Specificity in Recognition of Amyloid- (cid:98) Peptide by the Serpin-Enzyme Complex Receptor in Hepatoma Cells and Neuronal Cells*

The serpin-enzyme complex (SEC) receptor was orig- inally identified using a synthetic peptide (peptide 105Y) based on the sequence of a candidate receptor- binding domain of (cid:97) 1-antitrypsin (1-AT) and was subsequently shown to be a receptor on the surface of hepa- tocytes, monocytes, and neutrophils for recognition of (cid:97) 1-AT-elastase and several other serpin-enzyme complexes (Perlmutter, D. H., Glover, G. I., Rivetna, M., Schasteen, C. S., and Fallon, R. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3753–3757). Studies of the minimal re- quirements for binding to SEC receptor (SEC-R) showed that a pentapeptide FVFLM within the carboxyl-termi- nal tail of (cid:97) 1-AT was sufficient for binding to SEC-R and interacted with SEC-R in a sequence-specific manner (Joslin, G., Krause, J. E., Hershey, A. D., Adams, S. P., Fallon, R. J., and Perlmutter, D. H. (1991) J. Biol. Chem. 266, 21897–21902). Sequence motifs bearing homology with this pentapeptide domain were found in the amy- loid- (cid:98) peptide, and amyloid- (cid:98) peptide 1–42 was shown to compete for binding to SEC-R on hepatoma cells (Joslin, G., Fallon, In this study we examined the sequence specificity by which amyloid- (cid:98) peptide competes for binding to SEC-R and examined the possibility that SEC-R is expressed in cells of neuronal origin. The results show that amyloid-(cid:98) -(25–35) and amyloid- (cid:98) -(31–35) compete for binding to SEC-R as effectively as amyloid- (cid:98) -(1–39), amyloid- (cid:98) -(1– 40), and amyloid- (cid:98) -(1–42). Amyloid- (cid:98) -(1–16) does not compete for binding to SEC-R. There is cross-competi- tion for binding to the same site by 125 I-peptide 105Y and amyloid- (cid:98) -(25–35) as well as by 125 I-Y amyloid- (cid:98) -(25–35) and peptide 105Y. By deletions and substitutions within amyloid- (cid:98) -(25–35) and generation of chimeric amyloid-(cid:98) - (cid:97) 1-AT peptides, amyloid- (cid:98) -(31–35) is shown to be critical for binding to the SEC receptor. However, the up- stream region, amyloid- (cid:98) -(25–30), also contributes to recognition by SEC-R. The SEC-R is present on the sur- face of a neuronal cell line PC12 as well as that of murine cortical neurons in primary culture, and the specificity of neuronal SEC-R for amyloid- (cid:98) peptide is identical to that on hepatoma cells. Finally, SEC-R mediates inter- nalization and degradation of amyloid (cid:98) -peptide in PC12 cells. These results provide evidence that SEC-R plays a role in metabolism of amyloid- (cid:98)

The serpin-enzyme complex receptor (SEC-R) 1 is a cell surface protein of hepatoma cells, mononuclear phagocytes, and neutrophils that has a ligand binding subunit of ϳ70 -78 kDa (reviewed in Ref. 1). It was originally identified as the receptor responsible for feed-forward regulation of ␣1-AT synthesis by complexes of neutrophil elastase and ␣1-AT. A synthetic peptide, peptide 105Y (SIPPEVKFNKPFVYLI), based on the sequence of a candidate receptor-binding domain in the carboxylterminal tail of ␣1-AT itself, was shown to mediate increases in synthesis of ␣1-AT in human monocytes and human hepatoma HepG2 cells (2). Radioiodinated peptide 105Y bound to HepG2 cells with specific, saturable, and reversible characteristics. Scatchard analysis predicted a k d of ϳ40 nM and ϳ450,000 plasma membrane receptors/cell (2). Binding of 125 I-peptide 105Y to HepG2 cells was blocked by unlabeled elastase-␣1-AT, cathepsin G-␣1-antichymotrypsin, thrombin-antithrombin III, thrombin-heparin cofactor II, and, to a lesser extent, C1s-C1 inhibitor and tissue plasminogen activator-plasminogen activator inhibitor I complexes but not by the corresponding native proteins (2,3). Binding of radioiodinated elastase-␣1-AT, cathepsin G-␣1-antichymotrypsin, thrombin-antithrombin III, thrombin-thrombin-heparin cofactor II, and tissue plasminogen activator-plasminogen activator inhibitor I to HepG2 cells was also partially, but specifically, blocked by peptide 105Y (2,3). Other studies have shown that SEC-R mediates endocytosis and intracellular catabolism of elastase-␣1-AT complexes in HepG2 cells (4) and mediates directed migration of neutrophils toward ␣1-AT-elastase complexes (5).
In studies designed to determine the minimal requirements for binding to SEC-R, a series of smaller peptides based on the sequence of peptide 105Y were synthesized (6). The studies showed that the carboxyl-terminal pentapeptide FVYLI and its counterpart in the native ␣1-AT sequence, FVFLM, were sufficient for binding to SEC-R and that interaction between these pentapeptides and SEC-R was sequence-specific. A search for homologous sequences in other eukaryotic proteins showed similarities in the carboxyl-terminal regions of substance P, several other tachykinins, bombesin, and amyloid-␤ peptide (6). In fact, the homologous region of amyloid-␤ peptide, amyloid-␤- (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35), is toxic to neurons (7). It constitutes the minimal peptide sequence for the neurotoxic effect of amyloid-␤-(1-42) (7) and, therefore, has been considered a possible common final pathway for the neuronal degeneration in Alzheimer's disease. Studies in HepG2 cells have subsequently shown that amyloid-␤ peptide competes for binding of radioio-dinated peptide 105Y and radioiodinated elastase-␣1-AT complexes to SEC-R (8). Moreover, binding of amyloid-␤ peptide to SEC-R in neutrophils results in chemotactic activity and confers homologous desensitization to the chemotactic activity of peptide 105Y (5).
In this study we examined the sequence specificity for binding of amyloid-␤ peptide to SEC-R of the model hepatoma cell line HepG2 and examined the possibility that SEC-R is expressed on the surface of neurons in primary culture and in a transformed neuronal cell line.
Since many of these peptides, particularly the amyloid-␤ peptides, are not soluble in H 2 0, all were prepared in Me 2 SO to ensure that they were completely dissolved and could be compared with each other.
Cell Culture-Maintenance of HepG2 cells has been previously described (4 (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35), and amyloid-␤-(1-40) were radioiodinated by the chloramine T method. 125 I-peptide 105Y and 125 I-amyloid-␤-(1-40) were purified on Biogel P2 and 125 I-Yam␤-(25-35) (YGSNKGAIIGLM) was purified on Sephadex G10. Specific radioactivity for 125 I-peptide 105Y and 125 I-Yam␤-(25-35) varied from 25,000 to 35,000 cpm/ng and 125 Iamyloid-␤-(1-40) from 6,000 to 10,000 cpm/ng. For binding studies in HepG2 cells, separate monolayers in 24-well plates were dunked in phosphate-buffered saline and incubated for 2 h at 4°C in 125 I-labeled ligand in the absence or presence of competitor and diluted in binding medium (Dulbecco's modified Eagle's medium containing 10 mM Hepes, 0.1 mg/ml cytochrome c, 0.01% Tween 80, 1 mg/ml bovine serum albumin). The cells were then rinsed in phosphate-buffered saline, and cell-associated radioactivity was determined in 1 N NaOH homogenates. For binding studies in PC12 cells and murine cortical neurons, cells were detached from tissue culture flasks, washed with phosphate-buffered saline supplemented with 3 mM EDTA, and then, in suspension, incubated for 2 h at 4°C with a 125 I-labeled ligand with and without competitors diluted in suspension binding medium (phosphate-buffered saline, 3 mM EDTA, 1 mg/ml bovine serum albumin, and 0.01% Tween 80). At the end of this time interval, cells were lysed in 1 N NaOH and cell-associated radioactivity was determined. Specific binding was defined as the difference between total binding in the absence of unlabeled competitor and nonspecific binding in the presence of unlabeled competitor. Nonspecific binding varied from 8 to 30% depending on individual radioligand and individual radioligand preparations. Methods for Scatchard analysis have been previously described (2).
Determination of Cellular Uptake, Internalization, and Degradation-HepG2 cells were washed with PBS and then incubated at 37°C for time intervals up to 6 h with 125 I-ligand diluted in uptake medium (Dulbecco's modified Eagle's medium containing 10 mM Hepes, 0.1 mg/ml cytochrome c, 1 mg/ml bovine serum albumin). At specific time intervals, cell culture fluid was harvested, and cells were homogenized in 1 N NaOH. Homogenates were then subjected to scintillation counting to determine "total uptake." Specific uptake was defined as the difference between total and nonspecific uptake. In other experiments, cell surface and internal fractions of cell-associated radioactivity were determined by incubating cell monolayers or cell pellets for an additional 1 h at 4°C in PBS with proteinase K (0.5 mg/ml). The effect of proteinase K was terminated by the addition of 1 mM phenylmethylsulfonyl fluoride. Cells were then detached by gentle agitation and pelleted by centrifugation. Radioactivity in these cell pellets was defined as proteinase K-resistant or internal fraction, and that in the supernatants was defined as proteinase K-sensitive or cell surface fraction. Radioactivity in the cell culture fluid was determined before and after precipitation with 20% trichloroacetic acid, 4% phosphotungstic acid. This procedure precipitated greater than 97% of the radioactivity present at time 0 in medium containing 125 I-amyloid-␤- .
In order to determine the distribution of 125 I-amyloid-␤ peptide-(1-40) during a single cycle of endocytosis we used a previously described protocol (10,11). HepG2 cells or PC12 cells were incubated for 2 h at 4°C with 125 I-amyloid-␤-(1-40) in saturating concentrations (100 nM). Cell monolayers were rinsed and then incubated at 37°C for several different time intervals in uptake medium alone. Cell culture fluid was harvested, and cell monolayers were rinsed and incubated for an additional 60 min in PBS alone or PBS containing proteinase K as described above to determine proteinase K-resistant uptake (internal fraction) and proteinase K-sensitive uptake (surface-bound fraction). Cell culture fluid was counted before (total extracellular ligand) and after acid precipitation (acid-soluble extracellular ligand).
In order to provide further evidence that amyloid-␤-(25-35) was involved in SEC-R binding and to do cross-competition studies, we first examined the direct binding of 125 I-Yam␤-(25-35) (YGSNKGAIIGLM) to HepG2 cells (Fig. 2a). There was specific and saturable binding. Scatchard analysis predicted a k d of ϳ32.1 nM and ϳ430,000 plasma membrane receptors/cell. This is very similar to the characteristics by which 125 I-peptide 105Y binds to HepG2 cells (2). In separate experiments using 125 I-amyloid-␤-(1-40) and unlabeled peptide 105Y in HepG2 cells, binding characteristics were also similar to those of 125 Ipeptide 105Y (k d of ϳ35.3 nM and ϳ2.3 ϫ 10 5 plasma membrane receptors/cell). Finally, in cross-competition studies, there was no significant difference between unlabeled Yam␤-(25-35) and peptide 105Y in competition for binding of 125 I-Yam␤-(25-35) to HepG2 cells (Fig. 2b).
Evidence for Expression of the SEC Receptor in Neuronal Cells-First, we examined the possibility that 125 I-peptide 105Y binds to the neuronal cell line PC12 (Fig. 3a). There was specific and saturable binding with a k d of ϳ32.3 nM and ϳ180,000 plasma membrane receptors/cell. The results were almost identical when 125 I-amyloid-␤-(1-40) and unlabeled peptide 105Y were used in PC12 cells (data not shown). Moreover, cross-competition studies showed that unlabeled amyloid- ␤-(25-35) and peptide 105Y were similar in efficacy of competition for binding of 125 I peptide 105Y and 125 I-Yam␤-(25-35) to PC12 cells (Fig. 3b). These results indicated that the SEC-R was expressed in at least one neuronal cell line and that it had similar characteristics in rat and human cells. In order to exclude the possibility that it was only present on transformed neuronal cells, we also examined binding of 125 I-peptide 105Y to cortical neurons in primary culture (Fig. 3c). The results indicate that there is specific and saturable binding. The point of half-maximal binding at ϳ30 -40 nM is similar to that seen for binding of peptide 105Y to HepG2 cells, neutrophils, and PC12 cells. Taken together, these results indicate that the SEC-R is expressed in neuronal cells.
Sequence Specificity by Which Amyloid-␤- (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) Binds to the SEC Receptor-On the basis of previous experience with the sequence specificity by which peptide 105Y and ␣1-AT interact with SEC-R (2-6, 8), we generated synthetic peptides based on the sequence of amyloid-␤-(25-35) but engineered with selected substitutions and deletions. First, these substituted and deleted peptides were examined as competitors for binding of 125 I-peptide 105Y to HepG2 cells (Fig. 4a). The results show that substitution of alanine for the carboxyl-terminal methionine (peptide am␤-(25-34A)) and deletion of carboxyl-terminal leucine and methionine residues (peptide am␤-(25-33)) markedly reduces competitive binding efficacy, but substitution of alanine for the amino-terminal residue (peptide am␤-(25A-35)) or deletion of two amino-terminal residues (peptide am␤-(27-35)) do not reduce competitive binding efficacy. Deletion of the carboxyl-terminal glycine-leucine-methionine sequence (peptide Yam␤-(22-32)) completely abrogates binding even if the overall length of the peptide remains similar by adding residues from the amyloid-␤ peptide sequence at the amino terminus. Again, the carboxyl-terminal pentapeptide amyloid-␤-(31-35) is almost as effective as amyloid-␤-(25-35) as a competitor. These data show that the carboxyl-terminal residues of amyloid-␤-(25-35) play an important role in binding to SEC-R. The results were similar when the same peptides were used as competitors for binding of 125 I-Yam␤-(25-35) (Fig.  4b), except that peptides with carboxyl-terminal substitutions and deletions were slightly more effective as competitors and the carboxyl-terminal pentapeptide was slightly less effective as a competitor. This suggests that the amino-terminal region of amyloid-␤-(25-35) may play a more important role than the amino-terminal region of peptide 105Y in recognition by SEC-R.
Next, we examined several chimeric amyloid-␤/␣1-AT peptides as competitors for binding of 125 I-peptide 105Y (Fig. 5a) and 125 I-Yam␤-(25-35) (Fig. 5b) to HepG2 cells. The amyloid-␤/␣1-AT chimeric had the amino-terminal domain of amyloid-␤ and carboxyl-terminal domain of ␣1-AT, and the ␣1-AT/amyloid-␤ chimera had the reverse. There were no significant differences in competitive binding efficacy of these chimeric peptides as compared with each other and as compared with the original peptides 105Y and amyloid-␤-(25-35). In this design, then, one could not discern a greater role for the amino-terminal domain of amyloid-␤-(25-35) than the corresponding domain of peptide 105Y in recognition by SEC-R.
Comparison of the amino-terminal region of amyloid-␤-(25-35) with the corresponding region of peptide 105Y reveals a conserved asparagine-lysine sequence. In peptide 105Y this is separated from the carboxyl-terminal pentapeptide by a single proline residue, whereas the sequence in amyloid-␤-(25-35) is separated from the carboxyl-terminal pentapeptide by two residues, glycine-arginine. We examined the significance of this difference by generating synthetic peptides in which the intervening proline and glycine-arginine residues were swapped-i.e. GSNK-P-IIGLM; VKFNK-GA-FVFLM (Fig. 6). The results show no significant differences from peptide 105Y or amyloid-␤-(25-35) as competitors for binding of 125 I-peptide 105Y (Fig.  6a) or of 125 I-Yam␤-(25-35) (Fig. 6b). In contrast, however, substitution of two threonine residues for the two isoleucine residues at amyloid-␤ 31-32 (peptide am␤-(25-35)TT) markedly decreases the efficacy of competition for binding to SEC-R. These data provide further evidence that the carboxyl-terminal pentapeptide is critical for binding to SEC-R but also suggest that relative hydrophobicity is required even at residues 31-32.
Using the same deleted, substituted, chimeric, and swapped peptides, the sequence specificity of recognition of amyloid-␤ peptide by SEC-R was similar, if not identical, in PC12 cells and HepG2 cells (data not shown). Taken together, these data show that the carboxyl-terminal residues of amyloid-␤-(25-35) are particularly important in binding to SEC-R in hepatoma and neuronal cells.
Internalization of Amyloid-␤ Peptide Mediated by the SEC Receptor-Next, we examined the possibility that SEC-R mediates endocytosis and intracellular catabolism of amyloid-␤ peptide. First, we examined internalization of 125 I-amyloid-␤-(1-40) in the absence or presence of excess unlabeled peptide 105Y in HepG2 cells (Fig. 7). There was specific, time-dependent, and saturable internalization with steady state reached by 120 min. Kinetics of internalization of 125 I-Yam␤- (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) in HepG2 cells and internalization of 125 I-Yam␤-(25-35) and 125 Iamyloid-␤-(1-40) in PC12 cells were similar (data not shown). We also subjected 125 I-amyloid-␤-(1-40) to single cohort studies in PC12 cells (Fig. 8) to determine the distribution of amyloid-␤ peptide during a single cycle of endocytosis. PC12 cells were incubated for 2 h at 4°C with 125 I-amyloid-␤-(1-40) in saturating concentrations. Cell monolayers were rinsed extensively and then incubated at 37°C in uptake medium without any labeled ligand to examine the fate of the prebound cohort of labeled ligand. At several different time intervals, cell culture fluid was harvested, and cell monolayers were rinsed extensively again and incubated at 4°C for an additional 1 h in PBS alone or PBS containing proteinase K to determine total uptake, proteinase K-resistant uptake (internal fraction), and proteinase K-sensitive uptake (surface-bound fraction). Cell culture fluid was subjected to scintillation counting before and after acid precipitation to determine the kinetics of dissociation of prebound ligand and the kinetics of appearance of acidsoluble ligand degradation products, respectively. The results show that once bound to cell surface receptor, labeled amyloid-␤-(1-40) disappears from the cell surface within 5-10 min (proteinase K-sensitive fraction) coincident with its accumulation within the cells (proteinase K-resistant fraction) and dissociation from cell surface into the extracellular fluid (total extracellular ligand). During the next 40 min the intracellular concentration of labeled amyloid-␤-(1-40) decreases coincident with the appearance of ligand degradation products in the extracellular fluid (acid-soluble extracellular ligand).
The data provide evidence that amyloid-␤-(1-40), when soluble and recognized by SEC-R, is internalized and degraded in cells of neuronal origin. Furthermore, the kinetics of endocytosis and degradation are similar to that for another SEC-R ligand, the ␣1-AT-elastase complex (4).

I-Yam␤-(25-35) (b) in HepG2 cells by swapped amyloid-␤/
␣1-AT peptides. The procedure was exactly as described in the legend to Fig. 1.   FIG. 7. Internalization of 125 I-amyloid-␤ peptide-(1-40) in HepG2 cells. Cells were incubated for several different time intervals (horizontal axis) at 37°C in uptake medium, 125 I-amyloid-␤ peptide at saturating concentrations (100 nM), in the absence of competitor (Total), or in the presence of unlabeled peptide 105Y in 200-fold molar excess (Nonspecific). At the end of each time interval the cells were washed and incubated for 1 h at 4°C in phosphate-buffered saline supplemented with proteinase K. The effect of proteinase K was terminated by the addition of phenylmethylsulfonyl fluoride (1 mM). Cells were then detached by gentle agitation, pelleted by centrifugation, and washed, and the pellets were lysed for ␥ counting. Specific internalization represents the difference between total and nonspecific.
There is an asparagine-lysine sequence within the aminoterminal domain of amyloid-␤- (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) and within the corresponding region of peptide 105Y, but it is separated from the carboxyl-terminal pentapeptide by two amino acids, glycinealanine, in amyloid-␤-(25-35) as compared with one amino acid, proline, in peptide 105Y. The length or sequence in this region does not appear to affect receptor binding for either ligand. In contrast, substitution of the two isoleucine residues at positions 31 and 32 of amyloid-␤-(25-35) with two threonine residues significantly decreases binding to SEC-R. These data on sequence specificity will be useful in designing receptor agonists and antagonists.
Taken together with our previous studies (5,8), the data reported here provide extensive pharmacological evidence that soluble amyloid-␤ peptide and peptide 105Y, based on the sequence of ␣1-AT, bind to the same site, called SEC-R, on HepG2 cells, PC12 cells, and neutrophils. First, amyloid-␤-(1-42) competes for cross-linking of an ϳ70 -76-kDa radiolabeled polypeptide in HepG2 cells by a photoreactive derivative of peptide 105Y (8). The cross-linking of this ϳ76-kDa polypeptide is highly specific as shown by positive and negative control competitors and by the absence of any cross-linking in a SEC-Rnegative cell line CHO (8). Second, there is cross-competition for binding to HepG2 cells by 125 I-peptide 105Y and unlabeled amyloid-␤-(1-42) and by 125 I-amyloid-␤-(1-42) and unlabeled peptide 105Y (8). Third, we show here that the region within amyloid-␤-(1-42) responsible for this cross-competition is amyloid-␤-(25-35) (Fig. 1, a and b) and that there is crosscompetition for binding to HepG2 cells (Fig. 2b) and PC12 cells (  (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) to HepG2 to a similar extent (Fig. 5). Seventh, receptor-mediated endocytosis of 125 I-Yam␤-(25-35) is blocked by peptide 105Y (Fig. 6). Eighth, peptide 105Y completely abrogates the neutrophil chemotactic effect of amyloid-␤-(25-35) by homologous desensitization (3), providing additional pharmacological evidence that peptide 105Y and amyloid-␤ peptide bind to the same receptor in neu- At the end of each time interval the extracellular medium was harvested, and the cells were incubated for another 1 h at 4°C in PBS with proteinase K. Phenylmethylsulfonyl fluoride was added to 1 mM, and the cells were pelleted by centrifugation. The supernatant of this initial centrifugation was counted as the proteinase-K-sensitive cell fraction (cell surface-bound radioactivity). The cell pellet was washed extensively in PBS/1 mM phenylmethylsulfonyl fluoride, and then lysed and counted as the proteinase K-resistant cell fraction (internalized radioactivity). The extracellular medium was counted first (total extracellular ligand-dissociated ligand). This medium was then subjected to trichloroacetic acid-phosphotungstic acid precipitation, and the supernatant was counted as the acid-soluble extracellular ligand (ligand degradation products). The results are representative of three separate experiments.
trophils. It will be necessary, however, to isolate cDNA clones for SEC-R and express SEC-R cDNA in a heterologous cell to provide definitive evidence that peptide 105Y and soluble amyloid-␤-peptide both bind to it.
The results of this study may also have important implications for the pathogenesis of Alzheimer's disease (17)(18)(19)(20). Amyloid-␤ peptide is known to be a major constituent of the amyloid plaques found in the brains of individuals with Alzheimer's disease. There is a growing body of evidence that the amyloid-␤ peptide is generated as a free soluble peptide under physiologic circumstances as a result of an alternative post-translational processing pathway (12)(13)(14)(15)(16). In Alzheimer's disease there is greater use of this alternative processing pathway and more amyloid-␤ peptide generated (21)(22)(23). Presumably, this amyloid-␤ peptide accumulates in the brain and over time undergoes aggregation. A structure termed the "mature plaque" is found in brain during aging. Ultimately there is ingrowth of neurites, neurofibrillary degeneration, and ingrowth/activation of microglia into the plaque to form the "senile plaque" associated with the development of dementia (17)(18)(19)(20). The results of the current experiments indicate that SEC-R can mediate clearance and intracellular catabolism of soluble amyloid-␤ peptide in cells that are derived from the central nervous system. Perhaps, it functions to protect the CNS from excessive extracellular amyloid-␤ peptide accumulation but gets overwhelmed over time in individuals predisposed to Alzheimer's disease.
The results of this study are only applicable to amyloid-␤ peptide that is completely soluble. In a previous study using radioiodinated amyloid-␤ peptides there was no evidence for receptor-mediated endocytosis and degradation in human skin fibroblasts (24). These results could be due to the substantial degree of variability in expression of SEC-R in human skin fibroblast cell lines. Some skin fibroblasts do not express SEC-R. Even in human skin fibroblast cell lines that express SEC-R, there is a much lower number of SEC-R molecules/cell than in HepG2 cells or PC12 cells. 2 It will now be informative to compare the relative binding of insoluble amyloid-␤ peptides with that of soluble amyloid-␤ peptides, as studied here, in cell lines such as PC12 and HepG2, which express a substantial number of SEC-R molecules per cell. A number of studies over the last several years have shown that amyloid-␤ peptide- (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35), and peptides that contain the region corresponding to amyloid-␤-(25-35), when prepared in an insoluble, aggregated form, are toxic to neurons (7,(25)(26)(27)(28)(29). The most recent of these studies suggest that the toxic interaction of insoluble amyloid-␤ peptide-(25-35) with cells is not sequence-specific but is corre-lated with formation of insoluble fibrils (30) or correlated with the sequence specificity required for formation of insoluble fibrils (31).