©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Recognition of -Antitrypsin-Elastase and -Antichymotrypsin-Cathepsin G Complexes by the Low Density Lipoprotein Receptor-related Protein (*)

(Received for publication, June 10, 1994; and in revised form, November 9, 1994)

Wolfgang Poller (§) Thomas E. Willnow (¶) Jan Hilpert (**) Joachim Herz (§§)

From the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9046

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two multifunctional receptors, low density lipoprotein receptor-related protein (LRP) and gp330, have been implicated in the cellular uptake and degradation of a wide spectrum of functionally diverse ligands including plasma lipoproteins, proteases, and proteinase-inhibitor complexes. The two receptors show distinct tissue-specific expression patterns, suggesting different physiological functions. We have examined the cellular degradation of two serine proteinase inhibitor (serpin)-protease complexes, alpha(1)-antitrypsin-neutrophil elastase (alpha(1)AT bullet NEL) and alpha(1)-antichymotrypsin-cathepsin G (alpha(1)ACT bullet CathG) by normal murine fibroblasts (MEF) expressing LRP, and by a mutant fibroblast cell line (PEA13) which is genetically deficient for LRP. alpha(1)AT bullet NEL complexes bound to LRP on ligand blots and were degraded efficiently by the MEF cells, but not by PEA13 cells. Degradation of the complexes was also significantly reduced by antibodies directed against LRP, further suggesting that fibroblasts require LRP for the cellular uptake and degradation of alpha(1)AT bullet NEL complexes. In contrast to alpha(1)AT bullet NEL, MEF cells did not degrade alpha(1)ACT bullet CathG complexes. However, these complexes were rapidly degraded by the rat embryonal carcinoma cell line L2p58 which abundantly expresses gp330, raising the possibility that the alpha(1)ACT bullet CathG complex might be recognized by gp330. Both complexes were efficiently metabolized by the hepatoma cell line HepG2, presumably involving the serpin-enzyme complex receptor. The differential recognition of serpin-protease complexes by fibroblasts and hepatoma cells, however, indicates that LRP, gp330, and the serpin-enzyme complex receptor are distinct proteins.


INTRODUCTION

The low density lipoprotein receptor-related protein (LRP) (^1)(1) and the large glycoprotein gp330 (2) are complex, multifunctional endocytic receptors which mediate the cellular uptake of a heterogeneous spectrum of ligands, including apoE-rich remnant lipoproteins(3, 4) , lipoprotein lipase (5) , proteinase- or methylamine-activated alpha(2)-macroglobulin (alpha(2)M)(6, 7) , plasminogen activator-inhibitor complexes(4, 8, 9) , the active protease tissue-type plasminogen activator(10) , pro-urokinase(11) , and exotoxin A from Pseudomonas aeruginosa (PEA)(12) . Plasminogen activator inhibitor-1 which apparently binds to an independent site on LRP (13) belongs to the serine proteinase inhibitor (serpin) superfamily. Recently, a role for LRP in the removal of tissue factor pathway inhibitor has also been reported, thus further underscoring the role of this receptor in the regulation of extracellular protease activity(14) .

The spectrum of ligands that bind to LRP and gp330 is very similar(4) , although binding of alpha(2)M and PEA to gp330 has so far not been reported. LRP and gp330 both belong to the LDL receptor gene family which also includes a recently described very low density lipoprotein receptor(15) , and the LDL receptor itself(16) . The activity of LRP and gp330 has been proposed to be modulated in vivo by a receptor-associated protein (RAP) of approximately 39 kDa which blocks the binding of all known ligands to either receptor (4, 8, 9, 10, 11, 12, 17, 18, 19) .

The distinct tissue-specific expression patterns of the two receptors suggest different physiological functions. Whereas gp330 is highly expressed in the glomerular and proximal tubular epithelia of the kidney (20) and in the type II pneumocytes of the lung(21) , LRP is predominantly expressed in the hepatocytes, the neurons of the brain, fibroblasts, and macrophages(22, 23) . Targeted disruption of the LRP gene in mice prevents development of homozygous defective embryos beyond day 13.5 post coitum(24, 25) . Complete loss of all functions associated with LRP is obviously incompatible with survival beyond the embryonic stage, but loss or impairment of individual functions of LRP concerning a specific ligand might cause more limited damage in organs or tissues most dependent on this specific function. Identification of differential and/or tissue-specific recognition of a ligand by the two receptors LRP and gp330 might thus allow conclusions relating to their specific functions in vivo.

The high expression of gp330 in type II pneumocytes (21) suggests a specific role for this receptor in the lung and especially in the alveolar space. In contrast, in the lung LRP is preferentially expressed in the alveolar macrophages(23) . Because LRP and gp330 are expressed by defined cell populations within the lung, we have examined their possible role in the removal of complexes of the two closely related serpins alpha(1)-antitrypsin (alpha(1)AT) and alpha(1)-antichymotrypsin (alpha(1)ACT), both of which are expressed by lung alveolar macrophages and detectable in the bronchoalveolar lavage fluid in variable concentrations(26, 27) . alpha(1)AT and alpha(1)ACT are 42% homologous to each other on the amino acid level(28, 29) , and both are encoded by genes which lie in close proximity to each other on chromosome 14q32.1(30) . alpha(1)AT is known to protect lung structures against proteolytic damage by neutrophil elastase (NEL), which is specifically complexed and inactivated by alpha(1)AT(31) . Severe genetic alpha(1)AT deficiency is associated with a 20-30-fold increased risk for the development of chronic obstructive pulmonary disease, due to insufficient inhibition of elastolytic activity(32, 33) . Recently, defective mutants of alpha(1)ACT have also been described in patients with chronic obstructive pulmonary disease(34, 35) . Both inhibitors, alpha(1)AT and alpha(1)ACT, are apparently taken up and degraded together with the complexed proteases via a distinct SEC receptor which has been described in hepatoma cells and monocytes(36, 37) .

In the present study we have investigated possible roles of LRP and gp330 for the binding and cellular uptake of alpha(1)ATbulletNEL and alpha(1)ACTbulletCathG complexes by exploring the recognition of these ligands by cell types expressing gp330, LRP, or neither receptor, as well as by HepG2 cells which contain the SEC receptor.


MATERIALS AND METHODS

Wild type (MEF) and LRP-deficient (PEA13) murine embryonic fibroblasts were isolated as described earlier(38) . The L2p58 rat yolk sac carcinoma cell line was kindly provided by Dr. Marilyn Farquhar, UCSD, La Jolla. Human alpha(2)-macroglobulin activated with methylamine (alpha(2)M) was a gift from Dudley Strickland (American Red Cross, Rockville, MD). Recombinant glutathione S-transferase or a fusion protein of glutathione S-transferase and rat RAP were produced in DH5alpha bacteria as described(17) . Human NEL, human leukocyte cathepsin G (CathG), human alpha(1)AT, and human alpha(1)ACT were purchased from Sigma. Human plasma LDL was isolated and iodinated as described (39) . All other I-labeled ligands were radiolabeled by the IODO-GEN method(40) .

Proteinase-Inhibitor Complex Formation

10 µg of lyophilized NEL or CathG were dissolved in 100 µl of H(2)O and iodinated at 4 °C according to the manufacturer's protocol. The reaction was terminated by addition of sodium tyrosine to a final concentration of 0.25 mM and incubation for 2 min at 4 °C. Protease-inhibitor complexes were formed by addition of the protease inhibitor at a 3-5-fold molar excess and incubation at 22 °C for 30 min. After addition of phenylmethylsulfonyl fluoride to a final concentration of 2 mM, the complexes were separated from unincorporated I on a Sephadex G-25 column (Pharmacia LKB Biotechnology Inc.) equilibrated with phosphate-buffered saline. Complexes were stored at 4 °C for up to 48 h.

Ligand and Western Blotting

Partially purified membrane preparations from rat liver and kidney as well as from the various cell lines were prepared as described earlier (41) and separated by 3-10 or 4-15% SDS-gel electrophoresis under nonreducing conditions at 30 mA for 4 h at 4 °C. Proteins were transferred to nitrocellulose filters at 4 °C and processed for ligand blotting as follows: nitrocellulose strips were incubated in blotting buffer (50 mM Tris-HCl, 2 mM CaCl(2), 80 mM NaCl, 5% (w/v) bovine serum albumin, 0.1% (v/v) Triton X-100, pH 8) for 1 h at room temperature. The solution was replaced with blotting buffer containing the indicated iodinated ligands. After incubation for 1 h at room temperature, strips were washed 3 times for 10 min in blotting buffer without bovine serum albumin and exposed to DuPont Cronex film. The specific activities of the iodinated ligands were: I-GST-RAP, 1000-1200 cpm/ng; I-alpha(2)-macroglobulin, 350-1600 cpm/ng; I-LDL, 250 cpm/ng; alpha(1)AT/I-NEL, 3744-7100 cpm/ng; alpha(1)ACT/I-CathG, 6300-8200 cpm/ng. Western blots were performed using the ECL system (Amersham) according to manufacturer's recommendations.

Cellular Degradation of I-Labeled Ligands

All cells were grown in Dulbecco's modified Eagle's medium (DMEM, high glucose) supplemented with 10% fetal calf serum. 2 times 10^5 cells/well were seeded into 12-well plates and grown for 24 h prior to the addition of I-labeled ligands. For determination of LDL degradation, cells were grown for 48 h in medium containing 5% lipoprotein-deficient newborn calf serum prior to the degradation assay to induce expression of the LDL receptor. The medium was then replaced by DMEM (without glutamine) containing 0.2% (w/v) bovine serum albumin and the indicated iodinated ligands. Cellular degradation of I-labeled proteins was measured as described previously (39) and is expressed as nanograms of I-labeled trichloroacetic acid-soluble (non-iodide) material released into the culture medium per mg of total cell protein. Protein content of cell lysates was determined using the Coomassie Plus protein Assay Reagent (Pierce).


RESULTS

Complexes of I-labeled NEL and CathG with alpha(1)AT or alpha(1)ACT, respectively, were formed in vitro immediately following the radioiodination of the proteases as described under ``Materials and Methods'' and analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 1). In both cases the majority of the I-labeled proteases (lanes 1 and 3) was converted to an SDS-stable complex of higher molecular weight (lanes 2 and 4) as is typical for complexes of serpins with their target proteases. Preparations of rat liver (L) or rat kidney (K) membrane proteins were separated by electrophoresis on nonreducing 3-10% SDS-polyacrylamide gradient gels, transferred to nitrocellulose filters, and incubated with the labeled complexes or with I-labeled GST-RAP fusion protein (Fig. 2A, lanes 1 and 2) to demonstrate the presence of LRP and gp330 in the respective membrane preparations. alpha(1)ATbulletI-NEL complex bound only to LRP (lane 3) but not to gp330 (lane 4) on ligand blots. alpha(1)ACTbulletI-CathG complexes did not bind to either protein on ligand blots (not shown). The in vitro binding properties of the alpha(1)ATbulletI-NEL complex to LRP in partially purified liver membrane fractions were similar to those of other previously characterized LRP ligands (Fig. 2B), inasmuch as binding was dependent upon the presence of Ca and could be abolished by coincubation with EDTA (lane 2) or GST-RAP fusion protein (lane 4).


Figure 1: Protease-inhibitor complexes. 5 ng of I-elastase (I-NEL, lane 1), 20 ng of I-NEL complexed with alpha(1)-antitrypsin (alpha(1)ATbulletI-NEL, lane 2), 5 ng of I-cathepsin G (I-CathG, lane 3), and 5 ng of I-cathepsin G complexed with alpha(1)-antichymotrypsin (alpha(1)ACTbulletI-CathG, lane 4) were subjected to 4-15% SDS-gel electrophoresis, after which the proteins were transferred to nitrocellulose and exposed to DuPont Cronex film for 16 h (lane 2) or 5 days (lanes 1, 3, and 4) at -70 °C. The position of migration of the proteases, protease-inhibitor complexes, and molecular weight marker proteins (rainbow marker, Amersham) is indicated.




Figure 2: Binding of alpha(1)-antitrypsin-I-elastase to rat liver membranes. 700 µg of partially purified membrane proteins from rat liver and rat kidney were subjected to preparative nonreducing 3-10% SDS-gel electrophoresis and transferred to nitrocellulose membranes. The position of migration of LRP and gp330 (arrow), myosin (200 kDa), and phosphorylase b (97.4 kDa) in the gel is indicated. A, replicate nitrocellulose strips (approximately 50 µg of protein/strip) of liver (L) and kidney (K) membrane preparations were subjected to ligand blot analysis as described under ``Materials and Methods'' using 1 times 10^6 cpm/ml of I-labeled GST-RAP (lanes 1 and 2) or 1 times 10^6 cpm/ml alpha(1)ATbulletI-NEL complex (lanes 3 and 4). Blots were exposed to DuPont Cronex film for 24 h at -70 °C. B, replicate nitrocellulose strips (approximately 50 µg of protein/strip) of liver membrane proteins were preincubated at 22 °C for 30 min with either blotting buffer (lane 1) or blotting buffer containing 20 mM EDTA (lane 2), 30 µg/ml GST (lane 3), or 30 µg/ml GST-RAP (lane 4). Each strip was then incubated for 1 h with alpha(1)ATbulletI-NEL complex (1 times 10^6 cpm/ml), after which the strips were exposed to DuPont Cronex film for 24 h at -70 °C.



Cellular degradation experiments performed in cultured fibroblasts (Fig. 3) that either express LRP abundantly (MEF, open circles) or are genetically deficient for this receptor (PEA13, closed circles) confirmed the results obtained from the ligand blot experiments. alpha(1)ACTbulletI-CathG complexes that did not bind to LRP on ligand blots were not degraded by either cell line (Fig. 3A). In contrast, alpha(1)ATbulletI-NEL complexes which bound specifically to immobilized LRP were efficiently degraded by the LRP expressing MEF cells, but not by the PEA13 cells lacking LRP (Fig. 3B). As shown previously, PEA13 cells completely lack the ability to endocytose the LRP ligand alpha(2)-macroglobulin (Fig. 3C) due to absence of the receptor(38) , while they take up and degrade LDL normally (Fig. 3D). LDL does not bind to LRP, but to the related LDL receptor. Primary LRP-deficient fibroblasts isolated from 10.5 day mouse embryos homozygous for the LRP gene defect did also not degrade alpha(1)ATbulletI-NEL complexes, indicating that the inability of PEA13 cells to degrade this ligand resulted from the LRP gene defect and not from an unrelated clonal aberration (data not shown).


Figure 3: Degradation of multiple ligands by wild type and LRP-deficient murine embryonic fibroblasts. Replicate monolayers of wild type (MEF) or LRP-deficient (PEA13) murine embryonic fibroblasts received 1 ml of DMEM (without glutamine) containing 0.2% (w/v) bovine serum albumin and 500 ng/ml alpha(1)ACTbulletI-CathG complex (Panel A), 500 ng/ml alpha(1)ATbulletI-NEL complex (Panel B), 5 µg/ml I-alpha(2)M (Panel C), or 5 µg/ml I-LDL (Panel D). After incubation at 37 °C for the indicated time, the amount of radiolabeled degradation products secreted into the medium was determined. Each value represents the mean of duplicate incubations.



In contrast to the in vitro binding of alpha(1)ATbulletI-NEL complexes to LRP which was blocked by coincubation with GST-RAP fusion protein, degradation of alpha(1)ATbulletI-NEL by MEF cells (Fig. 4, lane 5) was reproducibly unaffected by the presence of GST-RAP in the culture medium (lane 6), while the degradation of I-alpha(2)M (lane 1) was completely blocked by GST-RAP (lane 2). Rabbit polyclonal antibodies directed against LRP (lane 3), but not preimmune IgG (lane 4), also substantially reduced the cellular degradation of I-alpha(2)M as well as of alpha(1)ATbulletI-NEL (lane 8), further indicating that LRP mediates the degradation of both these ligands. Degradation of alpha(1)ATbulletI-NEL (lane 7) and I-alpha(2)M (not shown) was largely inhibited by chloroquine, indicating that lysosomal processes are involved.


Figure 4: Inhibition of ligand degradation by anti-LRP IgG, GST-RAP, or chloroquine. Replicate monolayers of wild type murine embryonic fibroblasts received 1 ml of DMEM (without glutamine) containing 0.2% (w/v) bovine serum albumin and either 5 µg/ml I-alpha(2)M (lanes 1-4) or 500 ng/ml alpha(1)ATbulletI-NEL complex (lanes 5-9). In addition, the medium contained either 30 µg/ml GST (lanes 1 and 5), 30 µg/ml GST-RAP (lanes 2 and 6), 200 µg/ml anti-LRP IgG (lanes 3 and 8), 200 µg/ml preimmune IgG (lanes 4 and 9), or 100 µM chloroquine (lane 7). After 24 h incubation at 37 °C, the amount of radiolabeled degradation products secreted into the medium was determined. Each value represents the mean of duplicate incubations of multiple experiments. 100% values represent degradation rates in the absence of added reagents and range from 350 to 460 ng/mg of protein/h for alpha(2)M and from 7 to 14.6 ng/mg protein/h for alpha(1)ATbulletNEL complex.



We next explored the ability of four different cell lines, MEF, PEA13, HepG2, and the rat yolk sac carcinoma cell line L2p58, to degrade alpha(1)ACTbulletI-CathG complexes. HepG2 cells have been reported to express a not yet completely characterized SEC receptor (36, 37) and L2p58 cells have been found to express gp330 abundantly(42) . As shown in Fig. 5A, alpha(1)ACTbulletI-CathG complexes were not degraded by either MEF or PEA13 cells, however, they were efficiently metabolized by HepG2 cells and L2p58 cells. In contrast, I-alpha(2)M was degraded much more efficiently by MEF and by L2p58 than by HepG2 cells, indicating the presence of SEC receptors in HepG2 and L2p58 cells which are unrelated to LRP (Fig. 5B).


Figure 5: Degradation of alpha(1)ACTbulletI-CathG complex and I-alpha(2)M* by various cell lines. Replicate monolayers of HepG2 cells, L2p58 cells, and wild type (MEF) and LRP-deficient (PEA13) murine embryonic fibroblasts received 1 ml of DMEM (without glutamine) containing 0.2% (w/v) bovine serum albumin and either 500 ng/ml alpha(1)ACTbulletI-CathG complexes (Panel A) or 5 µg/ml I-alpha(2)M (Panel B). After incubation at 37 °C for the indicated time, the amount of radiolabeled degradation products secreted into the medium was determined. Each value represents the mean of duplicate incubations.



Similar to the degradation of alpha(1)ATbulletI-NEL complexes by murine fibroblasts (Fig. 4), GST-RAP also had no effect on the degradation of alpha(1)ACTbulletI-CathG complexes by L2p58 cells (Fig. 6, lane 5), while it effectively blocked I-alpha(2)M degradation (lane 2). As in MEF cells, degradation of these ligands by L2p58 cells was chloroquine sensitive (lanes 3 and 6).


Figure 6: Inhibition of ligand degradation in L2p58 cells by GST-RAP and chloroquine. Replicate monolayers of L2p58 cells received 1 ml of DMEM (without glutamine) containing 0.2% (w/v) bovine serum albumin and either 5 µg/ml I-alpha(2)M (1500 cpm/ng, lanes 1-3) or 500 ng/ml alpha(1)ACTbulletI-CathG complex (7000 cpm/ng, lanes 4-6). In addition, the medium contained 30 µg/ml GST (lanes 1 and 4), 30 µg/ml GST-RAP (lanes 2 and 5), or 100 µM chloroquine (lanes 3 and 6). After 9 h incubation at 37 °C, the amount of radiolabeled degradation products secreted into the medium was determined. Each value represents the mean of duplicate incubations. 100% values represent degradation rates in the absence of added reagents and are 20 ng/mg of protein/h for alpha(2)M and 8 ng/mg of protein/h for alpha(1)ACTbulletCathG complex.



L2p58 cells had been reported to abundantly express gp330. In an attempt to correlate the expression levels of LRP and gp330 in MEF cells and in L2p58 cells, respectively, we performed a semiquantitative Western blot analysis (Fig. 7). Crude membrane proteins from MEF (lanes 1 and 3) or L2p58 cells (lanes 2 and 4) were separated by SDS-gel electrophoresis and transferred to nitrocellulose membranes. LRP (lanes 1 and 2) and gp330 (lanes 3 and 4) were then detected by incubation of the filters with specific polyclonal antibodies. While MEF cells express LRP abundantly (lane 1) L2p58 cells express only low amounts of this protein (lane 2). In contrast, murine fibroblasts are completely devoid of gp330 (lane 3), whereas L2p58 cells contain this protein (lane 4).


Figure 7: Western blot analysis of L2p58 and MEF cells. Partially purified membrane proteins (40 µg in lanes 1 and 2, 20 µg in lanes 3 and 4) from MEF cells (M, lanes 1 and 3) and L2p58 cells (L, lanes 2 and 4) were subjected to nonreducing 4-15% SDS-gel electrophoresis and transferred to nitrocellulose membranes. The proteins were detected using 5 µg/ml anti-LRP IgG (lanes 1 and 2) or 5 µg/ml anti-gp330 IgG (lanes 3 and 4). Binding of the IgG was detected using the ECL system. The position of migration of LRP and gp330 (arrow), myosin (200 kDa), and phosphorylase b (97.4 kDa) in the gel is indicated.




DISCUSSION

We have studied the role of LRP in the cellular uptake and degradation of two homologous serpins, alpha(1)AT and alpha(1)ACT, complexed to their respective target proteases, NEL and CathG. We find that alpha(1)ATbulletNEL, but not alpha(1)ACTbulletCathG complexes, bind to LRP on ligand blots. Likewise, alpha(1)ATbulletI-NEL, but not alpha(1)ACTbulletI-CathG complexes, are efficiently internalized and degraded by fibroblasts expressing LRP. This degradation is reduced by polyclonal antibodies directed against LRP. Furthermore, murine embryonic fibroblasts genetically deficient for LRP did not degrade alpha(1)ATbulletNEL complexes indicating that this ligand is indeed endocytosed and degraded by normal murine fibroblasts in an LRP-dependent manner. HepG2 cells, in contrast, which have previously been reported to express a SEC receptor readily metabolized alpha(1)ACTbulletCathG (Fig. 5) and alpha(1)ATbulletNEL complexes (not shown) in agreement with earlier publications(36, 37, 43) . Both complexes were also taken up and degraded by L2p58 cells ( Fig. 5and data not shown) which besides LRP also express gp330 (Fig. 7). In contrast to other ligands recognized by LRP or gp330, degradation of neither alpha(1)ATbulletNEL nor of alpha(1)ACTbulletCathG complexes was affected by GST-RAP(4, 17) . Surprisingly, however, binding of alpha(1)ATbulletNEL complexes to LRP on ligand blots was effectively blocked by RAP. Although the reasons for this difference are not entirely clear, it is possible that the alpha(1)ATbulletNEL binding site of LRP immobilized on nitrocellulose filters may not be in its native conformation making it more sensitive to competition by RAP.

Our data support the existence of a SEC receptor on HepG2 cells which is unrelated to LRP, because HepG2 cells, but not fibroblasts, efficiently degrade alpha(1)ACTbulletCathG complexes. Although both cell types express LRP, as shown by their ability to degrade I-alpha(2)M, fibroblasts do so more effectively. This is a further indication for a physical distinction between the SEC receptor and LRP.

An involvement of gp330 in the metabolism of alpha(1)ACTbulletCathG complexes is possible, but not proven at the present time. Clearly, the SEC receptor in liver-derived cells is different from gp330 which is not expressed in the liver(4) . However, it is possible that the uptake and degradation of alpha(1)ACTbulletCathG complexes by L2p58 cells could involve gp330. alpha(1)AT and alpha(1)ACT both belong to the serpin class of protease inhibitors which includes about 20 different proteins including plasminogen activator inhibitor-1. The latter binds to both LRP and gp330 (4) and LRP can also bind alpha(1)ATbulletNEL complexes (this study). It is tempting to speculate that, for as yet unknown reasons, LRP and gp330 display a selective, but partially overlapping, ligand binding spectrum where some serpins might bind to one, but not to the other or to either receptor.

There is growing evidence that proteinase-antiproteinase complexes directly or indirectly initiate cellular signal transduction events. For example, alpha(1)AT-proteinase complexes mediate an increase of alpha(1)AT gene expression in human monocytes and macrophages (44) and are also a strong chemoattractant for neutrophils (45) . Similarly, alpha(1)ACTbulletCathG complexes stimulate the synthesis of acute phase proteins via interleukin-6(46) . Furthermore, receptor binding-competent alpha(2)M has been shown to elevate intracellular second messengers in murine peritoneal macrophages (47) and alpha(2)M has been reported to substantially increase the transforming growth factor-beta1 mediated proliferative response in smooth muscle cells(48) . There is no evidence that these signal transduction events are directly mediated by LRP, although such a possibility has been discussed for the SEC receptor. However, internalization and lysosomal degradation of these complexes by LRP and/or by another endocytic receptor (e.g. possibly gp330) could be an important regulatory mechanism which turns off the signal evoked by the binding of these complexes to another unrelated signaling receptor on the cell surface.

In summary, our data provide evidence for: 1) a role of LRP in the cellular metabolism of alpha(1)ATbulletNEL complexes, and 2) for differences in the mechanisms by which alpha(1)ATbulletNEL and alpha(1)ACTbulletCathG complexes are taken up and degraded by cells. alpha(1)ATbulletNEL, but not alpha(1)ACTbulletCathG complexes are bound and internalized by LRP which therefore appears to be distinct from the SEC receptor expressed on HepG2 cells. The availability of genetically defined cell lines which express or are devoid of LRP protein will continue to be valuable for future studies aiming at the analysis of receptor-ligand interactions of the two structurally and functionally closely related multifunctional receptors LRP and gp330.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant HL 20948 and a grant from the Perot Family Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Heisenberg Fellowship from the Deutsche Forschungsgemeinschaft. Current address: Medical University Clinic, Clinical Biochemistry and Pathobiochemistry, Versbacher Strasse 5, D-97080 Würzburg, Germany.

Recipient of a fellowship from the Deutsche Forschungsgemeinschaft during part of these studies.

**
Supported by the Biomedical Sciences Exchange Program and the Deutscher Akademischer Austauschdienst.

§§
Supported by the Syntex Scholar Program and is a Lucille P. Markey Scholar.

(^1)
The abbreviations used are: LRP, low density lipoprotein receptor-related protein; alpha(1)ACT, alpha(1)-antichymotrypsin; alpha(1)ACTbulletCathG, alpha(1)-antichymotrypsin-cathepsin G complexes; alpha(1)AT, alpha(1)-antitrypsin; alpha(1)ATbulletNEL, alpha(1)-antitrypsin-neutrophil elastase complexes; alpha(2)M, alpha(2)-macroglobulin activated with methylamine; CathG, cathepsin G; GST, glutathione S-transferase; LDL, low density lipoprotein; RAP, receptorassociated protein; NEL, neutrophil elastase; GST-RAP, GST-RAP fusion protein; DMEM, Dulbecco's modified Eagle's medium; serpin, serine proteinase inhibitor; SEC, serpin-enzyme complex.


ACKNOWLEDGEMENTS

We thank Wen-Ling Niu and John Dawson for excellent technical assistance and Edith Womack for assistance with cell culture. We are indebted to Mike Brown and Joe Goldstein for critical suggestions during the course of the study.


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