The ADAM10 Prodomain Is a Specific Inhibitor of ADAM10 Proteolytic Activity and Inhibits Cellular Shedding Events

Abstract

ADAM10 is a disintegrin metalloproteinase that processes amyloid precursor protein and ErbB ligands and is involved in the shedding of many type I and type II single membrane-spanning proteins. Like tumor necrosis factor-α-converting enzyme (TACE or ADAM17), ADAM10 is expressed as a zymogen, and removal of the prodomain results in its activation. Here we report that the recombinant mouse ADAM10 prodomain, purified from Escherichia coli, is a potent competitive inhibitor of the human ADAM10 catalytic/disintegrin domain, with a Ki of 48 nm. Moreover, the mouse ADAM10 prodomain is a selective inhibitor as it only weakly inhibits other ADAM family proteinases in the micromolar range and does not inhibit members of the matrix metalloproteinase family under similar conditions. Mouse prodomains of TACE and ADAM8 do not inhibit their respective enzymes, indicating that ADAM10 inhibition by its prodomain is unique. In cell-based assays we show that the ADAM10 prodomain inhibits betacellulin shedding, demonstrating that it could be of potential use as a therapeutic agent to treat cancer. ADAM10 is a disintegrin metalloproteinase that processes amyloid precursor protein and ErbB ligands and is involved in the shedding of many type I and type II single membrane-spanning proteins. Like tumor necrosis factor-α-converting enzyme (TACE or ADAM17), ADAM10 is expressed as a zymogen, and removal of the prodomain results in its activation. Here we report that the recombinant mouse ADAM10 prodomain, purified from Escherichia coli, is a potent competitive inhibitor of the human ADAM10 catalytic/disintegrin domain, with a Ki of 48 nm. Moreover, the mouse ADAM10 prodomain is a selective inhibitor as it only weakly inhibits other ADAM family proteinases in the micromolar range and does not inhibit members of the matrix metalloproteinase family under similar conditions. Mouse prodomains of TACE and ADAM8 do not inhibit their respective enzymes, indicating that ADAM10 inhibition by its prodomain is unique. In cell-based assays we show that the ADAM10 prodomain inhibits betacellulin shedding, demonstrating that it could be of potential use as a therapeutic agent to treat cancer. Members of the a disintegrin and metalloproteinase (ADAM) 3The abbreviations used are: ADAM, a disintegrin and metalloproteinase; aa, amino acid; APP, amyloid precursor protein; AR, amphiregulin; BTC, betacellulin; dabcyl, 4-(4-dimethylaminophenylazo)benzoyl; Me2SO, dimethyl sulfoxide; EGF, epidermal growth factor; EGFR, EGF receptor; IFNγ, interferon-γ; IMPE, immortalized pancreatic epithelial cells; Pro, prodomain; TACE, tumor necrosis factor-α converting enzyme; Ni-NTA, nickel-nitrilotriacetic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium; MMP, matrix metalloproteinase. family of proteolytic enzymes play important roles in many cellular signaling processes. They function as sheddases by cleaving type I and type II integral single membrane proteins to generate soluble mature forms (1Moss M.L. Bartsch J.W. Biochemistry. 2004; 43: 7227-7235Crossref PubMed Scopus (119) Google Scholar, 2Blobel C.P. Nat. Rev. Mol. Cell Biol. 2005; 6: 32-43Crossref PubMed Scopus (928) Google Scholar, 3Black R.A. Doedens J.R. Mahimkar R. Johnson R. Guo L. Wallace A. Virca D. Eisenman J. Slack J. Castner B. Sunnarborg S.W. Lee D.C. Cowling R. Jin G. Charrier K. Peschon J.J. Paxton R. Biochem. Soc. Symp. 2003; 70: 39-52Crossref PubMed Scopus (66) Google Scholar). ADAM family members typically contain several domains as follows: a prodomain, a catalytic and cysteine-rich disintegrin domain, a transmembrane region, and a cytoplasmic tail (4Blobel C.P. Wolfsberg T.G. Turck C.W. Myles D.G. Primakoff P. White J.M. Nature. 1992; 356: 248-252Crossref PubMed Scopus (612) Google Scholar). Prodomains are required for successful production of ADAM family members. For example, TACE and ADAM12 require their prodomains for efficient transport, folding, and expression (5Milla M.E. Leesnitzer M.A. Moss M.L. Clay W.C. Carter H.L. Miller A.B. Su J.L. Lambert M.H. Willard D.H. Sheeley D.M. Kost T.A. Burkhart W. Moyer M. Blackburn R.K. Pahel G.L. Mitchell J.L. Hoffman C.R. Becherer J.D. J. Biol. Chem. 1999; 274: 30563-30570Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 6Hougaard S. Loechel F. Xu X. Tajima R. Albrechtsen R. Wewer U.M. Biochem. Biophys. Res. Commun. 2000; 275: 261-267Crossref PubMed Scopus (52) Google Scholar, 7Loechel F. Overgaard M.T. Oxvig C. Albrechtsen R. Wewer U.M. J. Biol. Chem. 1999; 274: 13427-13433Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Similarly, the prodomain of ADAM10 acts as a molecular chaperone to allow the expression of catalytically active ADAM10 (8Fahrenholz F. Gilbert S. Kojro E. Lammich S. Postina R. Ann. N. Y. Acad. Sci. 2000; 920: 215-222Crossref PubMed Scopus (59) Google Scholar). ADAM family members are expressed as zymogens with the prodomains maintaining the enzymes in a latent state. Isolated prodomains have been shown to inhibit the proteolytic activity of ADAM family proteins in vitro (9Gonzales P.E. Solomon A. Miller A.B. Leesnitzer M.A. Sagi I. Milla M.E. J. Biol. Chem. 2004; 279: 31638-31645Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). However, not all prodomains are good inhibitors. For example, although the prodomain of TACE suppresses the activity of its catalytic domain with a Ki of 50 nm (9Gonzales P.E. Solomon A. Miller A.B. Leesnitzer M.A. Sagi I. Milla M.E. J. Biol. Chem. 2004; 279: 31638-31645Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), it is only a high micromolar inhibitor against the TACE construct, including both the catalytic and disintegrin domains. Because the catalytic and disintegrin domains are both retained in membrane-bound TACE, the prodomain, once removed, most likely does not negatively affect TACE activity in vivo. ADAM family members are zinc metalloproteinases, and their catalytic activity depends on the activation of the zinc-bound water and subsequent nucleophilic attack on the amide backbone of a protein substrate. The inhibitory mechanism, in which the prodomain interferes with metalloproteinase activity by replacing the zinc-bound water with a cysteine residue, is known as “the cysteine switch” and was originally described for members of the matrix metalloproteinase (MMP) family (10Van Wart H.E. Birkedal-Hansen H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5578-5582Crossref PubMed Scopus (1211) Google Scholar). For ADAM12, the cysteine is required for inhibition of the catalytic activity when an α2-macroglobulin assay is used to assess catalytic activity (7Loechel F. Overgaard M.T. Oxvig C. Albrechtsen R. Wewer U.M. J. Biol. Chem. 1999; 274: 13427-13433Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). However, mutation of the corresponding cysteine to alanine in the putative cysteine switch region of TACE does not diminish the inhibitory properties of the prodomain, suggesting that the cysteine switch hypothesis is invalid for this family member (9Gonzales P.E. Solomon A. Miller A.B. Leesnitzer M.A. Sagi I. Milla M.E. J. Biol. Chem. 2004; 279: 31638-31645Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Biologically important substrates of ADAM10 include epidermal growth factor (EGF) (11Sahin U. Weskamp G. Kelly K. Zhou H.M. Higashiyama S. Peschon J. Hartmann D. Saftig P. Blobel C.P. J. Cell Biol. 2004; 164: 769-779Crossref PubMed Scopus (794) Google Scholar), betacellulin (11Sahin U. Weskamp G. Kelly K. Zhou H.M. Higashiyama S. Peschon J. Hartmann D. Saftig P. Blobel C.P. J. Cell Biol. 2004; 164: 769-779Crossref PubMed Scopus (794) Google Scholar), Notch (12Pan D. Rubin G.M. Cell. 1997; 90: 271-280Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar), and amyloid precursor protein (APP) (13Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3922-3927Crossref PubMed Scopus (986) Google Scholar). Consequently, mis-regulation of ADAM10 catalytic activity is implicated in Alzheimer disease and carcinogenesis. For example, α-secretases such as ADAM10 and other metalloproteinases can carry out the first cleavage reaction of APP (14Kojro E. Fahrenholz F. Subcell. Biochem. 2005; 38: 105-127Crossref PubMed Google Scholar). Disruption of ADAM10 activity has been shown to decrease the level of soluble nonamyloidogenic APP both in vivo and in cell-based assays (15Postina R. Schroeder A. Dewachter I. Bohl J. Schmitt U. Kojro E. Prinzen C. Endres K. Hiemke C. Blessing M. Flamez P. Dequenne A. Godaux E. van Leuven F. Fahrenholz F. J. Clin. Investig. 2004; 113: 1456-1464Crossref PubMed Scopus (533) Google Scholar), suggesting that maintaining ADAM10 activity may play a protective role in Alzheimer disease for processing of APP via the α-secretase pathway. In contrast, excess ADAM10 activity may promote cell growth in cancer proliferation assays because of enhanced production of soluble mature forms of several ErbB ligands such as betacellulin (BTC) and EGF (2Blobel C.P. Nat. Rev. Mol. Cell Biol. 2005; 6: 32-43Crossref PubMed Scopus (928) Google Scholar, 11Sahin U. Weskamp G. Kelly K. Zhou H.M. Higashiyama S. Peschon J. Hartmann D. Saftig P. Blobel C.P. J. Cell Biol. 2004; 164: 769-779Crossref PubMed Scopus (794) Google Scholar). Blocking EGF receptor (EGFR) signaling in cancer by EGFR kinase inhibitors or that EGFR are to tumor growth M. I. Biochem. Sci. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, X. Investig. 2005; PubMed Scopus Google Scholar). to EGFR signaling could be inhibition of processing of soluble ErbB ligands because ADAM10 is a known of and EGF (11Sahin U. Weskamp G. Kelly K. Zhou H.M. Higashiyama S. Peschon J. Hartmann D. Saftig P. Blobel C.P. J. Cell Biol. 2004; 164: 769-779Crossref PubMed Scopus (794) Google Scholar), and soluble forms EGFR by receptor (2Blobel C.P. Nat. Rev. Mol. Cell Biol. 2005; 6: 32-43Crossref PubMed Scopus (928) Google Scholar). a and potent inhibitors of ADAM10 could be used to tumor in vivo. have to and a prodomain on the mouse of to high expression of soluble the cysteine switch mechanism, we a mutation by the cysteine to in the ADAM10 show that the mutation does not the inhibitory of the prodomain against of the inhibition of ADAM10 prodomains against and and that the type and prodomain are selective inhibitors against ADAM10 prodomain inhibits betacellulin shedding in cell-based assays in and may an for the of and TACE the catalytic/disintegrin domains from The enzymes to TACE catalytic domain from of and from and from of and from from or from from the of for and from of ADAM ADAM10 prodomain was the and to an or a modified and as a The modified and to a protein with a as follows: and and and and and and The type ADAM10 prodomain was used as a to cysteine to The for and For prodomains of TACE and ADAM8 mouse used as to that the as described for For prodomains and the was used as a and the the as described For prodomain the ADAM10 was used as a and or and Escherichia For a in of the The was for to protein by for in a The was removed, and or used Cell by in of 50 and or with the with cell from of a and For the type was to to of in a for The was to a with with a 50 and the protein was with a 50 and The protein was to an of from and purified with a on an a of or protein and an from to and as was on a for the prodomain the molecular of ADAM10 prodomain in was by and and on a The of the protein and of the to be and by the The protein was several and and was by the nm. The was by the of the ADAM10 in a and 50 was used to a on an from to nm was in a in with a of from the from the with TACE and of TACE TACE or ADAM10 catalytic/disintegrin was the M.L. Biochem. PubMed Scopus Google with nm and nm. The was from a in to in assay and in a with ADAM10 prodomain nm to or with of enzyme from to nm for TACE and from to nm for For assays with TACE and ADAM8 prodomain, the range of from to with N. L. G. C. Moss M.L. Biochemistry. 2004; 43: PubMed Scopus Google in assay 50 and was used to enzyme and with in the or of prodomain an of nm with M.L. Biochem. PubMed Scopus Google Scholar), in a was used to human catalytic/disintegrin domain The was as a from which was to the reaction in the or of ADAM10 prodomain and of and with ADAM catalytic domain was expressed and purified from E. as described S. S. D. S. Moss M.L. Bartsch J.W. Biol. Chem. PubMed Scopus Google Scholar). on the cleavage was used to enzyme activity with a a 50 in Me2SO, the was in of was of or of ADAM10 prodomain by of the reaction of in a of a of in was to The was to for in the nm and For assays with TACE and ADAM8 substrates used to activity of prodomains from to M.L. Biochem. PubMed Scopus Google Scholar). of of activity was by the prodomain from the prodomain with inhibition assays and the to is I is the inhibitor and Ki is the inhibition of was in and was to of M.L. Lambert M.H. Biochem. 38: PubMed Scopus Google with nm and nm. in and 50 was to the of The of prodomain from to nm. The was and from of The of the was the of because the The as a family of to several The to a competitive as described in (2Blobel C.P. Nat. Rev. Mol. Cell Biol. 2005; 6: 32-43Crossref PubMed Scopus (928) Google of inhibitor from to both In this is the from the is the is the I is the inhibitor is the for to enzyme; and Ki is the inhibition immortalized pancreatic epithelial cell K. D. S. E. S. K. M. Google was with the human with a as described J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google or with human in Dulbecco's modified Eagle's growth amino and and For shedding in and for 48 to in and for an in and For all shedding for with of and or of or ADAM10 For of shedding, with the and for shedding, with the and for and cell as described J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). human and human assays used to and in and cell to the The recombinant human and human used as for their respective with similar and a is for are expressed as the of against the prodomain and a the cytoplasmic domain of purified by protein Cell in and with the B. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). on and with the mouse 48 with the and used as we used or with to excess by with of the S. S. D. S. Moss M.L. Bartsch J.W. Biol. Chem. PubMed Scopus Google Scholar). 48 in CHAPS, inhibitor and to in of of to by and subsequent or and as by on the for the TACE prodomain (9Gonzales P.E. Solomon A. Miller A.B. Leesnitzer M.A. Sagi I. Milla M.E. J. Biol. Chem. 2004; 279: 31638-31645Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), we a amino of mouse ADAM10 the ADAM10 prodomain with the or to that the of the not with inhibition of of the region and the cleavage of the ADAM10 prodomain that the expression level was and that most of the protein was in an the from to we to of soluble ADAM10 prodomain for with The prodomain was purified via a by a The type prodomains the or both efficient inhibitors of ADAM10 with inhibition of and 48 that the ADAM10 prodomain inhibition does not on the of the properties of ADAM10 prodomain human human human human not inhibition was from the of prodomain not The inhibition was from the of prodomain in a metalloproteinases that as the of a cysteine in their prodomains is for inhibition of catalytic The cysteine is to the active zinc of the the enzyme to in an state. For of the ADAM family the cysteine is required for efficient inhibition of the the cysteine in the putative cysteine switch of the ADAM10 prodomain the of inhibition by a with the only cysteine to In is a the purified prodomain that the ADAM10 prodomain a and inhibitor of human ADAM10 with an inhibition of nm mouse ADAM10 catalytic activity is with a Ki of the hypothesis that are that for the of ADAM10 results that the cysteine in the ADAM10 prodomain is not required for efficient inhibition of catalytic and the inhibitory of ADAM10 prodomain is from the cysteine switch ADAM10 as a against ADAM and family members to the of the ADAM10 The inhibition was to The family matrix metalloproteinases and not by cysteine type or prodomains In TACE catalytic and TACE catalytic/disintegrin enzymes only by the prodomains of of ADAM8 was and and was with a Ki The potent inhibition of ADAM10 by its prodomain to be as the prodomains of TACE and ADAM8 only their respective enzymes micromolar The ADAM8 prodomain both TACE and only high of ADAM by mouse prodomains of TACE and human human human in a ADAM10 as a in of a protein on a its state. The prodomain of ADAM10 of and both the type and the to on the as a to the of the protein protein to this the of the by The molecular of the ADAM10 prodomain was in the range of the of as with a known molecular of The molecular not from to The for both the to and type that ADAM10 prodomain in as a under conditions. of the ADAM10 prodomain to of the of the ADAM10 prodomain and to that this protein is and to proteins and nm. The shown in a of and and that this is The nm However, the of a nm that a of this domain a of prodomain was to the of inhibition of a the was as a function of inhibitor for the a was used and a good with the the the that the prodomain of ADAM10 is a competitive inhibitor of ADAM10 catalytic and disintegrin The that ADAM10 has for the substrate. The inhibitor as a family of to the competitive inhibitor the from a of other The with this was 48 nm and the for was of the ADAM10 and to to the domain for inhibition of of the that the prodomain could most likely be the its with and of the prodomain via that the prodomain could be modified the type and and inhibition of ADAM10 The results are in potent inhibitors of ADAM10 with inhibition of and However, retained inhibitory of amino to the by to generate or not to affect suggesting that are not for prodomain to of the in a prodomain that was expressed as an in E. coli, and not The to was to amino this its inhibitory activity against ADAM10 and an inhibition of as a in out the that inhibition of ADAM10 with the of prodomain to of ADAM10 by prodomains of in a with ADAM10 the of of the ADAM10 prodomain on shedding from been A. Gilbert S. W. Postina R. Fahrenholz F. J. PubMed Scopus Google Scholar). has been that the of the ADAM10 prodomain shedding most likely by with mature ADAM10 in the or the cell B. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the ADAM10 prodomain could inhibit the shedding of ADAM10 we the of recombinant ADAM10 prodomain on the shedding of ErbB and AR, in cell-based cleavage ADAM10 is for and shedding of betacellulin (11Sahin U. Weskamp G. Kelly K. Zhou H.M. Higashiyama S. Peschon J. Hartmann D. Saftig P. Blobel C.P. J. Cell Biol. 2004; 164: 769-779Crossref PubMed Scopus (794) Google Scholar, J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), is for and shedding is of ADAM10 may only be on K. S. M. K. G. Y. Hartmann D. Saftig P. Blobel C.P. Mol. Biol. Cell. PubMed Scopus Google Scholar). Because shedding is by and not by J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), we to the of the ADAM10 prodomain on shedding of and in first that and shedding in we the of inhibitors selective ADAM10 and on shedding C. D. T.A. N. Saftig P. K. Hartmann D. Fahrenholz F. Postina R. S. A. 2003; PubMed Scopus Google Scholar, E. A. S. Saftig P. S. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, A. C. Lambert M.H. N. D.M. Leesnitzer M.A. Becherer J.D. Chem. 2005; PubMed Scopus Google Scholar). both and shedding in and shedding was in a by the inhibitor only inhibitory on shedding the in are in with that shedding is on ADAM10 J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). with the ADAM10 prodomain could shedding in the prodomain of ADAM10 was to inhibit betacellulin in a with inhibition and inhibition that the ADAM10 prodomain was to and shedding in and human not The ADAM10 prodomain that does not inhibit ADAM10 on betacellulin shedding, indicating that inhibition of ADAM10 activity depends on the inhibitory properties of the that the prodomain inhibits ADAM10 was with cell-based assays used to assess inhibition of shedding in was with the ADAM10 prodomain Moreover, the ADAM10 prodomain on other and shedding not that with recombinant ADAM10 prodomain can inhibit the shedding of an ADAM10 to the shedding from the cell and of and the ADAM10 prodomain as an inhibitor in cell-based is for an in vivo function of the prodomain, by ADAM10 we in with by against the prodomain and the cytoplasmic domain of The mouse ADAM10 was with against the prodomain or the cytoplasmic tail of ADAM10 in The and the ADAM10 prodomain and the prodomain or is to the with the that most of the protein is in the only a of prodomain can be to the cell Consequently, with a of ADAM10 in the that active and ADAM10 are in their cellular that active ADAM10 is with its prodomain under conditions. In with or with not The that prodomain is on the cell for a of ADAM10 activity by the of the prodomain ADAM10 this and we to and ADAM10 in out the hypothesis that of prodomain is the the prodomain is in Consequently, with to a of and the ADAM10 The that prodomain and mature ADAM10 are in that of the prodomain with ADAM10 the cell via is to that prodomain with mature ADAM10 the have that the prodomain of mouse ADAM10 is a inhibitor of human ADAM10 activity both in vitro and in cell-based this is the first report of a inhibitor of ADAM10 48 as other such as and inhibitors of metalloproteinases, inhibit as as other ADAM family members. For example, the ADAM10 prodomain does not inhibit the of other ADAM (8Fahrenholz F. Gilbert S. Kojro E. Lammich S. Postina R. Ann. N. Y. Acad. Sci. 2000; 920: 215-222Crossref PubMed Scopus (59) Google Scholar, P.E. Solomon A. Miller A.B. Leesnitzer M.A. Sagi I. Milla M.E. J. Biol. Chem. 2004; 279: 31638-31645Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, and X. Investig. 2005; PubMed Scopus Google under inhibition of ADAM10 is the prodomain can be used in cellular assays to assess ADAM10 the prodomains the inhibitory activity although are potent the of the inhibitory potential in a region to In the cysteine is for inhibition in the The prodomain as a of it is the to amino The cysteine is not required for that the prodomain and metalloproteinase domain is and we to this in However, we could not use the we used for the in vivo as the recombinant ADAM10 enzyme the domain is against the amino the recombinant ADAM10 is Moreover, a is not as both the recombinant ADAM10 and the prodomain contain this the only to the be or this is because of of the ADAM10 protein from a The mouse ADAM10 prodomain is in its to inhibit the ADAM10 catalytic/disintegrin In contrast, the mouse prodomain of TACE only inhibits the of human TACE catalytic domain and human ADAM10 catalytic/disintegrin domain, (9Gonzales P.E. Solomon A. Miller A.B. Leesnitzer M.A. Sagi I. Milla M.E. J. Biol. Chem. 2004; 279: 31638-31645Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In on activity is for micromolar of the prodomain for the human TACE catalytic/disintegrin domain Mouse ADAM8 prodomain only inhibits mouse ADAM8 catalytic domain, and does not inhibit the human ADAM8 catalytic/disintegrin However, it does human TACE and ADAM10 Because other ADAM family members do not to to their respective prodomains in an in vitro it is for other members to with mature in vivo. The of prodomains to inhibit enzymes does not to on with Mouse ADAM10 prodomain has the most to human ADAM10 prodomain by human TACE prodomain a could TACE prodomain inhibits to is when prodomain is that the cysteine switch region of mouse ADAM8 prodomain with TACE could mouse ADAM8 prodomain inhibits both TACE ADAM10 prodomain with ADAM10 the cell in In the prodomain with mature and ADAM10 when of prodomain with ADAM10 could proteins. However, that prodomain in vitro with ADAM10 with a inhibition that the ADAM10 prodomain is likely to be with ADAM10 in vivo or the has processing by a The and of the ADAM10 prodomain it a good for ADAM10 activity in vivo. In a betacellulin the prodomain was an Because the Ki in activity assays the it is micromolar of prodomain are required in this for this from inhibitors of ADAM10 used in this micromolar of inhibitor are required in cell-based assays that of proteins the inhibitors are against the enzyme in an in vitro is an of this as its is nm against TACE in an enzyme assay is micromolar in cell-based shedding have that inhibits ADAM10 in the range has an of micromolar range for betacellulin In inhibitors of metalloproteinases are required in micromolar in shedding assays are inhibitors of and S. M. S. H. K. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus (52) Google Scholar). that the prodomain of ADAM10 may be to inhibit ADAM10 in vivo micromolar are used in shedding although is that other ADAM10 are involved in processing of ErbB for example, EGF processing activity L. Blobel C.P. Res. 2005; PubMed Scopus Google Scholar), and other as may be to other processes are on The high expression level and of the prodomain in cell-based assays it for and Milla of for the TACE catalytic domain and and for