The Reversion-inducing Cysteine-rich Protein with Kazal Motifs (RECK) Interacts with Membrane Type 1 Matrix Metalloproteinase and CD13/Aminopeptidase N and Modulates Their Endocytic Pathways

Abstract

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is anchored to the cell surface via glycosylphosphatidylinositol. This molecule antagonizes the function of membrane type 1 matrix metalloproteinase (MT1-MMP) to promote proMMP-2 maturation. Here, we attempt to clarify the mechanism underlying RECK functions. First, we found that RECK forms a complex with MT1-MMP and inhibits its proteolytic activity. Notably, RECK increases the amount of MT1-MMP that associates with detergent-resistant membranes during sucrose gradient ultracentrifugation. Furthermore, perturbation of membrane cholesterol significantly affected the function of RECK in suppressing MT1-MMP function. These findings indicate that RECK possibly regulates MT1-MMP function by modulating its behavior on the cell surface as well as by enzymatic action; this prompted us to find another molecule whose behavior in detergent-resistant membranes is influenced by RECK. Subsequently, we found that RECK interacts with CD13/aminopeptidase N. Further, we found that RECK inhibits the proteolytic activity of CD13 in a cholesterol perturbation-sensitive manner. Finally, we examined whether RECK influences the behavior of MT1-MMP and CD13 during their internalization from the cell surface. In the absence of RECK, MT1-MMP and CD13 were internalized along with the markers of clathrin- or caveolae-dependent endocytosis. However, interestingly, in the presence of RECK these molecules were internalized preferentially with an endocytic marker that is neither clathrinnor caveolae-dependent, indicating that RECK modulates endocytic pathways of MT1-MMP and CD13. This modulation was correlated with the accelerated internalization and decay of MT1-MMP and CD13. This study unveils the novel function and target molecules of RECK. The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is anchored to the cell surface via glycosylphosphatidylinositol. This molecule antagonizes the function of membrane type 1 matrix metalloproteinase (MT1-MMP) to promote proMMP-2 maturation. Here, we attempt to clarify the mechanism underlying RECK functions. First, we found that RECK forms a complex with MT1-MMP and inhibits its proteolytic activity. Notably, RECK increases the amount of MT1-MMP that associates with detergent-resistant membranes during sucrose gradient ultracentrifugation. Furthermore, perturbation of membrane cholesterol significantly affected the function of RECK in suppressing MT1-MMP function. These findings indicate that RECK possibly regulates MT1-MMP function by modulating its behavior on the cell surface as well as by enzymatic action; this prompted us to find another molecule whose behavior in detergent-resistant membranes is influenced by RECK. Subsequently, we found that RECK interacts with CD13/aminopeptidase N. Further, we found that RECK inhibits the proteolytic activity of CD13 in a cholesterol perturbation-sensitive manner. Finally, we examined whether RECK influences the behavior of MT1-MMP and CD13 during their internalization from the cell surface. In the absence of RECK, MT1-MMP and CD13 were internalized along with the markers of clathrin- or caveolae-dependent endocytosis. However, interestingly, in the presence of RECK these molecules were internalized preferentially with an endocytic marker that is neither clathrinnor caveolae-dependent, indicating that RECK modulates endocytic pathways of MT1-MMP and CD13. This modulation was correlated with the accelerated internalization and decay of MT1-MMP and CD13. This study unveils the novel function and target molecules of RECK. The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) has been identified as a negative transcriptional target for various oncogene products, including activated Ras (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13221-13226Crossref PubMed Scopus (418) Google Scholar, 2Sasahara R.M. Takahashi C. Noda M. Biochem. Biophys. Res. Commun. 1999; 264: 668-675Crossref PubMed Scopus (95) Google Scholar). Overexpression of this molecule partially suppressed phenotypes induced by ras transformation in murine fibroblasts (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13221-13226Crossref PubMed Scopus (418) Google Scholar) and efficiently blocked matrix invasion and remote metastasis of malignant tumor cells (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13221-13226Crossref PubMed Scopus (418) Google Scholar). Subsequently, it was demonstrated that RECK functionally antagonizes multiple matrix metalloproteinases (MMPs) 3The abbreviations used are: MMP, matrix metalloproteinase; MT1-MMP, membrane type 1 MMP; GPI, glycosylphosphatidylinositol; APN, aminopeptidase N; TIMP, tissue inhibitor of metalloproteinase; GEEC, GPI-anchored protein enriched early endosomal compartment; MBCD, methyl-β cyclodextrin; MEF, mouse embryonic fibroblast; HUVEC, human umbilical vein endothelial cell; Tf, transferrin; TRITC, tetramethylrhodamine isothiocyanate; PBS, phosphate-buffered saline; DRM, detergent-resistant membrane; FACS, fluorescence-activated cell sorter; F-RECK, FLAG-tagged RECK; siRNA, short interference RNA.3The abbreviations used are: MMP, matrix metalloproteinase; MT1-MMP, membrane type 1 MMP; GPI, glycosylphosphatidylinositol; APN, aminopeptidase N; TIMP, tissue inhibitor of metalloproteinase; GEEC, GPI-anchored protein enriched early endosomal compartment; MBCD, methyl-β cyclodextrin; MEF, mouse embryonic fibroblast; HUVEC, human umbilical vein endothelial cell; Tf, transferrin; TRITC, tetramethylrhodamine isothiocyanate; PBS, phosphate-buffered saline; DRM, detergent-resistant membrane; FACS, fluorescence-activated cell sorter; F-RECK, FLAG-tagged RECK; siRNA, short interference RNA. including MMP-9, MMP-2, and MT1-MMP (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13221-13226Crossref PubMed Scopus (418) Google Scholar, 3Oh J. Takahashi R. Kondo S. Mizoguchi A. Adachi E. Sasahara R.M. Nishimura S. Imamura Y. Kitayama H. C. Horan T.P. Arakawa T. H. S. Y. M. S. Takahashi C. Noda M. PubMed Scopus Google Scholar). The phenotypes by as of proMMP-2 and of type this J. Takahashi R. Kondo S. Mizoguchi A. Adachi E. Sasahara R.M. Nishimura S. Imamura Y. Kitayama H. C. Horan T.P. Arakawa T. H. S. Y. M. S. Takahashi C. Noda M. PubMed Scopus Google we that the that in these J. Takahashi R. Kondo S. Mizoguchi A. Adachi E. Sasahara R.M. Nishimura S. Imamura Y. Kitayama H. C. Horan T.P. Arakawa T. H. S. Y. M. S. Takahashi C. Noda M. PubMed Scopus Google Scholar) by the with target and C. and C. it has been that in of the of RECK is correlated with of the tumor and of the tumor in A. M. M. M. H. K. PubMed Scopus Google Scholar). RECK was found to in cell Sci. PubMed Scopus Google Scholar). In RECK has been in PubMed Scopus Google Scholar, N. A. R. PubMed Scopus Google Scholar). The of RECK function is RECK that to by the of the the in these of RECK is from that of and is of inhibitor function of RECK (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13221-13226Crossref PubMed Scopus (418) Google Scholar). RECK has been to the of (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13221-13226Crossref PubMed Scopus (418) Google Scholar) and the proteolytic activity of (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13221-13226Crossref PubMed Scopus (418) Google Scholar) and J. Takahashi R. Kondo S. Mizoguchi A. Adachi E. Sasahara R.M. Nishimura S. Imamura Y. Kitayama H. C. Horan T.P. Arakawa T. H. S. Y. M. S. Takahashi C. Noda M. PubMed Scopus Google Scholar). Subsequently, the that RECK functionally to the of and C. and C. as in the of is a inhibitor of J. Sci. PubMed Scopus Google Scholar). with by tissue inhibitor of metalloproteinases J. Sci. PubMed Scopus Google the of RECK is its to to the membrane surface via a that is (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13221-13226Crossref PubMed Scopus (418) Google Scholar). In to of on embryonic J. Takahashi R. Kondo S. Mizoguchi A. Adachi E. Sasahara R.M. Nishimura S. Imamura Y. Kitayama H. C. Horan T.P. Arakawa T. H. S. Y. M. S. Takahashi C. Noda M. PubMed Scopus Google Scholar, J. Sci. PubMed Scopus Google Scholar). In this we that the membrane of RECK to this molecule in to it to In a of RECK that the the to the proteolytic activity of MT1-MMP that the membrane is for RECK to its function to MT1-MMP various of the matrix Y. M. J. PubMed Scopus Google Scholar). and of the of these molecules significantly cell various Y. M. J. PubMed Scopus Google Scholar). Furthermore, MT1-MMP proMMP-2 and and their Y. M. J. PubMed Scopus Google Scholar, M. R. J. PubMed Scopus Google Scholar). has been identified as its inhibitor J. Sci. PubMed Scopus Google Scholar). Further, the of proMMP-2 by MT1-MMP via complex J. PubMed Scopus Google Scholar). RECK significantly suppressed of proMMP-2 in cells J. Takahashi R. Kondo S. Mizoguchi A. Adachi E. Sasahara R.M. Nishimura S. Imamura Y. Kitayama H. C. Horan T.P. Arakawa T. H. S. Y. M. S. Takahashi C. Noda M. PubMed Scopus Google RECK has been to an inhibitor of and mechanism been of the of MT1-MMP, it a of that is by indicate that MT1-MMP internalization is by a clathrin- or caveolae-dependent endocytic or by a of these pathways M. N. M. R. Biochem. J. PubMed Scopus Google Scholar, A. C. J. Sci. PubMed Scopus Google Scholar, A. K. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, T. Y. H. M. J. PubMed Scopus Google Scholar, S. M. M. PubMed Scopus Google Scholar, Y. PubMed Scopus Google Scholar). These to the internalization of MT1-MMP from a of the cell membrane and its to for or Y. M. J. PubMed Scopus Google Scholar, M. R. J. PubMed Scopus Google Scholar). This to in the of proMMP-2 and cell A. K. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, T. Y. H. M. J. PubMed Scopus Google Scholar). is with MT1-MMP on the surface of human endothelial This to the mechanism of MT1-MMP internalization the cells were to S. M. J. PubMed Scopus Google Scholar). These findings indicate that cell surface molecules in MT1-MMP internalization and cell CD13/aminopeptidase is another membrane whose is by the ras oncogene N. Y. PubMed Scopus (95) Google Scholar). This molecule as a that is in protein and protein and it as a for including in by human A. J. 1999; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). this molecule is to in tumor the and in The of or cells of CD13 and or the in in various cell and and A. J. 1999; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. R. R. PubMed Scopus Google Scholar). the mechanism is the type by tumor cells and inhibits their invasion H. J. M. T. J. PubMed Scopus Google Scholar). MT1-MMP, inhibitor of CD13 has been Here, we that RECK forms complex with MT1-MMP and inhibits their proteolytic and influences their behavior on the cell surface. In this study that RECK is internalized via a identified novel endocytic that the GPI-anchored protein enriched early endosomal S. S. PubMed Scopus Google Scholar). that the internalization of MT1-MMP is clathrin- caveolae-dependent A. C. J. Sci. PubMed Scopus Google Scholar, S. M. M. PubMed Scopus Google Scholar) and that of CD13 is caveolae-dependent R. A. E. K. Y. K. T. T. J. PubMed Scopus Google Scholar). This study that endocytic pathways for these molecules to that is by RECK. and cells were with the human RECK (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13221-13226Crossref PubMed Scopus (418) Google or or were for in the presence of or The cells were or used for the mouse embryonic fibroblasts were J. Takahashi R. Kondo S. Mizoguchi A. Adachi E. Sasahara R.M. Nishimura S. Imamura Y. Kitayama H. C. Horan T.P. Arakawa T. H. S. Y. M. S. Takahashi C. Noda M. PubMed Scopus Google Scholar). were from from M. embryonic with by cells with and with J. Takahashi R. Adachi E. Kondo S. S. A. H. A. M. T. S. Takahashi C. Noda M. PubMed Scopus Google Scholar). was by M. T. Y. H. M. J. PubMed Scopus Google Scholar). umbilical vein endothelial cells were from to the with and by of or FLAG-tagged a was induced in M. Kondo S. Takahashi R. J. S. Kitayama H. Takahashi C. Noda M. PubMed Scopus Google Scholar) to novel and that the of a a or a and in The of (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13221-13226Crossref PubMed Scopus (418) Google Scholar). and cells were in a and 1 in the presence of inhibitor FLAG-tagged RECK was with MT1-MMP with mouse a from M. N. H. Y. M. PubMed Scopus Google and CD13 with from S. J. PubMed Scopus Google Scholar). The were on protein with and The cell or were by and used for as (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13221-13226Crossref PubMed Scopus (418) Google Scholar). and of cells were with and the were as the protein of the was by a and The of this was of MT1-MMP study the of MT1-MMP of of MT1-MMP was with various of a and RECK protein in of and The was for by The was as (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13221-13226Crossref PubMed Scopus (418) Google Scholar). by sucrose gradient was as J. C. PubMed Scopus Google Scholar). The and were The of the of to detergent-resistant membrane was by the of the by In cells were to cells were on a and for of that were by the cells with for and with for another The from the were as (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13221-13226Crossref PubMed Scopus (418) Google Scholar). The cell the was by the cell that was used for of was by as A. H. H. S. M. A. K. H. K. H. T. PubMed Scopus Google Scholar). and cells were in a for and with or of Y. K. Y. Y. H. PubMed Scopus Google Scholar) was to the as the a of The cells were for another and the was for The was by and to of Y. K. Y. Y. H. PubMed Scopus Google Scholar). The was by the in the presence of a amount of and this was to cells were from RECK or type as J. Takahashi R. Kondo S. Mizoguchi A. Adachi E. Sasahara R.M. Nishimura S. Imamura Y. Kitayama H. C. Horan T.P. Arakawa T. H. S. Y. M. S. Takahashi C. Noda M. PubMed Scopus Google Scholar). cells were in a for and the CD13 activity was the of was with various of and RECK protein in of and The was for by The was as (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13221-13226Crossref PubMed Scopus (418) Google Scholar). cells that been with and were on for with or in and with phosphate-buffered to the with the cells were to internalization of the cell surface to the for The cells were to the internalization and a cells that been with were for with and for in with The was and and The cells were with and for and blocked with in to with and the of of to and was examined by with were The of the mouse to CD13 was examined by its on cells to that by to CD13 from S. J. PubMed Scopus Google were In CD13 in cells that were to CD13 Z. H. K. H. M. S. PubMed Scopus Google Scholar). The of the to was examined by cells with as demonstrated in for RECK, and were from for for and cells or were and with to the by the and cells that were with T. Y. H. M. J. PubMed Scopus Google Scholar) and were with and with the and were and enriched The cells were with or for in on and for in The were and and The cells were with and a and of cells were and with PBS, The cell surface were by the cells with of A. C. J. Sci. PubMed Scopus Google Scholar). for the was and and the with the cells were to the cell surface that were by the internalization was by the cells with and on for cell surface was by the cells with in with 1 and The that been internalized during the were by the cell with by RECK with MT1-MMP and the mechanism underlying the of MT1-MMP activity by RECK, we to whether RECK interacts with FLAG-tagged RECK was human cells that of RECK (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13221-13226Crossref PubMed Scopus (418) Google and it was The an and a of MT1-MMP This that RECK forms a complex with MT1-MMP MT1-MMP is in an activated and a In we found that the of RECK cells significantly the of MT1-MMP This us to whether RECK the proteolytic activity of we a of RECK its that is for membrane Subsequently, the was to by protein This the of a by the of MT1-MMP in a with the These findings the that RECK is the inhibitor of MT1-MMP J. Takahashi R. Kondo S. Mizoguchi A. Adachi E. Sasahara R.M. Nishimura S. Imamura Y. Kitayama H. C. Horan T.P. Arakawa T. H. S. Y. M. S. Takahashi C. Noda M. PubMed Scopus Google Scholar). RECK the of MT1-MMP with is to the cell surface by a (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13221-13226Crossref PubMed Scopus (418) Google MT1-MMP is by a that with this their behavior on the cell we cells that RECK, the cells in and on a sucrose gradient in to the in from in with its of in cells the of RECK was with In the was was perturbation that was by the of the cells with methyl-β a the of RECK and with The of a RECK and MT1-MMP prompted us to whether the presence of RECK influenced the behavior of MT1-MMP in the In the absence of RECK, an of MT1-MMP was to with in the presence of RECK, the of MT1-MMP that was to with to an of perturbation the function of RECK in the of MT1-MMP with The of that were from and that RECK significantly to the of MT1-MMP with This the of cells in the of RECK in these cells an activity to that of RECK in the of MT1-MMP on the of RECK with this us to that RECK the of MT1-MMP with The of RECK on MT1-MMP to we whether modulates the of RECK on MT1-MMP function. that the function of RECK in proMMP-2 The of that we used in of cell These findings that the of RECK in functionally suppressing MT1-MMP is to cholesterol perturbation in the RECK the of CD13 in the of the of of its MT1-MMP, with This for that with RECK. that behavior in the on the presence of RECK. that were from or cells were by and by the of that in of cells of these were identified as by However, the of the and were significantly the of CD13 of CD13 has been that to the of CD13 we that the and forms of CD13. an study of the sucrose gradient that forms of CD13 in the of cells MT1-MMP, the CD13 was enriched in of the RECK their in in the presence of RECK. The of these forms was by with a inhibitor that RECK CD13 in a manner. The of MT1-MMP by interference to the of CD13 that the MT1-MMP activity is for CD13 RECK with CD13 and the RECK and CD13. RECK was with CD13 and Furthermore, we found that complex MT1-MMP the MT1-MMP and CD13 that on the of RECK the of this has been in this we whether RECK the aminopeptidase activity of CD13. of RECK the cell surface aminopeptidase activity to a with that in cells that were with a of a aminopeptidase inhibitor A. H. H. S. M. A. K. H. K. H. T. PubMed Scopus Google Scholar) these RECK and were to in an In the presence of MBCD, this of RECK on the aminopeptidase activity was to a MT1-MMP, RECK inhibits the proteolytic activity of CD13 in a cholesterol perturbation-sensitive manner. whether the enzymatic RECK and CD13 is to that RECK and MT1-MMP, we the of RECK on the aminopeptidase activity of CD13. that RECK the aminopeptidase activity of CD13 in a with the of the cells RECK and on CD13 in an In the the RECK the aminopeptidase activity of the CD13 with a Further, the in of this function of RECK was demonstrated in cells from whose aminopeptidase activity was significantly that of the type These findings indicate that RECK is an inhibitor of CD13. RECK with MT1-MMP and CD13 in a to the behavior of MT1-MMP and CD13 on the cell membrane as well as their proteolytic activity. This prompted us to whether RECK has on the by MT1-MMP and CD13 internalized from the cell this cells were with RECK and FLAG-tagged These cells were with and with cells to endocytosis. the the of the used in this was examined of RECK and MT1-MMP and CD13 were significantly on the cell surface cells were to to endocytosis. early these molecules were a with The of was significantly on the surface of cells that been with to that in MT1-MMP was internalized by RECK in these cells was The on the internalization of is a marker of These findings that the of RECK, MT1-MMP, and CD13 on the cell surface and during internalization is to cholesterol The for RECK to by the endocytic for RECK, FLAG-tagged RECK was and the early of its was in cells with and to to endocytosis. of with during we cells with these cells were and with the to The of with was by cells with and these cells were and In RECK and of during early Further, by interference on RECK internalization is to to caveolae-dependent M. M. M. M. J. C. A. H. PubMed Scopus Google Scholar). the of RECK and in early was of RECK was with was RECK internalization was by by and that is for the of H. 1998; PubMed Scopus Google Scholar). These findings that the endocytic by the of RECK is internalized is from clathrin- and caveolae-dependent and S. S. PubMed Scopus Google Scholar) identified a novel endocytic by multiple GPI-anchored This is of clathrin- and endocytic and it is by the of markers by S. S. PubMed Scopus Google Scholar). found that in cells with and with and a of was with during early internalization and with the of RECK and was by the of was by with K. PubMed Scopus Google Scholar). was by this the of internalized the internalization of RECK in cells was and well as by the of the novel endocytic by and S. S. PubMed Scopus Google Scholar) was by to C. or of and to of or activity. Further, it was by the that blocked this novel endocytic the internalization of the GPI-anchored protein was S. S. PubMed Scopus Google to the of RECK that this identified as S. S. PubMed Scopus Google Scholar, S. H. PubMed Scopus Google Scholar, M. Biophys. PubMed Scopus Google Scholar). that the endocytic by RECK is internalized is to the However, in the of a negative of to and the This from the cell in the of of the endocytic for RECK that it was to that by RECK the of MT1-MMP and CD13 the endocytic used for RECK we whether the of MT1-MMP and CD13 internalization was influenced by RECK. this we used cells that been with FLAG-tagged MT1-MMP, with or RECK. These cells were with and as was In the absence of RECK, was preferentially with during However, in the presence of RECK, was preferentially with and in the endocytic was in the of CD13 and These findings indicate that RECK the of MT1-MMP and CD13 internalization to that is by RECK. RECK the of the for MT1-MMP and we the internalization of MT1-MMP and CD13 that were by cells RECK. whether RECK the function by RECK. this FLAG-tagged MT1-MMP was in RECK was by of These cells were with or and as The of and was significantly in cells as with that in the cells and This that RECK to the of the internalization for MT1-MMP and CD13. RECK the and of MT1-MMP and we whether of the MT1-MMP and CD13 endocytic pathways is correlated with in their the internalization of RECK, MT1-MMP, and CD13 in cells that were the cell surface by the cells with This type of from by the in the that the the cell it is to the been internalized the A. C. J. Sci. PubMed Scopus Google Scholar). In we internalized in the molecules of RECK, MT1-MMP, or CD13 were internalized and a amount of these molecules were internalized and Notably, the internalization of MT1-MMP and CD13 was significantly accelerated in the presence of RECK and The of multiple that RECK significantly accelerated the internalization of MT1-MMP and CD13 by cells cell we the of MT1-MMP and CD13 and the of RECK on The that RECK significantly accelerated the decay of MT1-MMP and CD13 and These findings indicate that in the endocytic of its target molecules is significantly correlated with the in their of GPI-anchored efficiently with during their from the to the cell membrane by that in cholesterol and PubMed Scopus Google Scholar). In this we on this of GPI-anchored and the novel function of RECK in to its enzymatic with its and identified target we found that the presence of RECK significantly influences the of the endocytic pathways by MT1-MMP and CD13 The mechanism the complex and behavior of these molecules on the cell membrane; this to the of the as or the mechanism to S. H. PubMed Scopus Google Scholar, M. Biophys. PubMed Scopus Google Scholar). to the of GPI-anchored is in a clathrin- and S. S. PubMed Scopus Google Scholar). this that has been by and S. S. PubMed Scopus Google Scholar) with the endocytic RECK. the of by cholesterol is S. PubMed Scopus Google it is that the endocytic S. S. PubMed Scopus Google Scholar) and the RECK to that in the presence of RECK the endocytic mechanism to MT1-MMP function. However, we to in the MT1-MMP and CD13 endocytic pathways is correlated with their accelerated internalization and that RECK is internalized via the endocytic early the endosomal RECK and its target molecules is to the endosomal in a to However, it the endosomal a that from S. S. Sci. PubMed Scopus Google Scholar). this RECK the the and the decay of the the we that RECK modulates the function of MT1-MMP in is by its proteolytic activity and the is by its by modulating the endocytic of these to the of MT1-MMP function in study has that the RECK a to the behavior of tumor cells MT1-MMP activity to promote proMMP-2 (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13221-13226Crossref PubMed Scopus (418) Google that the function of RECK is and the the RECK inhibits MT1-MMP was significantly the inhibits MT1-MMP Z. Res. Google Scholar). This that as an inhibitor of MT1-MMP RECK is this for the of RECK to MT1-MMP activity in we that in the of MT1-MMP, of the endocytic is to the of its activity the of as a target of RECK. demonstrated that in the presence of RECK this molecule is internalized in a to the internalization of The of the complex CD13 and MT1-MMP was partially of CD13 on MT1-MMP function in proMMP-2 in and C. CD13 is in various human it is of to the of the RECK and CD13. S. and M. for A. for of the S. E. H. A. and S. for H. T. and A. for and A. for