Sher Karki

Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fcgamma receptors with both activating and inhibitory activities. By using computational design algorithms and high-throughput screening, we have engineered a series of Fc variants with optimized Fcgamma receptor affinity and specificity. The designed variants display >2 orders of magnitude enhancement of in vitro effector function, enable efficacy against cells expressing low levels of target antigen, and result in increased cytotoxicity in an in vivo preclinical model. Our engineered Fc regions offer a means for improving the next generation of therapeutic antibodies and have the potential to broaden the diversity of antigens that can be targeted for antibody-based tumor therapy.

The identification of optimal target antigens on tumor cells is central to the advancement of new antibody-based cancer therapies. We performed suppression subtractive hybridization and identified nectin-4 (PVRL4), a type I transmembrane protein and member of a family of related immunoglobulin-like adhesion molecules, as a potential target in epithelial cancers. We conducted immunohistochemical analysis of 2,394 patient specimens from bladder, breast, lung, pancreatic, ovarian, head/neck, and esophageal tumors and found that 69% of all specimens stained positive for nectin-4. Moderate to strong staining was especially observed in 60% of bladder and 53% of breast tumor specimens, whereas the expression of nectin-4 in normal tissue was more limited. We generated a novel antibody-drug conjugate (ADC) enfortumab vedotin comprising the human anti-nectin-4 antibody conjugated to the highly potent microtubule-disrupting agent MMAE. Hybridoma (AGS-22M6E) and CHO (ASG-22CE) versions of enfortumab vedotin (also known as ASG-22ME) ADC were able to bind to cell surface-expressed nectin-4 with high affinity and induced cell death in vitro in a dose-dependent manner. Treatment of mouse xenograft models of human breast, bladder, pancreatic, and lung cancers with enfortumab vedotin significantly inhibited the growth of all four tumor types and resulted in tumor regression of breast and bladder xenografts. Overall, these findings validate nectin-4 as an attractive therapeutic target in multiple solid tumors and support further clinical development, investigation, and application of nectin-4-targeting ADCs. Cancer Res; 76(10); 3003-13. ©2016 AACR.

Since the initial discovery of cytoplasmic dynein, it has become apparent that this microtubule-based motor is involved in several cellular functions including cell division and intracellular transport. Another multisubunit complex, dynactin, may be required for most, if not all, cytoplasmic dynein-driven activities and may provide clues to dynein's functional diversity. Recent genetic and biochemical findings have illuminated the cellular roles of dynein and dynactin and provided insight into the functional mechanism of this complex motor.

We used affinity chromatography to probe for a direct binding interaction between cytoplasmic dynein and dynactin. Purified cytoplasmic dynein was found to bind to an affinity column of p150Glued, the largest polypeptide in the dynactin complex. To test the specificity of the interaction, we loaded rat brain cytosol onto the p150Glued affinity column and observed that cytoplasmic dynein from cytosol was specifically retained on the column. Preincubation of the p150Glued affinity matrix with excess exogenous dynein intermediate chain resulted in a significant reduction of dynein binding, suggesting that p150Glued may be interacting with dynein via this polypeptide. Therefore we constructed an affinity column of recombinant dynein intermediate chain and observed that dynactin was retained from rat brain cytosol. These results demonstrate that the native dynein and dynactin complexes are capable of direct in vitro interaction mediated by a direct binding of the dynein intermediate chain to the p150Gluedcomponent of the dynactin complex. We have mapped the site of this interaction to the amino-terminal region of p150Glued, which is predicted to form an α-helical coiled-coil. Regulation of the dynein-dynactin interaction may prove to be key in the control mechanism for cytoplasmic dynein-mediated vesicular transport. We used affinity chromatography to probe for a direct binding interaction between cytoplasmic dynein and dynactin. Purified cytoplasmic dynein was found to bind to an affinity column of p150Glued, the largest polypeptide in the dynactin complex. To test the specificity of the interaction, we loaded rat brain cytosol onto the p150Glued affinity column and observed that cytoplasmic dynein from cytosol was specifically retained on the column. Preincubation of the p150Glued affinity matrix with excess exogenous dynein intermediate chain resulted in a significant reduction of dynein binding, suggesting that p150Glued may be interacting with dynein via this polypeptide. Therefore we constructed an affinity column of recombinant dynein intermediate chain and observed that dynactin was retained from rat brain cytosol. These results demonstrate that the native dynein and dynactin complexes are capable of direct in vitro interaction mediated by a direct binding of the dynein intermediate chain to the p150Gluedcomponent of the dynactin complex. We have mapped the site of this interaction to the amino-terminal region of p150Glued, which is predicted to form an α-helical coiled-coil. Regulation of the dynein-dynactin interaction may prove to be key in the control mechanism for cytoplasmic dynein-mediated vesicular transport. INTRODUCTIONCoordinated trafficking of organelles along microtubules is central to the viability of cell and is powered by the mechanochemical ATPases kinesin and cytoplasmic dynein. While the mechanisms which govern the specificity and regulation of this transport remain to be determined, there is growing evidence for the role of accessory factors in the function of the molecular motors involved. Recently, an integral membrane protein, kinectin, was found to be the essential anchor for kinesin-driven vesicle motility(1Toyoshima I. Yu H. Steuer E.R. Sheetz M.P. J. Cell Biol. 1992; 118: 1121-1131Crossref PubMed Scopus (185) Google Scholar, 2Kumar J. Hanry Y. Sheetz M.P. Science. 1995; 267: 1834-1837Crossref PubMed Scopus (133) Google Scholar). Although no membrane receptor for cytoplasmic dynein has been described yet, a distinct 20 S complex, dynactin, was shown to differentially co-purify with cytoplasmic dynein from a variety of sources(3Paschal B.M. Shpetner H.S. Vallee R.B. J. Cell Biol. 1987; 105: 1273-1282Crossref PubMed Scopus (412) Google Scholar, 4Steuer E. Wordeman L. Schroer T.A. Sheetz M.P. Nature. 1990; 345: 266-268Crossref PubMed Scopus (397) Google Scholar, 5Holzbaur E.L.F. Hammerback J.A. Paschal B.M. Kravit N.G. Pfister K.K. Vallee R.B. Nature. 1991; 351: 579-583Crossref PubMed Scopus (153) Google Scholar). Also, in an in vitro motility assay the dynactin complex was required to reconstitute dynein-mediated vesicular motility(6Gill S.R. Schroer T.A. Szilak I. Steuer E.R. Sheetz M.P. Cleveland D.W. J. Cell Biol. 1991; 115: 1639-1650Crossref PubMed Scopus (393) Google Scholar). These observations suggest that dynactin may interact with cytoplasmic dynein transiently or in a regulatory manner. However, the mechanism of interaction is not clearly understood.Dynactin is a macromolecular oligomeric complex of at least 10 different polypeptides(6Gill S.R. Schroer T.A. Szilak I. Steuer E.R. Sheetz M.P. Cleveland D.W. J. Cell Biol. 1991; 115: 1639-1650Crossref PubMed Scopus (393) Google Scholar, 7Paschal B.M. Holzbaur E.L.F. Pfister K.K. Clark S. Meyer D. Vallee R.B. J. Biol. Chem. 1993; 268: 15318-15323Abstract Full Text PDF PubMed Google Scholar, 8Schafer D.A. Gill S.R. Cooper J.A. Heuser J.E. Schroer T.A. J. Cell Biol. 1994; 126: 402-412Crossref Scopus (242) Google Scholar). The two best characterized components of the dynactin complex are p150Glued and a 45-kDa protein, centractin. cDNA cloning and amino acid sequence analysis revealed that rat p150Glued is 32% identical to the product of the Drosophila gene Glued(5Holzbaur E.L.F. Hammerback J.A. Paschal B.M. Kravit N.G. Pfister K.K. Vallee R.B. Nature. 1991; 351: 579-583Crossref PubMed Scopus (153) Google Scholar, 9Swaroop A. Swaroop M. Garen A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6501-6505Crossref PubMed Scopus (52) Google Scholar). It has been shown previously that the null mutation of Glued is embryonic lethal(10Harte P.J. Kankel D.R. Genetics. 1982; 101: 477-501Crossref PubMed Google Scholar), thus suggesting that the p150Glued polypeptide has a role in an essential cell function such as mitosis or vesicular transport. Centractin (also known as Arp1) ( 1The abbreviations used are: Arp1actin-related protein 1DHCdynein heavy chainDICdynein intermediate chainLIClight intermediate chainPIPES1,4-piperazinediethanesulfonic acidBSAbovine serum albuminPAGEpolyacrylamide gel electrophoresis.) is a novel form of actin that to Schroer T.A. Nature. 1992; PubMed Scopus Google Scholar, Meyer Nature. 1992; PubMed Scopus Google Scholar). of the dynactin complex by revealed from the of D.A. Gill S.R. Cooper J.A. Heuser J.E. Schroer T.A. J. Cell Biol. 1994; 126: 402-412Crossref Scopus (242) Google Scholar). of and are to be components of the dynactin the polypeptide has been as and the and are the and of B.M. Holzbaur E.L.F. Pfister K.K. Clark S. Meyer D. Vallee R.B. J. Biol. Chem. 1993; 268: 15318-15323Abstract Full Text PDF PubMed Google Scholar, 8Schafer D.A. Gill S.R. Cooper J.A. Heuser J.E. Schroer T.A. J. Cell Biol. 1994; 126: 402-412Crossref Scopus (242) Google of have evidence for an in interaction between cytoplasmic dynein and dynactin in that in components of of the complexes to which in and have been shown to with to the cytoplasmic dynein heavy chain p150Glued and M. J. Cell Biol. 1994; PubMed Scopus Google Scholar). S. in a in in and which are to the observed in dynein heavy chain L. Cooper J.A. 1994; Full Text PDF PubMed Scopus (185) Google Scholar, D. S. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar, E. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar, Meyer J. Cell Biol. 1994; PubMed Scopus Google Scholar). has been that dynein heavy as or of the Glued M. J. A. S. M. J. Cell Biol. 1995; PubMed Scopus Google Scholar). observations suggest that dynein and dynactin interact in or the to the that there is a direct interaction between cytoplasmic dynein and the dynactin complex, we constructed affinity of p150Glued or dynein intermediate chain of dynein or of rat brain cytosol and for of cytoplasmic dynein on a p150Glued affinity column or of the dynactin complex on a affinity column. The results that the cytoplasmic dynein complex with the dynactin complex via a direct binding of the dynein intermediate chain to p150Gluedcomponent of the dynactin complex. direct binding between cytoplasmic dynein and dynactin evidence in of of an accessory in dynein These results suggest that of the dynein-dynactin interaction in may be a key in the mechanism of regulation of cytoplasmic dynein-mediated trafficking the and from cytosol was by the of brain and a of dynein in the that by the of Paschal B.M. Shpetner H.S. Vallee R.B. 1991; PubMed Scopus Google is dynein from rat brain was as described by Paschal B.M. Vallee R.B. Nature. 1987; PubMed Scopus Google Scholar, B.M. Shpetner H.S. Vallee R.B. 1991; PubMed Scopus Google Scholar), a of brain to The 20 S from the which the of the the dynein and dynactin E.L.F. Hammerback J.A. Paschal B.M. Kravit N.G. Pfister K.K. Vallee R.B. Nature. 1991; 351: 579-583Crossref PubMed Scopus (153) Google Scholar). These on and used The in the from the was a gel column and of amino of rat E.L.F. Hammerback J.A. Paschal B.M. Kravit N.G. Pfister K.K. Vallee R.B. Nature. 1991; 351: 579-583Crossref PubMed Scopus (153) Google and rat B.M. A. Pfister K.K. Vallee R.B. J. Cell Biol. 1992; 118: PubMed Scopus Google the and The are predicted to recombinant with in to the amino in of the with the and and in The was to cell and the was loaded on a column The column was in 20 with of to of and was with in The was of and in by in The with the at a protein of at on the with for at The in and in to The of was the assay and to the of was to of 20 S from the of the of which dynein and dynactin, or brain was loaded on the affinity column. The with or in The with a of of and The in the with acid on or with 10 of The by Nature. PubMed Scopus Google and or onto and with the that the to a of in 20 S and column in was loaded with of or 20 S and was loaded with of the column The of the loaded with of a of of acid or from the or the to p150Glued and on a The of the is ( D. S. and E. L. for an to was an polypeptide to the of S. Holzbaur E.L.F. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar), and an to p150Glued was a recombinant polypeptide which amino from the rat cDNA E.L.F. Hammerback J.A. Paschal B.M. Kravit N.G. Pfister K.K. Vallee R.B. Nature. 1991; 351: 579-583Crossref PubMed Scopus (153) Google Scholar). The to p150Glued and from serum the polypeptide on The used was a a of Pfister E.L.F. Hammerback J.A. Paschal B.M. Kravit N.G. Pfister K.K. Vallee R.B. Nature. 1991; 351: 579-583Crossref PubMed Scopus (153) Google Scholar). The is a by E. M.P. J. Cell Biol. 1993; PubMed Scopus Google to have an in interaction between cytoplasmic dynein and the dynactin complex, evidence a direct interaction between the two has been to a direct binding of cytoplasmic dynein to the dynactin complex, we constructed an affinity column on which an amino-terminal of the largest polypeptide in the dynactin complex, was to The 20 S from of from a affinity from rat which is in cytoplasmic dynein and dynactin, was the p150Glued affinity column the column was with and The by and for dynein binding to p150Glued to in that the 20 S which loaded on to the p150Glued affinity column to the The was from the column with a of and and column which was constructed not with the of dynein loaded there is no in the from the p150Glued affinity column that dynein in the loaded to the column. Although in two at with the of Paschal B.M. A. Pfister K.K. Vallee R.B. J. Cell Biol. 1992; 118: PubMed Scopus Google Scholar), of dynein intermediate the of on is Pfister K.K. J. Cell Biol. 1994; PubMed Scopus Google to test the specificity of the interaction, we a column specifically cytoplasmic dynein from brain cytosol. brain cytosol was loaded on p150Glued affinity column as as a control column. The with with a of and The by and by The gel of the that of the of molecular and which with components of the cytoplasmic dynein complex and to B.M. Shpetner H.S. Vallee R.B. 1991; PubMed Scopus Google Scholar), are specifically retained by the p150Glued affinity are not retained by the control cytoplasmic dynein to p150Glued affinity column. brain cytosol was loaded on a column of of p150Glued or on The with and with of and The to and the gel with and and and and The from the column of and which on to the and of cytoplasmic of the specifically retained from cytosol by the p150Glued column by with for the dynein intermediate chain and heavy chain the dynein heavy chain and intermediate chain retained specifically by the p150Glued column and not by the control column. of the to native p150Glued in the cytosol is by the of the at in and in as was observed E.L.F. Hammerback J.A. Paschal B.M. Kravit N.G. Pfister K.K. Vallee R.B. Nature. 1991; 351: 579-583Crossref PubMed Scopus (153) Google cytoplasmic dynein complex to brain cytosol was loaded on a column of of p150Glued or on The with and with of and The by with or chain and and and and and are have the of the shown in and that was from the p150Glued affinity column with It is that this is to the of the as with the is that there may be a of dynein intermediate chain in the as was observed previously by Paschal B.M. Holzbaur E.L.F. Pfister K.K. Clark S. Meyer D. Vallee R.B. J. Biol. Chem. 1993; 268: 15318-15323Abstract Full Text PDF PubMed Google Scholar). may bind to dynactin with a affinity the dynein thus at a from the affinity the the interaction between dynein and dynactin be mediated by the binding of p150Glued to the or to the dynein However, by to the intermediate chain of dynein which may the binding to J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar), the of cytoplasmic dynein may to the of the dynein and the interaction of with binding of cytoplasmic dynein to the may be direct J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google or may be mediated by a vesicle may function to dynein to the as p150Glued has been to to organelles and the on by S.R. Schroer T.A. Szilak I. Steuer E.R. Sheetz M.P. Cleveland D.W. J. Cell Biol. 1991; 115: 1639-1650Crossref PubMed Scopus (393) Google Scholar, 7Paschal B.M. Holzbaur E.L.F. Pfister K.K. Clark S. Meyer D. Vallee R.B. J. Biol. Chem. 1993; 268: 15318-15323Abstract Full Text PDF PubMed Google Scholar). Also, D.R. J. Cell Biol. 1994; 126: PubMed Scopus Google have that p150Glued with from test the dynein-dynactin interaction is mediated by an interaction between p150Glued and we p150Glued affinity as the matrix with excess of the cytosol onto the column control column was with excess the dynein-dynactin interaction, excess the binding on p150Glued, and dynein no bind to the column. the from or p150Glued affinity for we observed a significant reduction as by in the observed binding of to the the p150Glued column of binding and with and that the binding of cytoplasmic dynein to the p150Glued of the dynactin dynein intermediate chain dynein-dynactin p150Glued affinity in or brain cytosol was loaded and as described in the to The for with that excess dynein binding to p150Glued affinity column. and and and and to we that exogenous dynein binding to the p150Glued we the that dynein-dynactin binding may be mediated by components the cytoplasmic dynein complex. To test the that p150Glued may bind to we constructed affinity of brain cytosol was loaded onto an affinity column of The column was and with and The by and for the binding of dynactin complex to by to p150Glued and centractin. p150Glued and are components of the dynactin complex, the of components suggest that the dynactin complex is binding to the column. shown in p150Glued and found to be retained by the affinity column and we have that to S. Holzbaur E.L.F. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). the of by the column is mediated by with the p150Glued or of the dynactin complex found to be retained on a constructed control column and dynactin complex to dynein intermediate brain cytosol was loaded on a column of dynein intermediate chain or on The with and with of and The by with or and and and and and are have the or have found that we the of affinity chromatography by the of the column to the by is not retained by the column. The to an form of a interaction of with and that may the affinity of cytoplasmic dynein and dynactin the of the p150Glued or in the cytosol was observed to bind to the affinity column. The of the column may have been a significant of p150Glued and in brain cytosol was retained by the affinity column. of p150Glued and by the column that the dynactin complex as a to dynein and dynactin have been to interact in vitro and in an interaction is this the for the regulation of cytoplasmic dynein. is a 20 S oligomeric complex that with cytoplasmic E.L.F. Hammerback J.A. Paschal B.M. Kravit N.G. Pfister K.K. Vallee R.B. Nature. 1991; 351: 579-583Crossref PubMed Scopus (153) Google Scholar, S.R. Schroer T.A. Szilak I. Steuer E.R. Sheetz M.P. Cleveland D.W. J. Cell Biol. 1991; 115: 1639-1650Crossref PubMed Scopus (393) Google Scholar, 7Paschal B.M. Holzbaur E.L.F. Pfister K.K. Clark S. Meyer D. Vallee R.B. J. Biol. Chem. 1993; 268: 15318-15323Abstract Full Text PDF PubMed Google Scholar, T.A. Sheetz M.P. J. Cell Biol. 1991; 115: PubMed Scopus Google Scholar). However, the for this has been from a direct interaction or the two complexes such as affinity and between the two complexes has been by in vitro in which the of vesicular motility mediated by cytoplasmic dynein required a that S.R. Schroer T.A. Szilak I. Steuer E.R. Sheetz M.P. Cleveland D.W. J. Cell Biol. 1991; 115: 1639-1650Crossref PubMed Scopus (393) Google Scholar). While has been observed that to dynein to components of dynactin complex, and B.M. Holzbaur E.L.F. Pfister K.K. Clark S. Meyer D. Vallee R.B. J. Biol. Chem. 1993; 268: 15318-15323Abstract Full Text PDF PubMed Google Scholar), a direct in interaction has been by evidence in components of the dynein or the dynactin complex to M. J. Cell Biol. 1994; PubMed Scopus Google Scholar, L. Cooper J.A. 1994; Full Text PDF PubMed Scopus (185) Google Scholar, D. S. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar, E. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar, Meyer J. Cell Biol. 1994; PubMed Scopus Google we the of to interact with native protein The results in this clearly demonstrate that native cytoplasmic dynein to p150Glued and that the dynactin complex to We that this interaction is mediated by the direct binding of the to p150Glued, we dynein binding to the p150Glued column by excess exogenous interaction between of cytoplasmic dynein and the dynactin complex has been observed by and ( and J. Cell in the have mapped the binding to the amino-terminal of p150Glued, between amino and region is predicted to form an α-helical E.L.F. Hammerback J.A. Paschal B.M. Kravit N.G. Pfister K.K. Vallee R.B. Nature. 1991; 351: 579-583Crossref PubMed Scopus (153) Google Scholar). results from demonstrate that the p150Glued of the dynactin complex to the and to S. Holzbaur E.L.F. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar), an protein which is a of the dynactin S.R. Schroer T.A. Szilak I. Steuer E.R. Sheetz M.P. Cleveland D.W. J. Cell Biol. 1991; 115: 1639-1650Crossref PubMed Scopus (393) Google Scholar, 7Paschal B.M. Holzbaur E.L.F. Pfister K.K. Clark S. Meyer D. Vallee R.B. J. Biol. Chem. 1993; 268: 15318-15323Abstract Full Text PDF PubMed Google Scholar, 8Schafer D.A. Gill S.R. Cooper J.A. Heuser J.E. Schroer T.A. J. Cell Biol. 1994; 126: 402-412Crossref Scopus (242) Google Scholar, Schroer T.A. Nature. 1992; PubMed Scopus Google Scholar, Meyer Nature. 1992; PubMed Scopus Google Scholar). suggest that p150Glued is a polypeptide with at least interacting as in of p150Glued and a dynein-dynactin results and S. Holzbaur E.L.F. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google have of interaction on is a at the amino of J. J.E. 1992; Full Text PDF PubMed Scopus Google which is to the of J. J.E. 1992; Full Text PDF PubMed Scopus Google Scholar), and the there is a of amino that p150Glued binding to centractin. has an in the amino-terminal of p150Glued that a for the interaction p150Glued, and has been analysis of dynactin D.A. Gill S.R. Cooper J.A. Heuser J.E. Schroer T.A. J. Cell Biol. 1994; 126: 402-412Crossref Scopus (242) Google revealed a of p150Glued with to the as as as the of of the the dynactin complex. heavy are as two interacting with and the E.L.F. Vallee R.B. Cell Biol. 1994; PubMed Scopus Google Scholar). that for cytoplasmic dynein have been in the of dynein-dynactin is not that this in are to be and is that dynein and the dynactin complex as we have not to components of complex are B.M. Holzbaur E.L.F. Pfister K.K. Clark S. Meyer D. Vallee R.B. J. Biol. Chem. 1993; 268: 15318-15323Abstract Full Text PDF PubMed Google Scholar). However, this may from the of the of interaction by the M. S. S. M. D. and E. L. for observed that two distinct to p150Glued the interaction between dynein and dynactin. to the that used previously to dynein and dynactin with the dynein-dynactin interaction and to the to that the binding of cytoplasmic dynein to p150Glued is mediated by is in of the of this polypeptide to the intermediate chain of dynein from on dynein suggest that the intermediate chain is in the binding of dynein to the J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). Paschal B.M. A. Pfister K.K. Vallee R.B. J. Cell Biol. 1992; 118: PubMed Scopus Google have predicted that the cytoplasmic may function in an to cytoplasmic dynein to organelles or p150Glued is to the in the is that the dynactin complex on the of the binding of the dynein via the interaction between p150Glued and has been that p150Glued to microtubules of with cytoplasmic dynein S. Holzbaur E.L.F. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). with a in the protein S. Holzbaur E.L.F. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, J. J.E. 1992; Full Text PDF PubMed Scopus Google Scholar). has been to as a protein for the binding of to the J. J.E. 1992; Full Text PDF PubMed Scopus Google Scholar). with we that p150Glued may function to organelles and to the and cytoplasmic dynein to a in which p150Glued is to the the dynein intermediate and centractin. a interaction with the 10 S. Holzbaur E.L.F. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google vesicle may of the vesicle that of the dynein are predicted to be E.L.F. PubMed Scopus Google for of the shown in may be a to the via the that results a direct binding between the cytoplasmic dynein and dynactin complexes and p150Glued, be to the dynactin, the and the are we are to the molecular mechanism of dynein-dynactin p150Glued that is to the in an motility is to dynein and is an and the that interaction of dynactin to the or is and of p150Glued may dynein function by the affinity of the polypeptide for the or the and Holzbaur ( and E. L. in have shown that p150Glued is differentially in to that have been to vesicle Sheetz M.P. J. Cell Sci. 1993; Google Scholar). has been shown to from microtubules J.E. J. Biol. Chem. 1991; Full Text PDF PubMed Google and by the interaction of p150Glued to microtubules may be in a to transport of organelles along microtubules by cytoplasmic dynein. of p150Glued may binding to dynein. this is to that and J. Cell Sci. 1993; 105: Google and J. Cell Biol. 1994; PubMed Scopus Google have that acid of and of cytoplasmic dynein from to the cytosol. the of this be to of p150Glued which the of from p150Glued and the of cytoplasmic dynein from the have and the interaction between cytoplasmic dynein and the dynactin complex, we not as the mechanism by which dynactin dynein this we have described a the dynein-dynactin interaction be observed in vitro and have the components in this These results the for the observed in the Glued mutation in as dynactin may be an essential of the transport INTRODUCTIONCoordinated trafficking of organelles along microtubules is central to the viability of cell and is powered by the mechanochemical ATPases kinesin and cytoplasmic dynein. While the mechanisms which govern the specificity and regulation of this transport remain to be determined, there is growing evidence for the role of accessory factors in the function of the molecular motors involved. Recently, an integral membrane protein, kinectin, was found to be the essential anchor for kinesin-driven vesicle motility(1Toyoshima I. Yu H. Steuer E.R. Sheetz M.P. J. Cell Biol. 1992; 118: 1121-1131Crossref PubMed Scopus (185) Google Scholar, 2Kumar J. Hanry Y. Sheetz M.P. Science. 1995; 267: 1834-1837Crossref PubMed Scopus (133) Google Scholar). Although no membrane receptor for cytoplasmic dynein has been described yet, a distinct 20 S complex, dynactin, was shown to differentially co-purify with cytoplasmic dynein from a variety of sources(3Paschal B.M. Shpetner H.S. Vallee R.B. J. Cell Biol. 1987; 105: 1273-1282Crossref PubMed Scopus (412) Google Scholar, 4Steuer E. Wordeman L. Schroer T.A. Sheetz M.P. Nature. 1990; 345: 266-268Crossref PubMed Scopus (397) Google Scholar, 5Holzbaur E.L.F. Hammerback J.A. Paschal B.M. Kravit N.G. Pfister K.K. Vallee R.B. Nature. 1991; 351: 579-583Crossref PubMed Scopus (153) Google Scholar). Also, in an in vitro motility assay the dynactin complex was required to reconstitute dynein-mediated vesicular motility(6Gill S.R. Schroer T.A. Szilak I. Steuer E.R. Sheetz M.P. Cleveland D.W. J. Cell Biol. 1991; 115: 1639-1650Crossref PubMed Scopus (393) Google Scholar). These observations suggest that dynactin may interact with cytoplasmic dynein transiently or in a regulatory manner. However, the mechanism of interaction is not clearly understood.Dynactin is a macromolecular oligomeric complex of at least 10 different polypeptides(6Gill S.R. Schroer T.A. Szilak I. Steuer E.R. Sheetz M.P. Cleveland D.W. J. Cell Biol. 1991; 115: 1639-1650Crossref PubMed Scopus (393) Google Scholar, 7Paschal B.M. Holzbaur E.L.F. Pfister K.K. Clark S. Meyer D. Vallee R.B. J. Biol. Chem. 1993; 268: 15318-15323Abstract Full Text PDF PubMed Google Scholar, 8Schafer D.A. Gill S.R. Cooper J.A. Heuser J.E. Schroer T.A. J. Cell Biol. 1994; 126: 402-412Crossref Scopus (242) Google Scholar). The two best characterized components of the dynactin complex are p150Glued and a 45-kDa protein, centractin. cDNA cloning and amino acid sequence analysis revealed that rat p150Glued is 32% identical to the product of the Drosophila gene Glued(5Holzbaur E.L.F. Hammerback J.A. Paschal B.M. Kravit N.G. Pfister K.K. Vallee R.B. Nature. 1991; 351: 579-583Crossref PubMed Scopus (153) Google Scholar, 9Swaroop A. Swaroop M. Garen A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6501-6505Crossref PubMed Scopus (52) Google Scholar). It has been shown previously that the null mutation of Glued is embryonic lethal(10Harte P.J. Kankel D.R. Genetics. 1982; 101: 477-501Crossref PubMed Google Scholar), thus suggesting that the p150Glued polypeptide has a role in an essential cell function such as mitosis or vesicular transport. Centractin (also known as Arp1) ( 1The abbreviations used are: Arp1actin-related protein 1DHCdynein heavy chainDICdynein intermediate chainLIClight intermediate chainPIPES1,4-piperazinediethanesulfonic acidBSAbovine serum albuminPAGEpolyacrylamide gel electrophoresis.) is a novel form of actin that to Schroer T.A. Nature. 1992; PubMed Scopus Google Scholar, Meyer Nature. 1992; PubMed Scopus Google Scholar). of the dynactin complex by revealed from the of D.A. Gill S.R. Cooper J.A. Heuser J.E. Schroer T.A. J. Cell Biol. 1994; 126: 402-412Crossref Scopus (242) Google Scholar). of and are to be components of the dynactin the polypeptide has been as and the and are the and of B.M. Holzbaur E.L.F. Pfister K.K. Clark S. Meyer D. Vallee R.B. J. Biol. Chem. 1993; 268: 15318-15323Abstract Full Text PDF PubMed Google Scholar, 8Schafer D.A. Gill S.R. Cooper J.A. Heuser J.E. Schroer T.A. J. Cell Biol. 1994; 126: 402-412Crossref Scopus (242) Google of have evidence for an in interaction between cytoplasmic dynein and dynactin in that in components of of the complexes to which in and have been shown to with to the cytoplasmic dynein heavy chain p150Glued and M. J. Cell Biol. 1994; PubMed Scopus Google Scholar). S. in a in in and which are to the observed in dynein heavy chain L. Cooper J.A. 1994; Full Text PDF PubMed Scopus (185) Google Scholar, D. S. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar, E. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar, Meyer J. Cell Biol. 1994; PubMed Scopus Google Scholar). has been that dynein heavy as or of the Glued M. J. A. S. M. J. Cell Biol. 1995; PubMed Scopus Google Scholar). observations suggest that dynein and dynactin interact in or the to the that there is a direct interaction between cytoplasmic dynein and the dynactin complex, we constructed affinity of p150Glued or dynein intermediate chain of dynein or of rat brain cytosol and for of cytoplasmic dynein on a p150Glued affinity column or of the dynactin complex on a affinity column. The results that the cytoplasmic dynein complex with the dynactin complex via a direct binding of the dynein intermediate chain to p150Gluedcomponent of the dynactin complex. direct binding between cytoplasmic dynein and dynactin evidence in of of an accessory in dynein These results suggest that of the dynein-dynactin interaction in may be a key in the mechanism of regulation of cytoplasmic dynein-mediated trafficking the