Cysteine Array Matrix Metalloproteinase (CA-MMP)/MMP-23 Is a Type II Transmembrane Matrix Metalloproteinase Regulated by a Single Cleavage for Both Secretion and Activation
2000; Elsevier BV; Volume: 275; Issue: 43 Linguagem: Inglês
10.1074/jbc.m006493200
ISSN1083-351X
AutoresDuanqing Pei, Tiebang Kang, QI Hua-xiong,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoMatrix metalloproteinases characterized so far are either secreted or membrane anchored via a type I transmembrane domain or a glycosylphosphatidylinositol linkage. Lacking either membrane-anchoring mechanism, the newly discovered CA-MMP/MMP-23 was reported to be expressed as a cell-associated protein. In this report, we present evidence that CA-MMP is expressed as an integral membrane zymogen with an N-terminal signal anchor, and secreted as a fully processed mature enzyme. We further demonstrate that L20GAALSGLCLLSALALL36 is required for this unique membrane localization as a signal anchor and its secretion is regulated by a proprotein convertase motif RRRR79sandwiched between its pro- and catalytic domains. Thus, CA-MMP is a type II transmembrane MMP that can be regulated by a single proteolytic cleavage for both activation and secretion, establishing a novel paradigm for protein trafficking and processing within the secretory pathway. Matrix metalloproteinases characterized so far are either secreted or membrane anchored via a type I transmembrane domain or a glycosylphosphatidylinositol linkage. Lacking either membrane-anchoring mechanism, the newly discovered CA-MMP/MMP-23 was reported to be expressed as a cell-associated protein. In this report, we present evidence that CA-MMP is expressed as an integral membrane zymogen with an N-terminal signal anchor, and secreted as a fully processed mature enzyme. We further demonstrate that L20GAALSGLCLLSALALL36 is required for this unique membrane localization as a signal anchor and its secretion is regulated by a proprotein convertase motif RRRR79sandwiched between its pro- and catalytic domains. Thus, CA-MMP is a type II transmembrane MMP that can be regulated by a single proteolytic cleavage for both activation and secretion, establishing a novel paradigm for protein trafficking and processing within the secretory pathway. extracellular matrix cysteine array matrix metalloproteinase matrix metalloproteinase membrane-type proprotein convertase green fluorescent protein phosphate-buffered saline endoplasmic reticulum polyacrylamide gel electrophoresis polyvinylidene difluoride Proteolysis mediated by metalloproteinases has been implicated in a diverse range of biological processes such as signal transduction (1Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1491) Google Scholar), precursor processings (2Black R.A. Rauch C.T. Kozlosky C.J. Peschon J.J. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Nelson N. Boiani N. Schooley K.A. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. 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Recently, localization and trafficking of MMPs have been recognized as important mechanisms of regulation, but remain poorly defined.MMPs discovered so far can be classified into two categories based on cellular localization: secreted or membrane-bound. The secreted MMPs are generally synthesized and discharged to the extracellular milieu where they are activated, bind to, and degrade their ECM substrates (9Birkedal-Hansen H. Moore W.G. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2630) Google Scholar,11Woessner Jr., J.F. FASEB J. 1991; 5: 2145-2154Crossref PubMed Scopus (3072) Google Scholar). On the other hand, members of the MT-MMP subgroup are limited to the cellular membranes, mostly the plasma membrane via a type I transmembrane domain or glycosylphosphatidylinositol anchor (13Nakahara H. Howard L. Thompson E.W. Sato H. Seiki M. Yeh Y. Chen W.T. Proc. Natl. Acad. Sci. U. S. 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Compared with their secreted counterparts, these membrane-bound MMPs may be able to express their activity with both precision and intensity against subjacent substrates, thus, exerting profound proteolytic activities in a wide range of biological and pathological processes such as cell migration, invasion, angiogenesis, and development (5Hiraoka N. Allen E. Apel I.J. Gyetko M.R. Weiss S.J. Cell. 1998; 95: 365-377Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar, 6Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar, 13Nakahara H. Howard L. Thompson E.W. Sato H. Seiki M. Yeh Y. Chen W.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7959-7964Crossref PubMed Scopus (360) Google Scholar, 17Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. 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Sato H. Seiki M. Yeh Y. Chen W.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7959-7964Crossref PubMed Scopus (360) Google Scholar, 22Hotary K. Allen E. Punturieri A. Yana I. Weiss S.J. J. Cell Biol. 2000; 149: 1309-1323Crossref PubMed Scopus (507) Google Scholar, 23Koshikawa N. Giannelli G. Cirulli V. Miyazaki K. Quaranta V. J. Cell Biol. 2000; 148: 615-624Crossref PubMed Scopus (548) Google Scholar, 24Lehti K. Valtanen H. Wickstrom S. Lohi J. Keski-Oja J. J. Biol. Chem. 2000; 275: 15006-15013Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). More dramatically, MT1-MMP-deficient mice are the first to display developmental abnormalities, in contrast to the apparent normal phenotypes reported for those from secreted MMPs, underscoring the significance of the membrane-bound MMPs (6Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar, 25Zhou Z. Apte S.S. Soininen R. Cao R. Baaklini G.Y. Rauser R.W. Wang J. Cao Y. Tryggvason K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4052-4057Crossref PubMed Scopus (682) Google Scholar, 26Shapiro S.D. Thromb. Haemostasis. 1999; 82: 846-849Crossref PubMed Scopus (90) Google Scholar).CA-MMP, a novel MMP identified recently (27Pei D. FEBS Lett. 1999; 457: 262-270Crossref PubMed Scopus (45) Google Scholar), is apparently the mouse orthologue of human MMP-23 (28Velasco G. Pendas A.M. Fueyo A. Knauper V. Murphy G. Lopez-Otin C. J. Biol. Chem. 1999; 274: 4570-4576Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Tissue distributions of this enzyme suggest that it may play an important role in the functions of lung, heart, as well as the reproductive organs such as ovary and prostate (27Pei D. FEBS Lett. 1999; 457: 262-270Crossref PubMed Scopus (45) Google Scholar, 28Velasco G. Pendas A.M. Fueyo A. Knauper V. Murphy G. Lopez-Otin C. J. Biol. Chem. 1999; 274: 4570-4576Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Enzymatically, purified CA-MMP behaves like a classic MMP: detectable on zymography and inhibited by tissue inhibitor of matrix metalloproteinases (TIMPs) (27Pei D. FEBS Lett. 1999; 457: 262-270Crossref PubMed Scopus (45) Google Scholar). Structurally, CA-MMP shares only two features with the rest of the MMP family, a conserved catalytic domain and a basic motif RRRR79 (27Pei D. FEBS Lett. 1999; 457: 262-270Crossref PubMed Scopus (45) Google Scholar, 28Velasco G. Pendas A.M. Fueyo A. Knauper V. Murphy G. Lopez-Otin C. J. Biol. Chem. 1999; 274: 4570-4576Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Otherwise, CA-MMP resembles little of a MMP. First, while all MMPs discovered so far are regulated by a latency mechanism based on a cysteine switch (29Van Wart H.E. Birkedal-Hansen H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5578-5582Crossref PubMed Scopus (1192) Google Scholar), CA-MMP may employ an entirely different mechanism for latency because there is no cysteine in its putative prodomain (27Pei D. FEBS Lett. 1999; 457: 262-270Crossref PubMed Scopus (45) Google Scholar). Second, we suggested that CA-MMP differ from the other MMPs by having two novel domains downstream of its catalytic domain: a cysteine-array (CA) and an Ig-like domain (27Pei D. FEBS Lett. 1999; 457: 262-270Crossref PubMed Scopus (45) Google Scholar), replacing the hinge and hemopexin-like domains implicated in interactions with substrates and intergrins (30Sanchez-Lopez R. Alexander C.M. Behrendtsen O. Breathnach R. Werb Z. J. Biol. Chem. 1993; 268: 7238-7247Abstract Full Text PDF PubMed Google Scholar, 31Brooks P.C. Silletti S. von Schalscha T.L. Friedlander M. Cheresh D.A. Cell. 1998; 92: 391-400Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar). Experimentally, CA-MMP has been shown as a cell-associated protein with no detectable secretion in COS cells (27Pei D. FEBS Lett. 1999; 457: 262-270Crossref PubMed Scopus (45) Google Scholar). Given its lack of a C-terminal transmembrane domain or glycosylphosphatidylinositol anchor found in the MT-MMPs, we hypothesize that CA-MMP may possess a novel mechanism for cellular localization. In this report, we present evidence that CA-MMP is localized as a type II transmembrane proteinase via a signal anchor. Furthermore, a specific cleavage at Arg79 releases active CA-MMP into the extracellular milieu. The apparent coupling of secretion and activation for a membrane-anchored zymogen defines an unprecedented strategy to regulate proteolytic activity.DISCUSSIONProteolysis is a basic mechanism of regulation in biology as exemplified by the caspase-mediated execution of cell death (48Salvesen G.S. Dixit V.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10964-10967Crossref PubMed Scopus (766) Google Scholar), proteosome-regulated progression of cell cycle (49Kirschner M. Trends Cell Biol. 1999; 9: M42-M45Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), and antigen processings (50Villadangos J.A. Bryant R.A. Deussing J. Driessen C. Lennon-Dumenil A.M. Riese R.J. Roth W. Saftig P. Shi G.P. Chapman H.A. Peters C. Ploegh H.L. Immunol. Rev. 1999; 172: 109-120Crossref PubMed Scopus (204) Google Scholar). The diversity and complexity of proteolysis may rival some of the better known biochemical processes such as phosphorylation/dephosphorylation. Irreversible and destructive in nature, proteolysis must be controlled tightly. With ECM components as substrates, members of the MMP family are encoded and synthesized as zymogens to be either secreted or displayed as surface proteinases via a type I transmembrane domain or glycosylphosphatidylinositol link, providing multiple steps for cells to regulate their activities under both physiological as well as pathological conditions (for reviews, see Refs. 9Birkedal-Hansen H. Moore W.G. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2630) Google Scholar and 10Massova I. Kotra L.P. Fridman R. Mobashery S. FASEB J. 1998; 12: 1075-1095Crossref PubMed Scopus (698) Google Scholar). Here we report the characterization of CA-MMP as a type II membrane-anchored zymogen that is proteolytically processed at a conserved motif and converted into a secretory and mature enzyme. This unique mechanism of localization and processing may allow cells to control the proteolytic activity of CA-MMP more efficiently in ECM remodeling events.CA-MMP is the first MMP to be demonstrated as a type II transmembrane protein. Among ∼25 MMPs discovered so far, they can be classified into two basic groups based on their cellular localizations. The first group is relatively large and made of secretory MMPs including collagenases, gelatinases, and stromeolysins (10Massova I. Kotra L.P. Fridman R. Mobashery S. FASEB J. 1998; 12: 1075-1095Crossref PubMed Scopus (698) Google Scholar). The second group is smaller and made of the 6 MT-MMPs (14Will H. Hinzmann B. Eur. J. Biochem. 1995; 231: 602-608Crossref PubMed Scopus (317) Google Scholar, 15Takino T. Sato H. Shinagawa A. Seiki M. J. Biol. Chem. 1995; 270: 23013-23020Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 16Puente X.S. Pendas A.M. Llano E. Velasco G. Lopez-Otin C. Cancer Res. 1996; 56: 944-949PubMed Google Scholar, 17Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar, 18Pei D. Cell Res. 1999; 9: 291-303Crossref PubMed Scopus (169) Google Scholar, 19Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). Based on the membrane anchoring mechanisms, these MT-MMPs may be further divided into type I transmembrane MMPs for MT1–3, 5-MMPs, and the glycosylphosphatidylinositol-anchored MMPs (MT4, 6-MMPs) (20Itoh Y. Kajita M. Kinoh H. Mori H. Okada A. Seiki M. J. Biol. Chem. 1999; 274: 34260-34266Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Thus, CA-MMP would establish the third subclass of membrane-bound MMPs as a novel type II transmembrane MMP. Without any precedence, we initially questioned the validity of the sequence-based prediction of CA-MMP being a type II transmembrane protein (Fig. 1). Although consistent with the finding that the majority of CA-MMP is cell-associated (27Pei D. FEBS Lett. 1999; 457: 262-270Crossref PubMed Scopus (45) Google Scholar), CA-MMP could be associated with the cells by means other than being transmembrane-anchored. Thus, we devoted our effort to (i) verify that CA-MMP is an integral membrane protein by cell fractionation and differential extractions with high salt, alkali, and Trition X-100 (Fig. 2) (35Wahlberg J.M. Spiess M. J. Cell Biol. 1997; 137: 555-562Crossref PubMed Scopus (117) Google Scholar, 39Gilmore R. Blobel G. Cell. 1985; 42: 497-505Abstract Full Text PDF PubMed Scopus (163) Google Scholar), (ii) demonstrate by N-terminal sequencing that L20GAALSGLCLLSALALL36 is retained with CA-MMP as a signal-anchor rather than being a cleavable signal peptide (41Rapoport T.A. Jungnickel B. Kutay U. Annu. Rev. Biochem. 1996; 65: 271-303Crossref PubMed Scopus (492) Google Scholar), and (iii) establish that the first 39 residues including the signal anchor is sufficient to localize a soluble reporter protein, GFP, to membrane and the signal anchor is required for the observed localizations. Taken together, these three independent lines of evidence argue strongly that CA-MMP is an integral membrane protein with L20GAALSGLCLLSALALL36 as the signal anchor. As for its topology, CA-MMP was predicted to be a type II transmembrane protein based on the charge rules (35Wahlberg J.M. Spiess M. J. Cell Biol. 1997; 137: 555-562Crossref PubMed Scopus (117) Google Scholar, 43von Heijne G. Nature. 1989; 341: 456-458Crossref PubMed Scopus (427) Google Scholar). The 19 residues that precede the signal anchor contain a net charge of +2 for CA-MMP and its orthologues whereas the downstream sequences contain a −2 for rat and mouse and 0 for human. This charge distribution would favor the transfer of the carboxyl side into the lumen (the positive inside rule) (43von Heijne G. Nature. 1989; 341: 456-458Crossref PubMed Scopus (427) Google Scholar). We proved this prediction by demonstrating that CA-MMP is N-glycosylated, a process unique for protein residing in the lumen of the secretory pathway (Fig. 4). Since the 4 likely N-glycosylation sites (Asn93, Asn149, Asn233, and Asn317, see Fig. 1 A) are located downstream of the signal anchor, the carboxyl side must reside in the lumen in order to beingN-glycosylated (Fig. 4). Together, this evidence argues that CA-MMP is a type II transmembrane protein.The coupled secretion and activation of CA-MMP is regulated by a conserved PC motif RRRR79. Being a type II transmembrane zymogen, CA-MMP assumes a reverse topology in comparison to the MT-MMPs (14Will H. Hinzmann B. Eur. J. Biochem. 1995; 231: 602-608Crossref PubMed Scopus (317) Google Scholar, 15Takino T. Sato H. Shinagawa A. Seiki M. J. Biol. Chem. 1995; 270: 23013-23020Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 17Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar, 19Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). The obligatory removal of its N-terminal prodomain would release the mature enzyme into the lumen and result in secretion. Sandwiched between its pro- and catalytic domains, there is a motif, RRRR79, which could be recognized by PCs (27Pei D. FEBS Lett. 1999; 457: 262-270Crossref PubMed Scopus (45) Google Scholar, 28Velasco G. Pendas A.M. Fueyo A. Knauper V. Murphy G. Lopez-Otin C. J. Biol. Chem. 1999; 274: 4570-4576Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). A similar RXKR motif has been demonstrated to be important for zymogen activation of MMP-11 through a process mediated by furin, a prototype PC (38Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (530) Google Scholar). Thus, a single cleavage at Arg79 could not only release CA-MMP from the membrane, but also remove its N-terminal prodomain. Indeed, we mapped the cleavage site for secreted CA-MMP at Arg79 (Figs. 3 and 8), thus, confirming a coupled process for both secretion and activation. This is an interesting strategy because cells secrete only active CA-MMP, not latent ones like other secretory MMPs (9Birkedal-Hansen H. Moore W.G. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2630) Google Scholar, 10Massova I. Kotra L.P. Fridman R. Mobashery S. FASEB J. 1998; 12: 1075-1095Crossref PubMed Scopus (698) Google Scholar). Accordingly, we speculate that CA-MMP functions in short distances from its origin of expression since its long distant efficacy would certainly be compromised by free tissue inhibitor of matrix metalloproteinases (TIMPs) in the extracellular milieu. Alternatively, the demand for CA-MMP activity is so acute that only an efficient and timely delivery of active species may meet the requirement. Both features would fit well with rapidity of the ovulation process where CA-MMP is expressed at relatively high levels (28Velasco G. Pendas A.M. Fueyo A. Knauper V. Murphy G. Lopez-Otin C. J. Biol. Chem. 1999; 274: 4570-4576Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). 3J. Nie and D. Pei, unpublished results. The identification of its target substrates during the ovulation process will shed light on its proposed role in breaking the wall of ovarian capsules to release oocytes (28Velasco G. Pendas A.M. Fueyo A. Knauper V. Murphy G. Lopez-Otin C. J. Biol. Chem. 1999; 274: 4570-4576Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar).The PC motif, RX(K/R)R, has been implicated in prohormone processing (47Steiner D.F. Curr. Opin. Chem. Biol. 1998; 2: 31-39Crossref PubMed Scopus (577) Google Scholar), the maturation of proproteins such as HIV gp-160 (51Hallenberger S. Bosch V. Angliker H. Shaw E. Klenk H.D. Garten W. Nature. 1992; 360: 358-361Crossref PubMed Scopus (476) Google Scholar), zymogen activations for MMPs (17Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar, 38Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (530) Google Scholar), tumor necrosis factor-α converting enzyme (2Black R.A. Rauch C.T. Kozlosky C.J. Peschon J.J. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Nelson N. Boiani N. Schooley K.A. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. Paxton R.J. March C.J. Cerretti D.P. Nature. 1997; 385: 729-733Crossref PubMed Scopus (2675) Google Scholar), and the newly identified aggrecan-cleaving ADAMTS-4 (7Tortorella M.D. Burn T.C. Pratta M.A. Abbaszade I. Hollis J.M. Liu R. Rosenfeld S.A. Copeland R.A. Decicco C.P. Wynn R. Rockwell A. Yang F. Duke J.L. Solomon K. George H. Bruckner R. Nagase H. Itoh Y. Ellis D.M. Ross H. Wiswall B.H. Murphy K. Hillman Jr., M.C. Hollis G.F. Arner E.C. et al.Science. 1999; 284: 1664-1666Crossref PubMed Scopus (618) Google Scholar). Proteolytic cleavage at the RX(K/R)R motif in these proproteins converts latent protein into active species, executing an irreversible step in biological regulations. In an evolutionary sense, the recruitment of a similar motif for the coupled secretion and activation of CA-MMP represents the first precedence with an unusual degree of degeneracy by converting a two-step process with implied flexibility to a single rigid one. The tradeoff may be efficiency and ease of control for dispensing the "right" amount of CA-MMP activity. It is not clear whether this strategy is advantageous over the standard processes of secretion mediated by signal peptidases and subsequent activation in the extracellular milieu for most of the secretory MMPs (10Massova I. Kotra L.P. Fridman R. Mobashery S. FASEB J. 1998; 12: 1075-1095Crossref PubMed Scopus (698) Google Scholar, 12Nagase H. Biol. Chem. 1997; 378: 151-160PubMed Google Scholar). Considering its divergent structure from the other MMPs, CA-MMP is certainly evolved with a dramatically different design (27Pei D. FEBS Lett. 1999; 457: 262-270Crossref PubMed Scopus (45) Google Scholar, 28Velasco G. Pendas A.M. Fueyo A. Knauper V. Murphy G. Lopez-Otin C. J. Biol. Chem. 1999; 274: 4570-4576Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). We can only speculate that such a unique design of domain structure warrants a coupled strategy for secretion and activation. Through a signal anchor, the cells retain latent CA-MMP within the cells, most likely in the ER network (Figs. 6 and 10), effectively eliminating any possibility of unwanted activation in the extracellular milieu. Since members of the PCs are known to be localized in the trans-Golgi network (47Steiner D.F. Curr. Opin. Chem. Biol. 1998; 2: 31-39Crossref PubMed Scopus (577) Google Scholar), trafficking of CA-MMP to the trans-Golgi complex would trigger the cleavage at Arg79 and result in the release of active CA-MMP (Fig.10). Thus, we predict that a key regulatory and rate-limiting step in controlling CA-MMP activity be the trafficking from ER to the trans-Golgi network (Fig. 10). Consequently, the observed low level of secretion in CA-MMP transfected MDCK cells may represent only the basal level of secretion in an overexpression system. It is possible that endocrine factors responsible for orchestrating the well executed turnover of ECM during ovulation may actually regulate the trafficking of CA-MMP from ER to the trans-Golgi network where the presumed cleavage by PCs takes place (Fig. 10). This attractive hypothesis is testable if cells from ovulating ovaries become available. Furthermore, the significance of the coupled secretion and activation could be tested in vivo by a "knock-in" approach to replace the signal anchor with a classic signal peptide. It would be interesting to see if such as a modified CA-MMP can substitute the wild type gene in vivo. Nonetheless, the discovery that CA-MMP/MMP-23 is a type II transmembrane proteinase with a coupled secretion and activation mechanism not only introduces a novel mechanism of regulation for MMP-mediated proteolysis but also establish an interesting paradigm for protein trafficking and processing within the secretory pathway. 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The matrix metalloprot
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