point is the average of duplicate determinations. The t1/2 (association) was determined. (Right) Plot of t1/2 (association) against 1251-apo-E HDLC concentration in the culture media (see Eq. 4). Slope (k 1) = 18 X 104 M-1 sec-1; y intercept (k-1) = 1.7 X 10-5 sec-1. The mean cellular protein was 0.5 mg/dish.

Half-Life of Lipoprotein-Receptor Complex. Confirmation of the dissociation rate constant (k-1) was undertaken by direct studies to determine the half-life of the LDL-receptor complex. A t 1/2 of 4.2 hr was obtained (Fig. 6). This was equivalent to a dissociation rate constant (k-1 = 0.693/t 1/2) of 4.6 X iO-5 seC1 as compared to 6.3 X 10-5 sec1 determined in the kinetic studies (Table 1). The t1/2 for LDL calculated from the k-1 from the kinetic studies was 3.1 hr. This reasonably good agreement provides confidence in the method. The half-life of dissociation of the HDLc-receptor complex could not be determined with confidence, and the t1/2 ranged from 8 to 16 hr. The variability of the data appeared to result from cell damage due to the excessively long incubations at 40C. Furthermore, the inability of heparin to release HDLC from the receptors proved to be an additional complication which has not been overcome. Calculations based on the dissociation rate constant from the kinetic studies gave a t 1/2 for apo-E HDLC of 11.3 hr.

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FIG. 4. Determination of the rates of association (k 0) and dissociation (k-1) of the 125I-LDL-receptor complex. (Left) Time course of 1251-LDL (180 cpm/ng) binding to cultured human fibroblasts as a function of concentration. Cells were incubated at 40C for the indicated times with: 1, 0.2; 2, 0.4; 3, 1.0; 4, 2.0; or 5, 3.0 ,Ug of 1251-LDL per ml. Receptor-bound 125I-LDL was determined by heparin release. Each point represents the average of duplicate determinations. The time at which half the equilibrium value of LDL was bound (t1/2) at each concentration was determined. (Right) Plot of tl/2(association) against the 1251-LDL concentration in the tissue culture media (see Eq. 4). Slope (k1) = 5.5 X 104 M-l sec1; y intercept (k.1) = 6.3 x 10-5 sec-1. The mean cellular protein was 0.5 mg/dish.

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FIG. 6. Time course of 125I-LDL (102 cpm/ng) dissociation from receptors of cultured human fibroblasts. Cells were incubated at 40C for 1 hr with 5 ,g of 125I-LDL per ml. Unbound 125I-LDL were removed and the plates were incubated with 5 gg of native LDL per ml for the time periods indicated. At each time point, the radioactivity of the medium was measured and the heparin-releasable and -nonreleasable 1251-LDL were determined. Each point is the average of triplicate determinations. The mean cellular protein was 0.5 mg/dish.

Total bound plus medium; 0, heparin-releasable 1251-LDL;*, heparin-nonreleasable 1251-LDL. A,

Cell Biology: Pitas et al. DISCUSSION lodinated apo-E HDLC and human LDL were used in equilibrium and kinetic experiments at 4VC to elucidate themechanism for the enhanced binding activity of the apo-E HDLC. Analysis of the equilibrium binding data by the method described by Scatchard (18) yielded three important observations. (i) The linearity of the plots for both LDL and apo-E HDLC indicated a lack of cooperativity among the receptors, the existence of only one class of binding sites, and the general validity of the method. (ii) Apo-E HDLC had a lower Kd (a 20-fold higher affinity) than did LDL for the receptors. (iii) When the receptors were saturated, 4 (3.6 + 0.4) times as many LDL particles as HDLC particles were bound to the cells. Kinetic studies confirmed that apo-E HDLC had a greater affinity for the cell surface receptors than did LDL and that the interaction of both lipoproteins with the receptors fit a simple bimolecular model. Apo-E HDLC bound more rapidly (higher ki) and dissociated more slowly (lower k-1) than LDL. The Kd derived from the rate constants for LDL and apo-E HDLC were in reasonable agreement with the results from the equilibrium data. The values obtained for the Kd of LDL in these experiments agreed with the Kd for the interaction of LDL with fibroblasts at 40C as determined by Brown and Goldstein, 4.5 X 10-9 M (16). Furthermore, the half-life of dissociation of LDL from the receptors, as determined by direct measurement (4.2 hr) or calculated from the kinetic data (3.1 hr), was consistent with the observation of Brown and Goldstein (21). Because evidence reported in this paper and elsewhere (4, 8, 20) indicates that LDL and HDLC are bound to the same receptors, the most feasible explanation for the lower number of HDLC particles bound to cells as compared to LDL at maximum receptor occupancy is that HDLC bind to approximately 4 times as many receptors as LDL. The conclusion that LDL and HDLC bind to the same receptors is supported by several observations. Native LDL and apo-E HDLC are competitive inhibitors of the binding of '25I-apo-E HDLC and 125I-LDL to the receptors of fibroblasts (8, 20). Furthermore, the high-affinity binding of either LDL or HDLC to the receptors initiates a similar series of intracellular events, including 3-hydroxy3-methylglutaryl CoA reductase suppression, lipoprotein degradation, and cholesteryl ester synthesis (4, 8, 20). Moreover, regardless of the total number of cell surface receptors available, the ratio of LDL to HDLC particles bound per cell remains constant. Cultured fibroblasts from patients with type II hyperlipoproteinemia, the heterozygous form of familial hypercholesterolemia, have approximately half as many LDL cell surface receptors as normal fibroblasts (22) and bind LDL and apo-E HDLC particles at a ratio of 4:1 per cell (unpublished observation). In addition, apo-E HDLC, like LDL, do not bind to fibroblast cells derived from patients with the homozygous form of familial hypercholesterolemia which lack functional LDL receptors (20). Thus, the evidence is consistent with the binding of LDL and HDLC to the same receptor and with the binding of HDLC to multiple receptors. For multiple receptor binding to occur, the apo-E HDLC must contain several (at least four) recognition sites per particle. One molecule of apo-E (Mr 37,000) could contain several recognition sites as a result of a repeating structural sequence, or more than one apo-E molecule

Proc. Natl. Acad. Sci. USA 76 (1979)

2315

could participate in multiple receptor binding. The apo-E

HDLc (Mr 3.6 X 106; 15% protein) possess 16 molecules of

apo-E per lipoprotein particle. Either mechanism is consistent with the data. In summary, the enhanced binding activity of apo-E UDLC can be accounted for by a greater affinity for the receptors associated with a more rapid rate of association and a slower rate of dissociation. In addition, apo-E HDLC appear to have several recognition sites per particle which are capable of interacting with multiple receptors. We thank Dr. C. Ronald Kahn, National Institute of Arthritis, Metabolism, and Digestive Diseases, and Dr. Donald L. Fry, National Heart, Lung and Blood Institute, for critical discussion of the data. We thank Mrs. K. S. Holcombe and Miss C. A. Groff for their assistance in preparation of the manuscript. Portions of the work were conducted under a National Heart, Lung and Blood Institute contract with Meloy Laboratories, Springfield, VA. 1. Goldstein, J. L. & Brown, M. S. (1977) Annu. Rev. Biochem. 46, 897-930. 2. Mahley, R. W. & Innerarity, T. L. (1977) J. Biol. Chem. 252, 3980-3986. 3. Innerarity, T. L., Mahley, R. W., Weisgraber, K. H. & Bersot, T. P. (1978) J. Biol. Chem. 253, 6289-6295. 4. Mahley, R. W. & Innerarity, T. L. (1978) in Sixth International Symposium on Drugs Affecting Lipid Metabolism, eds. Kritchevsky, D., Paoletti, R. & Holmes, W. L. (Plenum, New York), pp. 99-127. 5. Weisgraber, K. H., Innerarity, T. L. & Mahley, R. W. (1978) J.

Biol. Chem. 253,9053-9062. 6. Mahley, R. W., Innerarity, T. L., Pitas, R. E., Weisgraber, K. H., Brown, J. H. & Gross, E. (1977) J. Biol. Chem. 252, 7279-

7287. 7. Mahley, R. W. (1978) in Disturbances in Lipid and Lipoprotein Metabolism, eds. Dietschy, J. M., Gotto, A. M., Jr. & Ontko, J. A. (American Physiological Society, Bethesda, MD), pp. 181-

197. 8. Innerarity, T. L. & Mahley, R. W. (1978) Biochemistry 17, 1440-1447. 9. N4ahley, R. W., Innerarity, T. L., Weisgraber, K. H. & Fry, D. L. (1977) Am. J. Pathol. 87,205-226. 10. Mahley, R. W., Weisgraber, K. H. & Innerarity, T. L. (1974) Circ. Res. 35, 722-733. 11. Nahley, R. W., Weisgraber, K. H., Innerarity, T., Brewer, H. B., Jr. & Assmann, G. (1975) Biochemistry 14, 2817-2823. 12. Bolton, A. E. & Hunter, W. M. (1973) Biochem. J. 133, 529-

539. 13. Innerarity, T. L., Pitas, R. E. & Mahley, R. W. (1979) J. Biol. Chem., in press. 14. Bilheimer, D. W., Eisenberg, S. & Levy, R. I. (1972) Biochim. Biophys. Acta 260,212-221. 15. Goldstein, J. L., Basu, S. K., Brunschede, G. Y. & Brown, M. S.

(1976) Cell 7,85-95. 16. Brown, M. S. & Goldstein, J. L. (1974) Proc. NatI. Acad. Sci. USA 71, 788-792. 17. Kahn, C. R. (1975) in Methods in Membrane Biology, ed. Korn, E. D. (Plenum, New York), Vol. 3, pp. 81-146. 18. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51, 660-672. 19. Fisher, W. R., Hammond, M. G. & Warmke, G. L. (1972) Bio-

chemistry 11,519-525. 20. Bersot, T. P., Mahley, R. W., Brown, M. S. & Goldstein, J. L. (1976) J. Biol. Chem. 251, 2395-2398. 21. Brown, M. S. & Goldstein, J. L. (1976) Cell 9,663-674. 22. Goldstein, J. L., Brown, M. S. & Stone, N. (1977) Cell 12, 629641.