γ-Secretase-mediated Release of the Low Density Lipoprotein Receptor-related Protein 1B Intracellular Domain Suppresses Anchorage-independent Growth of Neuroglioma Cells

Abstract

The low density lipoprotein receptor related protein 1B (LRP1B) is a large endocytic receptor that was first identified as a candidate tumor suppressor gene. In the current investigation we demonstrate that LRP1B undergoes regulated intramembrane proteolysis in a γ-secretase-dependent process. The released intracellular domain (ICD) then translocates to the nucleus via a nuclear localization signal that is present within this domain. ICD release first requires shedding of the LRP1B ectodomain, which appears to be catalyzed by a member of the metalloproteinase family. Employing site-directed mutagenesis studies, we identified lysine residues 4432 and 4435 and arginine 4442 as key amino acids important for ectodomain shedding of LRP1B. We also demonstrate that an LRP1B minireceptor as well as the ICD domain alone suppresses anchorage-independent growth of LRP1B-deficient neuroglioma cells (H4 cells). Interestingly, abrogating ectodomain shedding resulted in a loss of the ability of LRP1B minireceptors to suppress anchorage-independent growth. Together, these studies reveal that LRP1B has tumor suppression function that is mediated by proteolytic processing of the receptor resulting in ICD release. The low density lipoprotein receptor related protein 1B (LRP1B) is a large endocytic receptor that was first identified as a candidate tumor suppressor gene. In the current investigation we demonstrate that LRP1B undergoes regulated intramembrane proteolysis in a γ-secretase-dependent process. The released intracellular domain (ICD) then translocates to the nucleus via a nuclear localization signal that is present within this domain. ICD release first requires shedding of the LRP1B ectodomain, which appears to be catalyzed by a member of the metalloproteinase family. Employing site-directed mutagenesis studies, we identified lysine residues 4432 and 4435 and arginine 4442 as key amino acids important for ectodomain shedding of LRP1B. We also demonstrate that an LRP1B minireceptor as well as the ICD domain alone suppresses anchorage-independent growth of LRP1B-deficient neuroglioma cells (H4 cells). Interestingly, abrogating ectodomain shedding resulted in a loss of the ability of LRP1B minireceptors to suppress anchorage-independent growth. Together, these studies reveal that LRP1B has tumor suppression function that is mediated by proteolytic processing of the receptor resulting in ICD release. The low density lipoprotein (LDL) 2The abbreviations used are: LDL, low density lipoprotein; LRP1B, LDL receptor-related protein (LRP) 1B; GV, Gal4 DNA binding domain fused to VP16 transactivating domain; ICD, intracellular domain; NLS, nuclear localization signal; EGFP, enhanced green fluorescent (EGF) protein; APP, amyloid precursor protein; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; HA, hemagglutinin; Tricine, N-[2-hydroxy-1, 1-bis(hydroxymethyl)ethyl]glycine; WT, wild type; RIP, regulated intramembrane proteolysis. receptor-related protein 1B (LRP1B) is a member of the LDL receptor gene family and was initially identified as a candidate tumor suppressor gene by positional cloning (1Liu C.X. Musco S. Lisitsina N.M. Forgacs E. Minna J.D. Lisitsyn N.A. Cancer Res. 2000; 60: 1961-1967PubMed Google Scholar, 2Liu C.X. Musco S. Lisitsina N.M. Yaklichkin S.Y. Lisitsyn N.A. Genomics. 2000; 69: 271-274Crossref PubMed Scopus (61) Google Scholar). The LRP1B gene is frequently inactivated by homozygous genomic deletions or by the expression of aberrant mRNA transcripts (1Liu C.X. Musco S. Lisitsina N.M. Forgacs E. Minna J.D. Lisitsyn N.A. Cancer Res. 2000; 60: 1961-1967PubMed Google Scholar). In human esophagus cancer cell lines and primary tumors the LRP1B gene was shown to be inactivated by transcriptional silencing through hypermethylation of its promoter CpG island (3Sonoda I. Imoto I. Inoue J. Shibata T. Shimada Y. Chin K. Imamura M. Amagasa T. Gray J.W. Hirohashi S. Inazawa J. Cancer Res. 2004; 64: 3741-3747Crossref PubMed Scopus (121) Google Scholar). Homozygous deletions of LRP1B have also been reported in human liver cancers (4Pineau P. Marchio A. Nagamori S. Seki S. Tiollais P. Dejean A. Hepatology. 2003; 37: 852-861Crossref PubMed Scopus (48) Google Scholar) and urothelial cancers (5Langbein S. Szakacs O. Wilhelm M. Sukosd F. Weber S. Jauch A. Lopez B.A. Alken P. Kalble T. Kovacs G. Lab. Investig. 2002; 82: 639-643Crossref PubMed Scopus (63) Google Scholar). By conventional and array-based comparative genomic hybridization analysis, loss of the LRP1B allele was noted in endocervical adenocarcinomas of uterus (6Hirai Y. Utsugi K. Takeshima N. Kawamata Y. Furuta R. Kitagawa T. Kawaguchi T. Hasumi K. Noda T. Am. J. Obstet. Gynecol. 2004; 191: 1173-1182Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Thus, collective data indicate that this gene is inactivated during cellular transformation, suggesting that LRP1B is a candidate tumor suppressor gene. Structurally, LRP1B is closely related to LRP1, a receptor that recognizes numerous ligands and is essential for normal development (7Herz J. Clouthier D.E. Hammer R.E. Cell. 1992; 71: 411-421Abstract Full Text PDF PubMed Scopus (522) Google Scholar). Like LRP1, LRP1B contains a furin cleavage site (REKR) within a β-propeller domain that is cleaved by furin during receptor trafficking (8Liu C.X. Li Y. Obermoeller-McCormick L.M. Schwartz A.L. Bu G. J. Biol. Chem. 2001; 276: 28889-28896Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 9Tanaga K. Bujo H. Zhu Y. Kanaki T. Hirayama S. Takahashi K. Inoue M. Mikami K. Schneider W.J. Saito Y. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1422-1428Crossref PubMed Scopus (33) Google Scholar, 10Cam J.A. Zerbinatti C.V. Knisely J.M. Hecimovic S. Li Y. Bu G. J. Biol. Chem. 2004; 279: 29639-29646Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The major structural differences between LRP1 and LRP1B occur within their intracellular domains (ICD). First, the LRP1B ICD contains an alternatively spliced exon (exon 90) that is not present in LRP1 (1Liu C.X. Musco S. Lisitsina N.M. Forgacs E. Minna J.D. Lisitsyn N.A. Cancer Res. 2000; 60: 1961-1967PubMed Google Scholar). Second, the LRP1B ICD contains a cluster of basic residues (KRKRRTK) that is similar to basic clusters in BRCA2 tumor suppressor which have been shown to function as a nuclear localization sequence (NLS) (11Yano K. Morotomi K. Saito H. Kato M. Matsuo F. Miki Y. Biochem. Biophys. Res. Commun. 2000; 270: 171-175Crossref PubMed Scopus (49) Google Scholar). Currently, the physiological function of LRP1B remains unknown. In mice, LRP1B expression is primarily restricted to the brain, adrenal glands, salivary gland, and testis (12Marschang P. Brich J. Weeber E.J. Sweatt J.D. Shelton J.M. Rich-ardson J.A. Hammer R.E. Herz J. Mol. Cell. Biol. 2004; 24: 3782-3793Crossref PubMed Scopus (56) Google Scholar). However, in humans, LRP1B is widely expressed (13Li Y. Lu W. Bu G. Biochem. Biophys. Res. Commun. 2005; 333: 868-873Crossref PubMed Scopus (12) Google Scholar). Like LRP1, LRP1B is expressed in vascular smooth muscle cells where it is thought to attenuate the migration of these cells by regulating activity and function of the PDGFR-β (9Tanaga K. Bujo H. Zhu Y. Kanaki T. Hirayama S. Takahashi K. Inoue M. Mikami K. Schneider W.J. Saito Y. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1422-1428Crossref PubMed Scopus (33) Google Scholar). Mice in which the LRP1B gene has been deleted develop normally (12Marschang P. Brich J. Weeber E.J. Sweatt J.D. Shelton J.M. Rich-ardson J.A. Hammer R.E. Herz J. Mol. Cell. Biol. 2004; 24: 3782-3793Crossref PubMed Scopus (56) Google Scholar), possibly due to its restricted expression pattern and functional compensation by other members of the LDL receptor superfamily. Studies using LRP1B minireceptors have revealed that this receptor recognizes ligands such as the receptor-associated protein, urokinase and tissue-type plasminogen activator (8Liu C.X. Li Y. Obermoeller-McCormick L.M. Schwartz A.L. Bu G. J. Biol. Chem. 2001; 276: 28889-28896Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), and Pseudomonas exotoxin A (15Pastrana D.V. Hanson A.J. Knisely J. Bu G. FitzGerald D.J. Biochim. Biophys. Acta. 2005; 1741: 234-239Crossref PubMed Scopus (26) Google Scholar) that are also recognized by LRP1, revealing that LRP1B and LRP1 exhibit overlapping ligand specificity. A number of transmembrane receptors have been found to undergo regulated intramembrane proteolysis (RIP). The common features of RIP involve ectodomain shedding of the transmembrane protein, generating a membrane-bound product with a short ectodomain, which is then cleaved within the transmembrane domain releasing the ICD. The first transmembrane proteins discovered to undergo RIP were Notch (16De Strooper B. Annaert W. Cupers P. Saftig P. Craessaerts K. Mumm J.S. Schroeter E.H. Schrijvers V. Wolfe M.S. Ray W.J. Goate A. Kopan R. Nature. 1999; 398: 518-522Crossref PubMed Scopus (1826) Google Scholar, 17Struhl G. 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J. 2002; PubMed Scopus Google Scholar). In to LRP1 P. Herz J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), several other members of the LDL receptor family have been to undergo include B. T. S. B. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) and U. Y. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar, P. J. Herz J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). to the function of LRP1B, we studies to LRP1B also undergoes revealed that LRP1B undergoes proteolytic processing via a release of the ICD. we demonstrate that LRP1B suppresses anchorage-independent growth of neuroglioma cells (H4 and that this function requires LRP1B proteolysis. LRP1B of the transmembrane of (8Liu C.X. Li Y. Obermoeller-McCormick L.M. Schwartz A.L. Bu G. J. Biol. Chem. 2001; 276: 28889-28896Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) were by using the with the as a The were the site and site of the by using the LRP1B and LRP1B and the signal of and an fused to the of the expressed In the of LRP1B ICD, the was the and of cloning with a gene. the the expression was for the sequence expression of the were by DNA for shedding of were using the site-directed of and were used of the the were by DNA and and cells were in with cells and cell lines were in with of the of these cells with the were with the in were with protein expression and expression contains sequence Gal4 binding of the and the is by this as an to for activity was the cell using the activity of the were using activity was by the of by the the the of cleavage the was to the cells and the gene were the for gene were in and in and cell were using cell with or were used for and were and was LRP1B cellular was using a the sequence of LRP1B the in the cells with wild and LRP1B were in with for was using the an of cell was and with were with cells in were with the the cells were with in The was using a by cell was used the cells were in and with and LRP1B using cells were in the of for surface expression of LRP1B the transmembrane domain in these cells was by using a the cell surface cells well were with the in with and to the with with and for were by for of LRP1B by an LRP1B undergoes RIP we a of of of LRP1B expression for a which contains the of LRP1B with the cluster of ligand binding that has been to and ligands (8Liu C.X. Li Y. Obermoeller-McCormick L.M. Schwartz A.L. Bu G. J. Biol. Chem. 2001; 276: 28889-28896Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). expression were that of and include LRP1B a protein that of the LRP1B the transmembrane and the ICD. LRP1B a receptor in which of the ectodomain is deleted and is similar to the G. A. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). a for we also expressed the LRP1B ICD. LRP1B LRP1 in the of an alternatively spliced exon within its ICD, and in to these we also LRP1B in which the sequence by the alternatively spliced exon was deleted LRP1B cleavage analysis, a gene was in which the Gal4 DNA binding domain and a VP16 domain was fused to the of the as P. Herz J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). cells were with the LRP1B and LRP1B ICD cleavage was by which is expressed the LRP1B ICD is released by proteolysis. The demonstrate that expression of LRP1B LRP1B and LRP1B resulted in the of activity and the a to that of the and The data also that the spliced of LRP1B in which the sequence by exon was deleted was also cleaved as as wild LRP1B revealing that of the receptor undergo similar proteolytic and that the cleavage site is not within the sequence by exon the cleavage was mediated by the was cells were with LRP1B and then with of the the activity was and the that the gene activity cleavage of LRP1B was by in a similar to the for the fused to the domains which as a in this that the release of the intracellular domain is mediated by were using human neuroglioma cell lines not Interestingly, the gene activity cells with LRP1B was that of LRP1B were not due to protein expression by the as of cell using the of the expressed protein or an that recognizes the the revealed that LRP1B was expressed or LRP1B in the in gene activity between these minireceptors that LRP1B is a of LRP1B an of ectodomain shedding to the the LRP1B ICD a signal in the the ICD was not by analysis, suggesting that it is The is a LRP1B we cleavage of LRP1B, we the processing of LRP1B and LRP1B in the of by using the the of the receptor as well as the of LRP1B with the of the protein and A furin LRP1B is the are with a and In the of LRP1B the the of this receptor was in the of LRP1B a the protein was the were with a a was to in the of and this is we that this a for that shedding of the LRP1B with the gene the data that the LRP1B ICD is released by a We also noted that the density of was in the of and suggesting that LRP1B be a of similar to the for of Notch G. A. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, S. K. H. T. G. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The of LRP1B by a the of for proteolytic processing of LRP1B, we the of the of the the cells were with an of we a in the of the in the of the that ectodomain shedding by or a related is to the with these we found that the metalloproteinase also the of the We also of other of the of the We found that the the of the suggesting an of LRP1B proteolytic is to which by processing of the this is the for its LRP1B LRP1B contains a ectodomain and is to the membrane-bound the proteolytic cleavage site resulting in shedding of the ectodomain be to the transmembrane domain. In LRP1B a of amino acids the transmembrane domain with the first contains a number of basic residues that are for proteolysis. We and as for site-directed analysis, and of were in which and were by and a in which was by the ectodomain of these receptors was the cell cells were with and and was to their surface not that receptor was expressed the cell We cell for the of the membrane-bound cells were in the of the and of this was by The reveal that cells with receptor the and that cells with receptor not this The also that the processing of the receptors by furin during trafficking through the was not with receptor ectodomain shedding of and cells were for expression of LRP1B by The reveal that an of LRP1B was in the of cells with receptor not in cells of the or was not related to expression as and receptors were similar in cell Together, these reveal that the ectodomain of LRP1B is and be in and that of or to receptors are not released the cell which in to proteolytic Like LRP1B also contains a furin cleavage site residues and we also the of the LRP1B furin cleavage site its proteolytic By and to we the furin cleavage of However, the was in the of not revealing that furin cleavage this site is not for ectodomain In we cells with and found that the was in the of the that furin activity is not for the ectodomain The in the of to the LRP1B ICD, we that this is in Thus, we an in the of the to we the LRP1B ICD of this cells were with and in the of we an that is the for the LRP1B ICD was not present the cells were with the revealing that the was the shedding of LRP1B were used for these and The LRP1B ICD to the LRP1B ICD contains a cluster of basic residues (KRKRRTK) that is similar to basic clusters in BRCA2 tumor which have been shown to function as a the that the within LRP1B ICD function as and the of the cleaved ICD to the nucleus for of with the LRP1B this is the the LRP1B ICD was fused to EGFP, and cells with this protein were by cells were also with and the ICD The that and are the cell A and the LRP1B ICD protein in the with in the of the sequence in the LRP1B ICD resulted in a cellular similar to that of alone of the in the to or of the sequence present in LRP1B not the nuclear localization of the proteins not reveal that the LRP1B ICD to the nucleus as a of a basic cluster that as a nuclear localization signal Interestingly, also indicate that the LRP1 ICD, which not the basic cluster found in LRP1B, not to the is in with a A. T. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar) in with the studies of D.E. E.H. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). is to be frequently inactivated by or in tumor cells (3Sonoda I. Imoto I. Inoue J. Shibata T. Shimada Y. Chin K. Imamura M. Amagasa T. Gray J.W. Hirohashi S. Inazawa J. Cancer Res. 2004; 64: 3741-3747Crossref PubMed Scopus (121) Google Scholar, P. Marchio A. Nagamori S. Seki S. Tiollais P. Dejean A. Hepatology. 2003; 37: 852-861Crossref PubMed Scopus (48) Google Scholar, S. Szakacs O. Wilhelm M. Sukosd F. Weber S. Jauch A. Lopez B.A. Alken P. Kalble T. Kovacs G. Lab. Investig. 2002; 82: 639-643Crossref PubMed Scopus (63) Google Scholar, Y. Utsugi K. Takeshima N. Kawamata Y. Furuta R. Kitagawa T. Kawaguchi T. Hasumi K. Noda T. Am. J. Obstet. Gynecol. 2004; 191: 1173-1182Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). We to LRP1B transcripts in neuroglioma cells and aberrant transcripts with deletions in exon to exon to a loss of protein not revealing that these LRP1B-deficient cells a to the of LRP1B to cellular Thus, cells were with wild minireceptor expression and were by the cells with for The cells were then used to anchorage-independent growth in We used to due to the of tumor cell in the R. Li Chin S. 2000; PubMed Scopus Google Scholar). of and the of this receptor was by in cells with the minireceptor was the cells with to the cells the of to suppress as by this we RIP processing of LRP1B to its tumor suppression function by using a shedding receptor that we to be to ectodomain release the ability of to suppress growth a of the as a function of and a of the number of of The LRP1B cleavage was in growth of the growth of the suggesting that cleavage of LRP1B in the the of LRP1B growth is related to RIP processing of LRP1B, we also the with cells with LRP1B ICD and found that the LRP1B ICD anchorage-independent growth of cells similar to that of and the of these reveal that expression of LRP1B suppresses tumor cell anchorage-independent growth by proteolytic release of the intracellular domain. of the LDL receptor a of ligands the and to of processing and within ligands include and and growth of these ligands is an important function for LDL receptor family is to a for members in signal and in regulating cell occur by regulated proteolysis of the receptor resulting in release of ICD. In the current we that the LRP1B ICD is released by First, using a gene we demonstrate that the LRP1B ICD is released in a γ-secretase-dependent of cell identified a the LRP1B domain that activity was with was found to cells were with the The of this was by that it is a product of proteolysis. Together, these data that the the LRP1B ICD. the LRP1B ICD within the was by cells with a the LRP1B ICD fused to localization the within the LRP1B ICD, as of this short cluster of basic residues resulted in of the LRP1B ICD. In to LRP1B, the LRP1 ICD was not in the is in to a using a A. T. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). The for this is not In regulated intramembrane proteolysis of transmembrane proteins that is mediated by ectodomain shedding is first proteolysis then the protein to undergo cleavage by resulting in a release of the ICD. that ectodomain shedding of LRP1B is also cleavage was by generating receptors that were not and were to be by In the for the candidate for the ectodomain we found that an and a of ectodomain shedding of LRP1B suggesting that a of the is in the shedding process. such as Notch and RIP processing is ligand which in ectodomain this we not ligand binding to LRP1B is for ectodomain shedding and ICD release. is to that ligand binding cluster LRP1B and LRP1B are LRP1B contains in the and ligands have been identified that to this of the the that ectodomain shedding of LRP1B be a that be by ligand as has been for A. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar), this to be in a candidate tumor suppressor LRP1B has not been for its tumor suppression function in or in anchorage-independent growth we found that a LRP1B minireceptor suppresses the anchorage-independent growth of LRP1B-deficient tumor cells in activity of LRP1B minireceptor appears related to its proteolysis and ICD as receptors in which the shedding is the suppression of LRP1B tumor cell growth In of the nuclear localization of the released intracellular the that to tumor suppression occur in the nucleus by expression of related to cell In this data a for LRP1B as a tumor suppressor and that this function requires proteolytic processing of LRP1B. the by which LRP1B as a tumor suppressor and the is to for the of human