Low Density Lipoprotein Receptor Activity in Homozygous Familial Hypercholesterolemia Fibroblasts*

We have identified specific low affinity low density lipoprotein (LDL) receptors in skin fibroblasts from two patients previously classified as having LDL recep- tor-negative homozygous familial hypercholesterolemia (FHC). K,,, and maximum capacity for cell-associ- ated and degraded were determined by two independent methods, a traditional technique in which increasing amounts of '"I-LDL were added until receptor saturation was achieved and a new technique in which the displacement of a small amount of l"I-LDL tracer was observed during the addition of variable amounts of unlabeled LDL. The K, for specific cell-associated 12'1-LDL in FHC cells was 3.5-7.3 times that of normal cells and the maximum specific capacity was reduced to 11% of normal. Thus, some FHC cells reduced affinity as well as reduced capacity for LDL. FHC cell LDL

We have identified specific low affinity low density lipoprotein (LDL) receptors in skin fibroblasts from two patients previously classified as having LDL receptor-negative homozygous familial hypercholesterolemia (FHC). K,,, and maximum capacity for cell-associated and degraded '"I-LDL were determined by two independent methods, a traditional technique in which increasing amounts of '"I-LDL were added until receptor saturation was achieved and a new technique in which the displacement of a small amount of l"I-LDL tracer was observed during the addition of variable amounts of unlabeled LDL. The K , for specific cellassociated 12'1-LDL in FHC cells was 3.5-7.3 times that of normal cells and the maximum specific capacity was reduced to 11% of normal. Thus, some FHC cells have reduced affinity as well as reduced capacity for LDL.
The FHC cell receptors share many but not all properties of the normal skin fibroblast LDL receptor. Specific degradation of bound lZ5I-LDL occurred concomitantly with LDL binding and was greatly reduced by the addition of chloroquine, an inhibitor of lysosomal function. Preincubation of FHC cells with cholesterol or LDL resulted in significant suppression of receptor function. Modification of lysine residues of LDL abolished receptor activity in both normal and FHC cells. Treatment of FHC cells with compactin, a cholesterol synthesis inhibitor, resulted in significant increases in specific '"1-LDL binding and degradation compared to FHC cells without compactin treatment. Normal cells also showed increases in '"I-LDL binding and degradation with compactin treatment, but the mean percentage increase in specific lZ5I-LDL degradation was significantly greater in FHC cells (strain GM 2000, 160 k 18%) than in normal cells (29 2 8%).
Homozygous familial hypercholesterolemia is a well described clinical phenotype which is accompanied by a deficiency in the cell surface receptor for LDL' and accelerated atherosclerosis with premature death (1,2) remains unknown. Goldstein and Brown ( 3 ) have postulated that the clinical phenotype can be accounted for by three disorders that are clinically similar but biochemically distinct. Thus, cultured fibroblasts from patients with homozygous FHC are classified as receptor-negative in which less than 21' of normal LDL receptor activity is detectable, receptor-defective in which 2-25% of normal LDL receptor activity is detectable, and "internalization-defective" in which LDL receptors are able to bind LDL particles normally but LDL is not taken up by the cell. This paper focuses on the receptornegative form of homozygous FHC.
LDL receptor-negative fibroblasts clearly have a gross reduction in "'I-LDL binding activity, but it is unresolved whether or not some residual activity is present. Several lines of evidence suggest the existence of significant LDL receptor activity in "receptor-negative'' cells. LDL receptor activity can be assessed either directly by the measurement of ""I-LDL bound to cultured fibroblasts or indirectly by a number of methods that measure the effects of LDL on cellular metabolic functions. Indirect methods include determinations of the effect of LDL on 3-hydroxy-3-methylglutaryl-Co A reductase activity, cholesteryl ester formation, and the release of amino acids by LDL degradation. Goldstein et al. (4) classify cells as "receptor-negative'' if LDL fails to stimulate any incorporation of oleate into cholesteryl esters. However, when receptor-specific "'I-LDL binding was studied in five fibroblast strains that were later classified as LDL receptornegative on the basis of oleate incorporation studies (4), these cells bound 11.2 +-3.7% of the expected amount of LDL ( 5 ) .
Breslow et al. (6) found 12% of normal '"I-LDL specific binding in fibroblasts from a patient apparently classified as LDL receptor-negative. Using a dose-response curve, measurable levels of specific degradation of LDL (15%' of normal) were demonstrated by Goldstein and Brown in fibroblasts from a typical homozygote with the receptor-negative form of FHC (4, 7 ) . Haba et al. (31) found from 0-7.54> of the expected specific LDL internalization and degradation in receptor-negative fibroblasts. Fung et al. (10,11) have repeatedly demonstrated LDL-mediated suppression of hydroxymethylglutaryl-CoA reductase activity and of acetate incorporation into sterols in receptor-negative fibroblasts. This is evidence for specific receptor activity since LDL entering fibroblasts by nonspecific pinocytosis evidently does not regulate sterol synthesis or esterification ( 7 , 12, 15). Using fluorescent and immunofluorescent microscopy to study individual fibroblasts, Kruth and Vaughn (35) reported that some homozygous receptor-negative cells bind small amounts of LDL and accumulate intracellular cholesterol.
The present study was undertaken to characterize receptor activity present in fibroblasts from patients with the receptornegative form of homozygous FHC. We have identified specific low affinity LDL receptor activity in these fibroblasts which 12857 is distinct from nonspecific uptake of LDL by cells and binding of LDL by blank culture dishes.

EXPERIMENTAL PROCEDURES
Materials-The diphosphate salt of chloroquine, Hepes buffer, and cholesterol were purchased from Sigma. Cholesterol was repurified twice by the method of Fieser (32). Calcium chloride was purchased from Mallinckrodt Chemical Works.
Dextran sulfate was purchased from Calbiochem-Behring. Compactin in the lactone form was obtained from Dr. A. Endo, Sankyo Co., Ltd., Tokyo, Japan. The lactone was converted to the sodium salt by heating in base; 0.385 mM compactin was subsequently adjusted to physiologic pH and stored a t -20 "C (14). Newborn calf serum was purchased from Gibco, Inc., Grand Island, NY. ''?I was purchased from Amersham. Radiation-sterilized plastic 35-mm diameter six-well cluster dishes were purchased from Costar Corp., Bedford, MA. LDL (density 1.019-1.063 g/ml) from normal, healthy donors was prepared by ultracentrifugation and iodinated as previously reported (7,15). Unlabeled LDL prepared from an unrelated patient with homozygous FHC was used for displacement of specifically bound "'I-LDL. This FHC LDL was indistinguishable from LDL prepared from normal donors in displacing "'I-LDL in cultured fibroblasts. Lipoprotein-deficient human serum was prepared from the bottom fraction after ultracentrifugation and washing a t a density of 1.215 g/ml (7). For the experiment presented in Table VIII, LPDS was shaken for 4 h at room temperature with 125 mg/ml of silicic acid (SI-R, Sigma) followed by sedimentation of the silicic acid. This reduced the total cholesterol content of the LPDS to less than 1 pg/ml (30). Stock LPDS solutions were adjusted to 70 mg of protein/ml (100% LPDS). All media were sterilized by filtration through 0.22-pm filters. Receptor grade mouse epidermal growth factor (Collaborative Research, Waltham, MA) was iodinated as previously described (8). Human "'I-thrombin (9) was a gift of Dr. Douglas Tollefsen and topical bovine thrombin was purchased from Parke-Davis.
Cells-Skin fibroblast strains from two patients with the "receptor- Puck's saline G (16) and 1 ml of MEM & 10% LPDS (7 mg of protein/ ml) was added to each well to induce receptor activity. After 72 h, the medium was removed and cells were then assayed for '""I-LDL binding and degradation as previously described (15, 23). Cells were incubated for 5 h a t 37 "C with "'I-LDL in MEM + 10% LPDS k unlabeled LDL. The medium was removed and assayed for trichloroacetic acid-soluble non-iodide degradation products. Degraded LDL was determined after subtraction of a blank measured in dishes without cells (15,23). Following aspiration of the medium, the dishes were immediately placed on ice and washed. In some experiments, the surface-bound LDL was eluted for 1 h at 4 "C with 4 mg/ml of dextran/S04 (Calbiochem) in Puck's saline G and internalized LDL was then determined by dissolving the cells in NaOH. Cell-associated "'I-LDL was determined either by dissolving the washed cells directly in 0.625 N NaOH for 20 min at room temperature without first eluting the surface LDL or as the sum of dextran/S04-releasable + internali :ed LDL in experiments where there was too little surface-bound :.DL to analyze separately.
Calculations-Statistical comparisons were performed by the Student's t-test with mean e standard error of the mean presented.
Unless otherwise stated, specific "'I-LDL binding or degradation was computed as the difference of values determined in the absence (total) and the presence (nonspecific) of 20-fold or greater excess of unlabeled LDL. The estimated standard error of this difference was computed as SEM (specific) = dSEM:,,L*~ + SEM~l,,,,,;,,,,f,, where the number of samples for total and nonspecific binding was identical. Experiments were routinely performed in triplicate. K,,, and maximum capacit.v for '"I-LDL were calculated from kinetic data determined in triplicate or sextuplicate by plotting according to Scatchard (17,18,34) the ratio of cell-associated (or degraded) ""I-LDL (nanograms of LDL protein/mg of cell protein) to free '2'I-LDL (micrograms LDL protein/ml) on the ordinate uersus the cell associated (or degraded) "'I-LDL (nanogramdmg) on the abscissa and fitting the points to a straight line by the method of least squares. The slope of the line is -l/K,,, (K,,, = Michaelis-Menten constant) and the intercept on the abscissa is the maximum capacity. Determinations of K,, and maximum capacity for ""I-LDL were also accomplished from analysis of independent data obtained via the displacement of a small and fixed amount of labeled LDL by variable quantities of unlabeled LDL. The rationale for this technique is given in the text. In calculating the K,,,, the following assumptions were made: 1) labeled and unlabeled LDL compete identically for binding sites; 2) binding follows Michaelis-Menten kinetics; 3 ) nonspecific binding of ""I-LDL is not a function of total (labeled + unlabeled) LDL concentration. The following definitions are given. Q = total concentration of LDL (labeled + unlabeled) added to the medium, pg/ml; T = concentration of "'I-LDL tracer in pg/ml; Bc2 = specific cell-associated LDL (labeled + unlabeled), ng of LDL/mg of cell protein; BT = specific cell-associated '"I-LDL tracer, ng of '"I-LDL/ mg of cell protein; Bmilr,, = maximum specific capacity for cell-associated LDL (labeled + unlabeled LDL), ng of LDL/mg of cell protein.
From the Michaelis-Menten theory: Since the amount of LDL bound is negligible with respect to the total amount of LDL present, the free LDL present in the medium = 4.

Characterization of LDL Receptor Activity in FHC Cells-
A dose-response curve for cell-associated '2511-LDL in LDL receptor-negative GM 2000 cells is presented in Fig. 1. Although the absolute level is low, appreciable cell-associated "'I-LDL displaceable by unlabeled LDL is apparent and easily distinguishable from binding to culture dishes without cells (blank dishes). In subsequent data, receptor-specific cell-associated or degraded '"I-LDL was calculated by subtracting nonspecific values (seen in the presence of cells but with excess unlabeled LDL) from total values (seen in the presence of cells without excess unlabeled LDL). The estimated standard error of this difference is routinely presented in the figures and tables. Blank culture dishes reproducibly manifested a minor degree of specific binding which has been disregarded in subsequent calculations. No LDL specific degradation products were observed in blank culture dishes. Data of the experiment of Fig. 1 were recalculated as nanograms of "'I-LDL specifically cell-associated and degraded per mg of cell protein and are displayed in Fig. 2. Specific LDL degradation occurred and increased in parallel with cell-associated LDL, suggesting that the cell-associated material was transferred to lysosomes. This was further supported by the data of Table I in which addition of 60 ~L M chloroquine (lysosomal enzyme inhibitor) to the assay solution during a 5-h incubation in the presence of 50 pg/ml of "'I-LDL resulted in the nearly complete inhibition of LDL degradation in FHC cells accompanied by a significant increase in the amount of internalized LDL.
The time course of specific cell-associated and degraded LDL in receptor-negative GM 1915 cells is shown in Fig. 3. The FHC cells qualitatively resembled normal cells. Cellassociated '"I-LDL values rose progressively after addition of '"I-LDL and no degradation was observed until 45 min after which degradation rose linearly with time. Maximum cell associated '251-LDL was observed at 5 h (data not shown).  Little LDL binding and no significant degradation were observed in blank culture dishes.
Specificity of FHC cell receptor for lipoproteins is demonstrated in Table 11. In both normal and FHC cells, unlabeled LDL reduced the specific degradation of ""I-LDL to a similar extent. However, methyl LDL and acetoacetyl LDL, lipoproteins modified a t critical lysine residues involved in receptor binding (27), did not compete significantly for either normal or FHC receptors.
Since only a very small per cent of the radioactivity added to the cultured cells became cell-associated, the nature of this material was further examined. The '"I-LDL tracer added contained 3.6% of material that remained in the organic phase after a Folch lipid extraction and wash (26) followed by evaporation of the organic solvent. Internalized "' I radioactivity dissolved in 0.5 ml of 0.625 N NaOH from an experiment identical with that described in Table IX was neutralized with 0.5 ml of 1 N HCl and immediately vortexed with 1.0 ml of chloroform. The chloroform phase was removed, dried, counted, and compared to that remaining in the aqueous phase. In GM 2000 cells, 7.5% of the radioactivity was chloroform-soluble, whereas in normal cells 3.6% was chloroformsoluble. Hence, this material did not appear to be primarily lipid in either normal or FHC cells. Since apoprotein B, the protein constituent of LDL, is insoluble in aqueous solutions of TMU whereas other apoproteins are soluble in TMU (25), the solubility of bound radioactivity in this solvent was tested (Table 111). In both normal and GM 2000 cells, over 87% of the bound counts were TMU-insoluble. Hence, the cell-associated radioiodinated material appears to be "'I-LDL apoprotein and not a contaminant.
Saturability of FHC cell receptor activity is demonstrated by the displacement curve shown in Fig. 4. In the presence of a constant amount of '"I-LDL (50 pg/ml) in the medium, the amount of cell-associated '"'I-LDL tracer decreased from 104 k 2.8 ng/mg of cell protein to 18.2 k 1.1 ng/mg as the concentration of unlabeled LDL in the assay medium was increased from 0 to 2000 pg/ml. In the same experiment, the amount of degraded tracer decreased from 223 +-15.4 ng/mg of cell protein to 51.9 k 12.9 ng/mg. The displacement of I2'I-LDL by large amounts of unlabeled LDL does not appear to be an artifact since similar amounts of unlabeled LDL did not displace specifically bound '"I-thrombin or I-mouse epidermal growth factor from normal cell receptors (Table IV).
LDL binding in FHC cells was calcium-sensitive (Table V). The effect of calcium on specific LDL binding was assessed by a modification of the technique used by Goldstein and Brown to characterize high affinity receptor activity (7). In the presence of 50 pg/ml of '"I-LDL, both specific dextran-SO4-releasable surface-bound and internalized LDL were significantly increased in GM 2000, GM1915, and normal cells after the addition of 2 mM calcium to the assay medium. This Cells were incubated at 37 "C and then harvested and processed for specific cell-associated ""I-LDL and degraded LDL a t specified time intervals.
effect of calcium on specific binding could not be accounted for by binding to blank culture dishes alone. Calcium sensitivity was the one property not observed by Dana et al. in studving the binding of "'I-LDL to glass beads (33).
Calculation of Binding Parameters-Two methods were used to calculate the apparent K,,, and maximum capacity for specific cell-associated '"'I-LDL and degraded LDL in GM 2000 cells (Table VI). First, a Scatchard analysis was applied to the equilibrium dose-response data of Fig. 2 in which increasing quantities of ""I-LDL were added with and without excess unlabeled LDL (Fig. 5). It is clear that only low affinity binding (flat slope) is present in GM 2000 cells. The apparent K,,, (reciprocal of affinity) of these cells was 127 pg/ml compared to 16.5 pg/ml in the normal cell type. The mean K,,, for receptor-negative cells (103 pg/ml) was 4.6 times that of normals, while the mean maximum binding capacity (364 ng/   A, normal human fibroblasts were plated at lO'/well in MEM + 15% NBCS. The medium was removed after 5 days, the cells were washed with saline G, and 25 mM Hepes-buffered MEM, pH 7.3, containing 0.1% bovine serum albumin was added for 5 min to decrease nonspecific binding. This medium was removed and 0.7 ml of the same medium containing 0.83 nM '"I-a-thrombin f 200-fold excess of unlabeled bovine thrombin or unlabeled LDL was added. The cells were incubated for 60 min at 37 "C in air and then washed as for LDL binding experiments. The cells were dissolved in 0.625 N NaOH and counted for total cell-associated binding. B, normal human fibroblasts were plated at 10r'/welL in MEM + 15% NBCS for 4 days. The medium was removed, the cells were washed with saline G, and 1 ml of 10% LPDS in MEM was added. This media were removed after 3 days and 0.7 ml of the same medium containing 16 ng/nd of '"I-mEGF f 20-fold excess of unlabeled mEGF or unlabeled LDL was added. The cells were incubated 40 min at 37 "C in 5% CO?, then washed as for LDL binding experiments. The cells were dissolved in 0.625 N NaOH and counted for total cell-associated bindine. mg) was 11.5% of normal (Table VI). Similar results were found for '"I-LDL degradation. The receptor-negative cells thus have both reduced affinity and capacity for LDL.
One limitation of the Scatchard method is that, it is not practical to use concentrations of added '"I-LDL significantly above the K,, of the receptor-negative cells. Nonspecific II"1-LDL binding rises in proportion to the added 12'II-LDL concentration, whereas the low level of specific binding in FHC cells is constant. Therefore, the data become less reliable as large quantities of '"I-LDL are employed. To circumvent this, an analysis of the I2'I-LDL displacement curve of Fig. 4 was performed as described under "Experimental Procedures." This technique has the advantage of a constant and low level of nonspecifically bound '"I-LDL counts. The analysis is based upon the principle that as cold LDL is added in increasing amounts to I2"I-LDL in the assay medium, the unlabeled material will compete for receptor binding. However, the degree of competition will be imperfect depending on the per days of receptor induction, the medium was removed. One group of cells was then washed with calcium and magnesium-free Puck's saline G and 1.5 ml of 50 mM Tris-CI (pH 7.5), 0.1 M NaCl buffer containing 50 pg/ml of "'I-LDL with or without 2 mg/ml of unlabeled LDL was added to each well. A second group of cells was treated identically except CaCL was added to the assay medium yielding a final calcium concentration of 2 mM. The cluster dishes were sealed with parafilm, incubated in air at 37 "C for 3 h. and then Drocessed for LDL bindine.    Fig. 6 in such a way as to linearize the data, analogous to the Lineweaver-Burk method of enzyme analysis. In Fig. 6, the x-intercept = -Km. It can be seen that the apparent affinities of FHC cells (48.7 pg/ml) and a normal cell type (6.6 pg/ml) differ markedly. By this method, maximum capacity for cell-associated '"'I-LDL of receptor-negative cells was 10.6% of normal (Table  VI). The above parameters were computed for cell-associated (surface-bound plus internalized) '"I-LDL and degraded LDL. Although it was possible to measure dextran-S04-releasable cell surface binding (Tables I and V), the level of binding was too low to determine reliable K , and maximum binding values.
More precise surface binding data could be obtained from the measurement of cell-associated '""I-LDL at 4 "C, a condition which prevents LDL internalization and degradation (1). Receptor-specific '"I-LDL binding at 4 "C in three normal cell strains performed as specified in the legend to   consistently to demonstrate specific le51-LDL binding a t concentrations of added '"I-LDL less than 10 pg/ml. However, at higher amounts of added '"I-LDL (Fig. 7), appreciable specific binding activity was observed and the binding capacity/mg of  (Table  VII). Specific degradation of '"I-LDL in FHC cells was 34% of that observed in normal cells. This experiment suggests that the receptor activity of FHC fibroblasts is relatively greater compared to normals when receptors are not artifi-cially induced by LPDS. The ability of LDL or cholesterol preincubation to down-regulate LDL receptors of normal and FHC cells grown in LPDS is presented in Table VIII. LDL receptor activity was reduced in both cell types, but the FHC cells demonstrated more resistance to down-regulation than normal cells.
The effect of 48-h preincubation with compactin, an inhibitor of cholesterol biosynthesis, was also studied. GM 2000 FHC and normal cells were prepared as described in the legend to Table IX by 2 days culture in growth medium followed by a 48-h incubation in 10% LPDS f 4 p~ compactin. Specific '"I-LDL binding was then determined in triplicate by incubation with 100 pg/ml of '"'I-LDL for 5 h at 37 "e.

DISCUSSION
The data presented in this paper concur with the studies of Brown and Goldstein (1, 5 , 7 ) in that no high affinity LDL receptor activity was found in skin fibroblasts from patients with the receptor-negative form of homozygous FHC (Fig. 5). However, specific but low affinity LDL receptor activity was identified which is distinct from the nonspecific bulk phase pinocytosis of '"I-LDL measurable in FHC cells in the presence of large amounts of unlabeled LDL (Fig. 4, Ref. 7) and from nonspecific binding of '"'1-LDL by culture dishes without cells (Figs. 1,3, and 7). The cell-associated "'I-labeled material appears to be apo-LDL since it is not extractable into chloroform and is insoluble in aqueous tetramethylurea (Table  111). Such specific low affinity receptor activity is consistent with previous findings of small amounts of specific binding of "'I-LDL (5,6), small amounts of specific degradation of '251-LDL ( 7 ) , and regulation of sterol synthesis by LDL (10,11) in receptor-negative FHC fibroblasts.
The LDL receptors in FHC cells demonstrated by our experiments resemble the normal high affinity LDL receptor in several ways. 1) The binding of "'I-LDL is accompanied by appropriate degradation (Fig. 2). During a 5-h incubation at 37 "C with 100 pg/ml of '"I-LDL, the ratio of dextran-S04releasable surface-bound LDLinternalized LDLdegraded LDL was 10.6:100:152 in GM 2000 cells and 10.0:100:182 in normal cells. The degradation of "'I-LDL was linear after a 45-min delay in both normal and receptor-negative cells (Fig.  3B), consistent with a requirement that LDL be transferred to lysosomes before degradation. Chloroquine, a lysosomal enzyme inhibitor, reduced l2'1-LDL degradation to 5% of normal in GM 2000 cells. Thus, the usual pathway of LDL uptake is apparently present in FHC cells. 2) The time course for cell-associated "'I-LDL was similar in normal and FHC cells (Fig. 3A). 3) Modification of lysine residues reduced the capacity of LDL to interact with both FHC and normal receptors (Table 11). 4) Binding to both cell types was calciumsensitive (Table V). 5) FHC cells demonstrated down-regulation of LDL receptors in response to pretreatment with cholesterol and LDL (Table VIII) and increased receptor activity in response to compactin, a cholesterol biosynthesis inhibitor (Table IX).
The LDL receptors of FHC cells are also easily distinguished from those of normal cells. The maximum specific capacity for cell-associated '"I-LDL in FHC cells was 9.8-13% of normal cells assayed in the same manner (Table VI). But receptor affinity of LDL was also reduced to 14-2870 of normal.
The K , for LDL in FHC cells (49-127 pg/ml) was 3.5-7.3 times that of normal cells and much nearer the estimated normal extracellular LDL apoprotein B concentration of 70 pg/ml. Thus, FHC cells should metabolize significant amounts of LDL when grown chronically under conditions in which large amounts of LDL are present in the medium. This was confirmed in Table VI1 in which FHC cells metabolized 34% of the normal amount of LDL when 80 pg/ml of LDL was present. Reduced affinity of l2'1-LDL in GM 2000 cells was also noted in assays conducted at 4 "C (Fig. 7), a condition preventing LDL internalization and degradation.
The data presented do not allow for a distinction between altered expression of the classic LDL receptor and a genetically separate class of low affinity LDL receptors that might be present in all cell types. It should be noted, however, that in another FHC mutation characterized by increased LDL receptor affinity but low capacity there is no evidence for a class of low affinity receptors even though they should have been more easily detected than in normal cells (17). Of interest is the recent report by Beisiegel et al. (36) that some LDL receptor-negative fibroblasts have much more binding of radiolabeled anti-LDL receptor antibody than of radiolabeled LDL, suggesting that the high affinity LDL receptor might be present but modified.
The regulation of receptors in GM 2000 cells was also altered. Preincubation of normal cells for 48 h with compactin in LPDS medium resulted in increased specific I2'II-LDL degradation compared to cells grown in LPDS without compactin (Table IX). This confirms similar previous work with compactin in normal skin fibroblasts (29). Thus, LPDS medium apparently is not a maximum stimulus to LDL receptor accumulation. GM 2000 cells also demonstrated increased receptor activity after compactin preincubation. The mean percentage increase in specific LDL degradation in this cell type after compactin preincubation was significantly greater (160 f 18%) than in normal cells from three individuals (29 f 8%). Since the absolute increase in specific receptor activity after compactin addition was considerably greater in normal cells than in FHC cells, the greater percentage increase in specific LDL degradation in GM 2000 cells may be physiologically insignificant and merely represent an artifact of experimental conditions or data treatment. An alternative explanation is that the greater percentage increase in degradation in GM 2000 cells after compactin preincubation represents the unmasking of specific LDL receptors usually down-regulated by endogenous cellular production of cholesterol. Prolonged preincubation with compactin appears important in order to demonstrate increased receptors since Haba et al. (31) did not observe any changes in LDL receptor activity in either normal or FHC fibroblasts incubated with both I2'II-LDL and compactin for only 6 h. Thus, our finding of specific low affinity LDL receptors and hyper-responsiveness to compactin in GM 2000 cells raises the possibility that the primary abnormality in FHC fibroblasts may not necessarily be a defect in the structural gene for the LDL receptor but rather a defect in the genes regulating LDL receptor expression or cholesterol metabolism. A mechanism for FHC implicating increased endogenous production of cholesterol has been proposed previously by Fogelman et al. (22).
Low affinity receptors may have been overlooked in previous experiments for several reasons. The curve describing "'I-LDL binding of fibroblasts is sigmoidal and very little binding is seen at '"I-LDL concentrations far below the K,. Thus, when '"I-LDL at concentrations of 5-20 pg/ml is used, normal cells have appreciable receptor occupancy whereas receptor-negative cells show very little occupancy. "'I-LDL concentrations above 50 pg/ml should be employed for FHC cells and, for this reason, 1-2 mg/ml of unlabeled LDL in alternate wells is required to detect specific binding of the labeled material. We have often used large quantities of LDL prepared from the pheresis plasma of an FHC homozygote for this purpose. We also induced LDL receptors by growing cells in 7 mg/ml of LPDS for 72 h, a larger amount and longer time than customarily used.
The major classification scheme for FHC fibroblasts has utilized principally an indirect method for determination of LDL receptor function, eg., radiolabeled oleate incorporation into cholesteryl esters after exposure of cells to LDL (2, 4). Receptor-negative cells revealed no oleate incorporation in response to LDL whereas receptor-defective cells had 5-20% of normal incorporation. These are measurements of the acute effect of metabolized LDL on cells, not the actual LDL processed by surface binding, internalization, and degradation. Since low levels of specific LDL receptor activity can be identified in at least some receptor-negative fibroblasts, we believe that the phenotype of homozygous FHC may be associated with a spectrum of qualitative and quantitative receptor abnormalities encompassing the functional categories receptor-negative and receptor-defective. Since it is agreed that almost all homozygous FHC fibroblasts are severely deficient in LDL receptor function, future studies should more clearly define the exact nature of the LDL binding abnormalities present in these cells.