Low Density Lipoprotein Receptor-related Protein-1 Promotes β1 Integrin Maturation and Transport to the Cell Surface
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m306625200
ISSN1083-351X
AutoresAna M. Salicioni, Alban Gaultier, Cristina Brownlee, Michael K. Cheezum, Steven L. Gonias,
Tópico(s)Peptidase Inhibition and Analysis
ResumoLow density lipoprotein receptor-related protein-1 (LRP-1) mediates the endocytosis of multiple plasma membrane proteins and thereby models the composition of the cell surface. LRP-1 also functions as a catabolic receptor for fibronectin, limiting fibronectin accumulation in association with cells. The goal of the present study was to determine whether LRP-1 regulates cell surface levels of the β1 integrin subunit. We hypothesized that LRP-1 may down-regulate cell surface β1 by promoting its internalization; however, unexpectedly, LRP-1 expression was associated with a substantial increase in cell surface β1 integrin in two separate cell lines, murine embryonic fibroblasts (MEFs) and CHO cells. The total amount of β1 integrin was unchanged because LRP-1-deficient cells retained increased amounts of β1 in the endoplasmic reticulum (ER). Expression of human LRP-1 in LRP-1-deficient MEFs reversed the shift in subcellular β1 integrin distribution. Metabolic labeling experiments demonstrated that the precursor form of newly synthesized β1 integrin (p105) is converted into mature β1 (p125) more slowly in LRP-1-deficient cells. Although low levels of cell surface β1 integrin, in LRP-1-deficient MEFs, were associated with decreased adhesion to fibronectin, the subcellular distribution of β1 integrin was most profoundly dependent on LRP-1 only after the cell cultures became confluent. A mutagen-treated CHO cell line, in which LRP-1 is expressed but retained in the secretory pathway, also demonstrated nearly complete ER retention of β1 integrin. These studies support a model in which LRP-1 either directly or indirectly promotes maturation of β1 integrin precursor and thereby increases the level of β1 integrin at the cell surface. Low density lipoprotein receptor-related protein-1 (LRP-1) mediates the endocytosis of multiple plasma membrane proteins and thereby models the composition of the cell surface. LRP-1 also functions as a catabolic receptor for fibronectin, limiting fibronectin accumulation in association with cells. The goal of the present study was to determine whether LRP-1 regulates cell surface levels of the β1 integrin subunit. We hypothesized that LRP-1 may down-regulate cell surface β1 by promoting its internalization; however, unexpectedly, LRP-1 expression was associated with a substantial increase in cell surface β1 integrin in two separate cell lines, murine embryonic fibroblasts (MEFs) and CHO cells. The total amount of β1 integrin was unchanged because LRP-1-deficient cells retained increased amounts of β1 in the endoplasmic reticulum (ER). Expression of human LRP-1 in LRP-1-deficient MEFs reversed the shift in subcellular β1 integrin distribution. Metabolic labeling experiments demonstrated that the precursor form of newly synthesized β1 integrin (p105) is converted into mature β1 (p125) more slowly in LRP-1-deficient cells. Although low levels of cell surface β1 integrin, in LRP-1-deficient MEFs, were associated with decreased adhesion to fibronectin, the subcellular distribution of β1 integrin was most profoundly dependent on LRP-1 only after the cell cultures became confluent. A mutagen-treated CHO cell line, in which LRP-1 is expressed but retained in the secretory pathway, also demonstrated nearly complete ER retention of β1 integrin. These studies support a model in which LRP-1 either directly or indirectly promotes maturation of β1 integrin precursor and thereby increases the level of β1 integrin at the cell surface. The β1 integrin subunit associates with multiple α-subunits to form transmembrane adhesion receptors for extracellular matrix proteins, including collagen, fibronectin, vitronectin, and laminin (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9235) Google Scholar). Once present at the cell surface, mature integrins anchor the plasma membrane to the actin cytoskeleton and promote cell signaling (2Schwartz M.A. Ginsberg M.H. Nat. Cell Biol. 2002; 4: E65-68Crossref PubMed Scopus (685) Google Scholar, 3Fassler R. Pfaff M. Murphy J. Noegel A.A. Johansson S. Timpl R. Albrecht R. J. Cell Biol. 1995; 12 8: 979-988Crossref Scopus (217) Google Scholar). Integrin and growth factor-initiated cell signaling responses are integrated by the cell to regulate gene expression, cell migration, cell growth, apoptosis, and development (2Schwartz M.A. Ginsberg M.H. Nat. Cell Biol. 2002; 4: E65-68Crossref PubMed Scopus (685) Google Scholar). Altered cell surface integrin expression may be particularly important in cancer, impacting on various aspects of cancer metastasis (4Varner J.A. Cheresh D.A. Curr. Opin. Cell Biol. 1996; 8: 724-730Crossref PubMed Scopus (469) Google Scholar, 5Mizejewski G.J. Proc. Soc. Exp. Biol. Med. 1999; 222: 124-138Crossref PubMed Scopus (381) Google Scholar). Understanding mechanisms that regulate the concentration of integrins in the plasma membrane is an important problem. The β1 integrin subunit is synthesized as an 87-kDa polypeptide that undergoes glycosylation in the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; LRP-1, low density lipoprotein receptor-related protein-1; RAP, receptor-associated protein; uPAR, urokinase receptor; APP, amyloid precursor protein; GST, glutathione S-transferase; Endo-H, endoglycosidase H; PNGase F; peptide-N-glycosidase F; EGFr, epidermal growth factor receptor; MEF, murine embryonic fibroblast; BSA, bovine serum albumin; FBS, fetal bovine serum; PBS, phosphate-buffered saline; CHO, Chinese hamster ovary. and in the Golgi apparatus (6Akiyama S.K. Yamada K.M. J. Biol. Chem. 1987; 262: 17536-17542Abstract Full Text PDF PubMed Google Scholar, 7Argraves W.S. Suzuki S. Arai H. Thompson K. Pierschbacher M.D. Ruoslahti E. J. Cell Biol. 1987; 105: 1183-1190Crossref PubMed Scopus (449) Google Scholar). In the ER, the most prevalent, incompletely glycosylated form of β1 has a mass of 105 kDa and is thus referred to as p105. Mature β1 has a mass of ∼125 kDa (p125). In some cells, p105 is the primary form of β1 integrin identified; however, p105 is not found at the cell surface and cannot function in cell adhesion or cell signaling (6Akiyama S.K. Yamada K.M. J. Biol. Chem. 1987; 262: 17536-17542Abstract Full Text PDF PubMed Google Scholar, 8De Strooper B. Van Leuven F. Carmeliet G. Van Den Berghe H. Cassiman J.J. Eur. J. Biochem. 1991; 199: 25-33Crossref PubMed Scopus (35) Google Scholar). Many factors control maturation of β1 and its transfer to the cell surface, including the availability of α-subunits, growth factors such as transforming growth factor-β (TGF-β), and the state of activation of Ras (9Ignotz R.A. Massague J. Cell. 1987; 51: 189-197Abstract Full Text PDF PubMed Scopus (418) Google Scholar, 10Heino J. Ignotz R.A. Hemler M.E. Crouse C. Massague J. J. Biol. Chem. 1989; 264: 360-368Google Scholar, 11Bellis S.L. Newman E. Friedman E.A. J. Cell. Physiol. 1999; 181: 33-44Crossref PubMed Scopus (38) Google Scholar). The membrane-bound protein chaperone, calnexin, associates with β1 in the ER, promoting integrin assembly but inhibiting integrin transfer to the cell surface (12Lenter M. Vestweber D. J. Biol. Chem. 1994; 269: 12263-12268Abstract Full Text PDF PubMed Google Scholar, 13Hotchin N.A. Gandarillas A. Watt F.M. J. Cell Biol. 1995; 128: 1209-1219Crossref PubMed Scopus (128) Google Scholar). Other protein chaperones, including calreticulin and receptor-associated protein (RAP), may associate with β1-containing integrins or integrin-based adhesion complexes; however, a role for these proteins in integrin maturation has not been defined (14Coppolino M. Leung-Hagesteijn C. Dedhar S. Wilkins J. J. Biol. Chem. 1995; 270: 23132-23138Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 15Tran H. Pankov R. Tran S.D. Hampton B. Burgess W.H. Yamada K.M. J. Cell Sci. 2002; 115: 2031-2040Crossref PubMed Google Scholar). Talin, which is best known for its role in focal adhesion assembly, and HEMCAM/gicerin, an immunoglobulin superfamily protein, also may regulate β1 maturation (16Martel V. Vignoud L. Dupe S. Frachet P. Block M.R. Albiges-Rizo C. J. Cell Sci. 2000; 113: 1951-1961Crossref PubMed Google Scholar, 17Alais S. Allioli N. Pujades C. Duband J.L. Vainio O. Imhof B.A. Dunon D. J. Cell Sci. 2001; 114: 1847-1859Crossref PubMed Google Scholar). LRP-1 is a receptor for over 40 soluble ligands, which undergoes rapid and constitutive endocytosis in clathrin-coated pits, delivering most bound ligands to lysosomes for degradation (18Strickland D.K. Gonias S.L. Argraves W.S. Trends Endocrinol. Metabol. 2002; 13: 66-74Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). In some cells, LRP-1 may also localize in caveolae/lipid rafts, where it functions in cell signaling (19Boucher P. Liu P. Gotthardt M. Hiesberger T. Anderson R.G. Herz J. J. Biol. Chem. 2002; 277: 15507-15513Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 20Herz J. Strickland D.K. J. Clin. Invest. 2001; 108: 779-784Crossref PubMed Scopus (911) Google Scholar). In addition to soluble ligands, LRP-1 mediates the endocytosis of other plasma membrane proteins, including the urokinase receptor (uPAR), tissue factor, and amyloid precursor protein (APP), and thereby down-regulates the plasma membrane levels of these proteins (21Conese M. Nykjaer A. Petersen C.M. Cremona O. Pardi R. Andreasen P.A. Gliemann J. Christensen E.I. Blasi F. J. Cell Biol. 1995; 131: 1609-1622Crossref PubMed Scopus (196) Google Scholar, 22Weaver A.M. McCabe M. Kim I. Allietta M.M. Gonias S.L. J. Biol. Chem. 1996; 271: 24894-24900Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 23Webb D.J. Nguyen D.H. Gonias S.L. J. Cell Sci. 2000; 113: 123-134Crossref PubMed Google Scholar, 24Ulery P.G. Beers J. Mikhailenko I. Tanzi R.E. Rebeck G.W. Hyman B.T. Strickland D.K. J. Biol. Chem. 2000; 275: 7410-7415Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 25Narita M. Bu G. Olins G.M. Higuchi D.A. Herz J. Broze Jr., G.J. Schwartz A.L. J. Biol. Chem. 1995; 270: 24800-24804Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Regulation of the concentration and activity of membrane proteins represents an indirect mechanism by which LRP-1 may control cell signaling. For example, by down-regulating cell surface uPAR in murine embryonic fibroblasts (MEFs), LRP-1 suppresses activation of the small GTPase, Rac1, and inhibits cell migration (22Weaver A.M. McCabe M. Kim I. Allietta M.M. Gonias S.L. J. Biol. Chem. 1996; 271: 24894-24900Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 26Ma Z. Thomas K.S. Webb D.J. Moravec R. Salicioni A.M. Mars W.M. Gonias S.L. J. Cell Biol. 2002; 159: 1061-1070Crossref PubMed Scopus (94) Google Scholar). We previously demonstrated that LRP-1 binds fibronectin and mediates its endocytosis, limiting fibronectin accumulation in association with cell surfaces (27Salicioni A.M. Mizelle K.S. Loukinova E. Mikhailenko I. Strickland D.K. Gonias S.L. J. Biol. Chem. 2002; 277: 16160-16166Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). LRP-1 also is an endocytic receptor for thrombospondin-1 (28Godyna S. Liau G. Popa H Stefansson S. Argraves W.S. J. Cell Biol. 1995; 129: 1403-1410Crossref PubMed Scopus (129) Google Scholar). By modifying the composition of the extracellular matrix, LRP-1 may regulate processes such as cell adhesion and migration. In this study, we sought to determine whether LRP-1 regulates cell surface integrin expression. We hypothesized that LRP-1 may down-regulate basal levels of integrin subunits, at the cell surface, by facilitating integrin endocytosis. In support of this hypothesis, Czekay et al. (29Czekay R.-P. Aergeerts K. Curriden S.A. Loskutoff D.J. J. Cell Biol. 2003; 160: 781-791Crossref PubMed Scopus (272) Google Scholar) recently reported that plasminogen activator inhibitor-1 promotes αv integrin endocytosis by forming an integrin-containing multiprotein complex that is recognized and internalized by LRP-1. In this report, we demonstrate the unanticipated finding that LRP-1 expression is associated with a substantial increase in cell surface β1 integrin. The effects of LRP-1 are observed principally in confluent cell cultures and reflect the ability of LRP-1 to directly or indirectly promote maturation of newly synthesized β1 integrin in the secretory pathway. Regulation of β1 integrin maturation represents a novel mechanism by which LRP-1 may influence interactions of the cell with its microenvironment. Reagents and Proteins—Purified fibronectin, vitronectin and α5β1 integrin were purchased from Chemicon International (Temecula, CA). Type I collagen was obtained from BD Biosciences (Palo Alto, CA). Glutathione S-transferase (GST)-RAP was expressed in bacteria and purified as previously described (27Salicioni A.M. Mizelle K.S. Loukinova E. Mikhailenko I. Strickland D.K. Gonias S.L. J. Biol. Chem. 2002; 277: 16160-16166Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) using a construct obtained from Dr. Joachim Herz (University of Texas Southwestern Medical Center, Dallas, TX). As a control, GST without fused RAP, was also expressed and purified from bacteria transformed with the empty vector, pGEX-2T. Endoglycosidase H (Endo-H) and peptide-N-glycosidase F (PNGase F) were purchased from Roche Applied Science (Mannheim, Germany) and Sigma, respectively. Polyclonal antibody PAB1952, which recognizes the C-terminal cytoplasmic domain of β1 integrin, was obtained from Chemicon International (Temecula, CA). β1 integrin-specific polyclonal antibody 363 was kindly provided by Dr. Douglas DeSimone (University of Virginia). Polyclonal anti-epidermal growth factor receptor (EGFr) antibody was purchased from Upstate Biotechnology (Lake Placid, NY) and polyclonal anti-extracellular signal-regulated kinase (ERK/MAPK) antibody was from Zymed Laboratories Inc. (San Francisco, CA). TGF-β1,2,3-neutralizing antibody 1D11 was from R&D Systems. The activity of this antibody was confirmed in endothelial cell growth assays, as previously described by our laboratory (30Arandjelovic S. Freed T.A. Gonias S.L. Biochemistry. 2003; 42: 6121-6127Crossref PubMed Scopus (31) Google Scholar). Streptavidin-Sepharose, peroxidase-conjugated donkey anti-rabbit IgG and Protein A-Sepharose were from Amersham Biosciences. Trans35S-label, for metabolic labeling, was from ICN Biochemicals (Irvine, CA). The membrane-impermeable biotinylation reagent, sulfo-NHS-LC-biotin, was from Pierce. Cell culture media was from Invitrogen Life Technologies, Inc. Cell Culture—MEFs that are genetically deficient in LRP-1 (interchangeably called MEF-2 or PEA 13 cells), LRP-1(+/–) MEFs (PEA10), and wild-type MEFs (MEF-1 cells) were obtained from the ATCC. PEA10 and MEF-2 cells were cloned from the same culture of MEFs, heterozygous for LRP-1 gene disruption, after selection with Pseudomonas exotoxin A (31Willnow T.E. Herz J. J. Cell Sci. 1994; 107: 719-726Crossref PubMed Google Scholar). MEF-1 cells were isolated from the same mouse strain. All MEFs were cultured in Dulbecco's modified Eagle's medium with 10% FBS (HyClone Laboratories, Logan, UT). B41 cells are MEF-2 cells that were transfected for stable expression of full-length human LRP-1 (27Salicioni A.M. Mizelle K.S. Loukinova E. Mikhailenko I. Strickland D.K. Gonias S.L. J. Biol. Chem. 2002; 277: 16160-16166Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Wild-type Chinese hamster ovary (CHO K1) cells, LRP-1-deficient CHO 13–5-1 cells, and CHO 14–2-1 cells, in which LRP-1 is expressed but retained intracellularly without transfer to the cell surface (32FitzGerald D.J. Fryling C.M. Zdanovsky A. Saelinger C.B. Kounnas M. Winkles J.A. Strickland D. Leppla S. J. Cell Biol. 1995; 129: 1533-1541Crossref PubMed Scopus (100) Google Scholar), were cultured in Ham's F-12 medium, supplemented with 5% FBS optimized for CHO cells (HyClone Laboratories, Logan, UT), 2 mml-glutamine, 100 units/ml of penicillin, and 100 μg/ml of streptomycin. HT-1080 fibrosarcoma cells were obtained from the ATCC and cultured in MEM supplemented with 10% FBS and penicillin/streptomycin. In some experiments, MEFs were cultured in serum-free medium in the presence of GST-RAP (200 nm) or an equivalent concentration of GST (negative control) for 3 days. The medium and GST-RAP were replaced daily. To test the effects of different plating substrata, MEFs were cultured in 6-well plates that were pre-coated with 20% FBS, 5 μg/ml purified vitronectin, 10 μg/ml fibronectin, 25 μg/ml type I collagen or 10 μg/ml poly-l-lysine for 2 h at 37 °C. After precoating and before adding cells, the wells were blocked with 5 mg/ml bovine serum albumin (BSA) for 1 h. To block the function of α5β1, MEFs were pre-treated with 10 μg/ml monoclonal antibody BMA5 (Chemicon). Preparation of Cell Extracts, SDS-PAGE, and Immunoblotting— Cells were extracted in 1% Triton X-100, 0.125% Tween-20, 0.5% deoxycholate, 50 mm HEPES, pH 7.5, 0.5 m NaCl, 10 μg/ml aprotinin, 10 μg/ml E64, and 10 μg/ml leupeptin. To detect β1 integrin glycoforms, equal amounts of cellular protein were subjected to SDS-PAGE under non-reducing conditions in 7.5% gels, transferred to nitrocellulose membranes, and probed with primary antibody PAB1952 for 12 h, followed by peroxidase-conjugated donkey anti-rabbit IgG. Secondary antibody was visualized by enhanced chemiluminescence (Renaissance-ECL, PerkinElmer Life Sciences). Biotinylation and Recovery of Cell Surface Integrin Subunits—Monolayer cultures of MEFs and CHO cells were washed three times with ice-cold 20 mm sodium phosphate, 150 mm NaCl, pH 7.4 (PBS) to remove contaminating FBS and other soluble proteins and then treated with the membrane-impermeable biotinylation reagent, sulfo-NHS-LC-biotin (0.1 mg/ml), for 15 min at 22 °C, as described previously (27Salicioni A.M. Mizelle K.S. Loukinova E. Mikhailenko I. Strickland D.K. Gonias S.L. J. Biol. Chem. 2002; 277: 16160-16166Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Biotinylation reactions were terminated by adding 50 mm Tris-HCl, 150 mm NaCl, 100 mm glycine, pH 7.5 for 15 min at 22 °C. After washing with PBS, the cells were counted and lysed in extraction buffer. Biotinylated cell surface proteins were precipitated with Streptavidin-Sepharose (Amersham Biosciences). The affinity precipitates were recovered by centrifugation, washed, boiled in SDS sample buffer, and subjected to SDS-PAGE and immunoblot analysis to detect β1 integrin. Endoglycosidase Digestion—For PNGase F treatment, MEF extracts were diluted to a concentration of 1 mg/ml in 100 mm Tris-Cl, pH 7.4 with protease inhibitors, 50 mm β-mercaptoethanol and 0.1% SDS. The samples were then boiled for 2 min. After neutralization with Triton X-100 (0.5%), the samples were incubated with PNGase F (0.025–2.5 units/ml) at 37 °C for 12 h. A second dose of enzyme was added for an additional 12 h. For Endo-H treatment, MEF extracts were diluted to a concentration of 1 mg/ml in 100 mm sodium acetate, pH 5.5, containing 0.02% SDS and protease inhibitors. Samples were treated with Endo-H (30 milliunits/ml) at 37 °C for 12 h and then with a second dose of Endo-H for an additional 12 h. Enzymatic reactions were terminated by protein precipitation with ice-cold acetone. Pellets were air-dried. The samples were then neutralized and resuspended in SDS sample buffer for SDS-PAGE and immunoblot analysis. Metabolic-labeling of β1 Integrin—MEFs were seeded in 60-mm dishes in serum-containing medium, and allowed to grow until 100% confluent. Depletion of intracellular methionine was achieved by culturing in l-methionine/l-cysteine-free Dulbecco's modified Eagle's medium containing 5% FBS for 1 h. The FBS was predialyzed to remove associated l-cysteine and l-methionine. The cells were then labeled with 0.15 mCi/ml of TRAN35S-Label (which includes radioactive l-methionine and l-cysteine) for 1 h in the same medium. After labeling, the cells were harvested immediately (time 0) or chased for the indicated times in DMEM supplemented with 10% FBS and excess of non-radioactive l-methionine/l-cysteine (2 mm). Cell extracts were prepared by boiling for 10 min in 1% SDS, 20 mm Tris-HCl, 150 mm NaCl (TBS), 5 mm EDTA. The extracts were diluted 1:10 in TBS with 1% (v/v) Triton X-100 and protease inhibitor mixture (Roche Applied Science), subjected to centrifugation, and precleared protein-A Sepharose (Amersham Biosciences). β1 integrin subunit was recovered by immunoprecipitation with polyclonal antibody 363. The precipitates were then subjected to SDS-PAGE on 6% gels. The gels were fixed, dried, and exposed to intensifying screens. Radiolabeled β1 integrin subunit was detected using a Storm 860 PhosphorImager and analyzed using ImageQuant software (Molecular Dynamics). Cell Adhesion—Costar 96-well plates were coated with various concentration of fibronectin in PBS overnight at 4 °C, rinsed, and then blocked with 2% (w/v) BSA (Sigma) in PBS for 2 h at room temperature. CHO K1, CHO 14-2-1, PEA10 and MEF-2 cells were harvested in calcium/magnesium-free PBS containing 0.5 mm EDTA. The cells were pelleted at 1000 × g for 5 min and re-suspended at a density of 106 cells/ml in Puck's saline medium A, supplemented with 10 mm HEPES, pH 7.4, 0.5 mm CaCl2, 0.5 mm MgCl2, and, when indicated, 1 mm MnCl2. Cells were allowed to adhere for different periods of time at 37 °C in a humidified atmosphere. Non-adherent cells were removed by washing with PBS. Adherent cells were fixed for 20 min with 4% formaldehyde in PBS, rinsed with PBS, and stained with 0.2% crystal violet in 2% ethanol for 30 min. Excess stain was washed away. Cell associated stain was then released in 1% SDS (50 μl per well). The absorbance at 595 nm was measured. Each value represents the mean of 18 separate replicates, divided among three different experiments. To evaluate cell spreading, cells were allowed to adhere for 30 min and then photographed using an Axiovert microscope with a Contax camera. LRP-1-deficient MEFs Demonstrate Decreased p125/Cell Surface β1 Integrin—β1 integrin was compared by immunoblot analysis in LRP-1-deficient MEF-2 cells and in two LRP-1-positive cell lines (MEF-1 and PEA10). PEA10 cells were cloned from cultures of LRP-1(+/–) embryonic fibroblasts that had been subjected to selection with Pseudomonas exotoxin A, as were MEF-2 cells (31Willnow T.E. Herz J. J. Cell Sci. 1994; 107: 719-726Crossref PubMed Google Scholar). MEF-1 cells are LRP-1(+/+). As shown in Fig. 1A, the LRP-1-positive MEF-1 and PEA10 cells showed similar amounts of p125 and p105. In both cases, p125 accounted for more than 50% of the total β1 integrin. By contrast, LRP-1-deficient MEF-2 cells demonstrated decreased p125 and increased p105. In seven separate experiments, the amount of p125, as a fraction of the total amount of glycosylated β1 (p125/p105+p125), was 2.3 ± 0.3 fold higher in MEF-1 cells than in MEF-2 cells (p < 0.01). B41 cells are MEF-2 cells that were transfected for stable expression of full-length human LRP-1. Expression of human LRP-1 in MEF-2 cells increased the fraction of p125 β1 integrin (Fig. 1B). In four separate experiments, p125/p125+p105 was increased by 2.5 ± 0.3-fold in the B41 cells compared with MEF-2 cells (p < 0.05). To confirm the identity of p105 as an intracellular β1 precursor in the ER, we treated MEF extracts with Endo-H. This enzyme dissociates N-linked mannose-rich glycans, which become Endo-H-resistant after modification by glycosyltransferases in the Golgi apparatus. As anticipated, Endo-H totally eliminated p105, replacing this band with a new band that migrated near unglycosylated β1 core protein. p125 was not modified by Endo-H, confirming that this species is the mature form of the integrin subunit. Because LRP-1-deficient MEF-2 cells have decreased amounts of p125, we hypothesized that these cells have decreased amounts of cell surface β1 integrin. To test this hypothesis, we biotinylated cell surface proteins in MEFs, using the membrane-impermeable biotinylation reagent, sulfo-NHS-LC-biotin. Biotinylated proteins were affinity-precipitated with streptavidin-Sepharose and probed for β1 integrin. As shown in Fig. 1C (representative of four separate experiments), the LRP-1-positive cells (MEF-1 and B41) had increased amounts of biotinylated β1 compared with MEF-2 cells. As anticipated, biotinylated p105 was never recovered in the affinity precipitates, confirming that p105 is not found on cell surfaces in MEFs. We considered a number of previously described mechanisms whereby LRP-1 may indirectly control maturation of p105. TGF-β establishes an autocrine pathway in some cell cultures (33Lysiak J.L. Hussaini I.M. Glass W.F. Allietta M. Gonias S.L. J. Biol. Chem. 1994; 270: 21919-21927Abstract Full Text Full Text PDF Scopus (45) Google Scholar) and has been shown to promote β1-chain maturation (10Heino J. Ignotz R.A. Hemler M.E. Crouse C. Massague J. J. Biol. Chem. 1989; 264: 360-368Google Scholar, 11Bellis S.L. Newman E. Friedman E.A. J. Cell. Physiol. 1999; 181: 33-44Crossref PubMed Scopus (38) Google Scholar). Furthermore, LRP-1 may function as a TGF-β receptor (34Huang S.S. Ling T.-Y. Tseng W.-F. Huang Y.-H. Tang F.-M. Leal S.M. Huang J.S. FASEB J. 2003; 17: 2068-2081Crossref PubMed Scopus (138) Google Scholar). To test whether alterations in endogenously produced TGF-β activity are responsible for differences in β1 integrin maturation in LRP-1-positive and -negative MEFs, we treated PEA10 and MEF-2 cells with TGF-β-neutralizing antibody (20 μg/ml) or with an equivalent concentration of preimmune mouse IgG for 12 h in serum-free medium. Fig. 1D shows that TGF-β-neutralizing antibody had no effect on β1 distribution between p125 and p105 forms. In separate experiments, plating LRP-1-positive and -negative cells on different substrata, including vitronectin, fibronectin, type I collagen, and poly-l-lysine (negative control) had no effect on the distribution of β1 integrin into the p125 and p105 bands (results not shown). Furthermore, α5β1 function-blocking antibody (10 μg/ml for 48 h) failed to alter p105 and p125 in LRP-1-positive and -negative MEFs. However, culture confluency did affect the distribution of β1 integrin into p125 and p105, and this result was only observed in LRP-1-positive cells. We applied two strategies to test the effects of culture confluency on β1 integrin maturation. In one set of experiments, cells were plated at different densities and cultured for 24 h. By visual inspection, cells plated at 105/well remained sub-confluent. Cells plated at 106/well or 2 × 106/well were confluent; however, as anticipated, the cells plated at 2 × 106/well became confluent sooner. In a second set of experiments, cells were plated at the equivalent density (2 × 106/well) and analyzed after culturing for increasing periods of time. In both experiments, the fraction of β1 integrin migrating as mature p125 increased under conditions that favored development of cell culture confluency only in LRP-1-expressing cells (Fig. 2). The results presented thus far suggest a model in which LRP-1 regulates the relative abundance of β1 integrin glycoforms in MEFs. To confirm that the total amount of β1 integrin is not altered, we treated extracts of MEF-1, MEF-2, and B41 cells with PNGase F, which hydrolyzes aspartyl-glycosamine bonds, releasing nearly all N-linked glycans. As shown in Fig. 3A, PNGase F modified both p125 and p105, converting both species into a common product that migrated near the mobility of core protein. The amount of product observed with each of the three cell lines was essentially identical, supporting the conclusion that LRP-1 does not regulate the total amount of β1 integrin in the cell. In control experiments, we also determined that the total amount of EGFr (Fig. 3B) and ERK/MAPK (Fig. 3C) were equivalent in the MEF protein extracts. Thus, the LRP-1-dependent changes in p125 and p105 β1 integrin are specific. LRP-1 Expression Regulates p125/Cell Surface β1 Integrin in CHO Cells—As a second model system to assess the effects of LRP-1 on the subcellular distribution of β1 integrin, we compared LRP-1-positive CHO K1 cells and LRP-1-deficient CHO 13-5-1 cells. We also studied CHO 14-2-1 cells, which have a mutation in either LRP-1 or an unidentified gene product that is necessary for LRP-1 transport in the secretory pathway so that, in this cell line, LRP-1 does not transfer to the cell surface. The CHO 13-5-1 and 14-2-1 cells were cloned from CHO K1 cells treated with the mutagen, ethyl methane sulfate, and selected based on resistance to Pseudomonas exotoxin A (32FitzGerald D.J. Fryling C.M. Zdanovsky A. Saelinger C.B. Kounnas M. Winkles J.A. Strickland D. Leppla S. J. Cell Biol. 1995; 129: 1533-1541Crossref PubMed Scopus (100) Google Scholar). As shown in Fig. 4A (left panel), the LRP-1-deficient CHO 13-5-1 cells demonstrated decreased p125 and increased p105, compared with CHO K1 cells. Thus, the effects of LRP-1 expression on β1 distribution into p105 and p125 were similar in CHO cells and MEFs. In the CHO 14-2-1 cells, p125 was almost entirely absent. To confirm these results, cell surface β1 integrin was compared in the three CHO cell lines using our surface protein biotinylation method (Fig. 4A, right panel). Once again, only p125 was detected in streptavidin-affinity precipitates, as anticipated. The amount of biotinylated β1 integrin was reduced by 60% in CHO 13-5-1 cells, compared with CHO K1 cells, and to trace levels in CHO 14-2-1 cells (n = 5, Fig. 4B). The results of our experiments with CHO 13-5-1 cells confirm, in a second model system, that cell surface β1 integrin is decreased when LRP-1 is not expressed. The profound results obtained with CHO 14-2-1 cells may suggest that either LRP-1 accumulation in the ER dramatically retards maturation of β1 integrin or that these cell
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