Rack1, a Receptor for Activated Protein Kinase C, Interacts with Integrin β Subunit

Abstract

The integrin β subunit cytoplasmic domains are important for activation-dependent cell adhesion and adhesion-dependent signaling events. We report an interaction between integrin β subunit cytoplasmic domain and Rack1, a Trp-Asp (WD) repeat protein that has been shown to bind activated protein kinase C. The Rack1-binding site on integrin β2subunit resides within a conserved, membrane-proximal region. In the yeast two-hybrid assay, WD repeats five to seven of Rack1 (Rack1-WD5/7) interact with integrin β1, β2, and β5 cytoplasmic domain. In eukaryotic cells, Rack1 co-immunoprecipitates with at least two different β integrins, β1 integrins in 293T cells and β2 integrins in JY lymphoblastoid cells. Whereas Rack1-WD5/7 binds integrins constitutively, the association of full-length Rack1 to integrinsin vivo requires a treatment with phorbol esters, which promotes cell spreading and adhesion. These findings suggest that Rack1 may link protein kinase C directly to integrins and participate in the regulation of integrin functions. The integrin β subunit cytoplasmic domains are important for activation-dependent cell adhesion and adhesion-dependent signaling events. We report an interaction between integrin β subunit cytoplasmic domain and Rack1, a Trp-Asp (WD) repeat protein that has been shown to bind activated protein kinase C. The Rack1-binding site on integrin β2subunit resides within a conserved, membrane-proximal region. In the yeast two-hybrid assay, WD repeats five to seven of Rack1 (Rack1-WD5/7) interact with integrin β1, β2, and β5 cytoplasmic domain. In eukaryotic cells, Rack1 co-immunoprecipitates with at least two different β integrins, β1 integrins in 293T cells and β2 integrins in JY lymphoblastoid cells. Whereas Rack1-WD5/7 binds integrins constitutively, the association of full-length Rack1 to integrinsin vivo requires a treatment with phorbol esters, which promotes cell spreading and adhesion. These findings suggest that Rack1 may link protein kinase C directly to integrins and participate in the regulation of integrin functions. Integrins are αβ heterodimeric adhesion receptors that mediate attachment of cells to the extracellular matrix and specific cell counter-receptors (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9014) Google Scholar). Various extracellular stimuli have been shown to affect the adhesiveness of integrins and regulate attachment of cells to the extracellular matrix (2Springer T.A. Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6400) Google Scholar). This process known as an activation-dependent cell adhesion is best illustrated in leukocytes where the attachment of integrin LFA1 1The abbreviations used are: LFA1, leukocyte function antigen-1; GST, glutathione S-transferase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; mAb, monoclonal antibody(ies); GST-Rack1, GST-tagged Rack1; WD, Trp-Asp.(αLβ2) to intercellular cell adhesion molecule-1 (ICAM-1) substrates can be promoted by cross-linking T cell receptors or stimulating cells with phorbol 12-myristate 13-acetate (PMA) (3Dustin M.L. Springer T.A. Nature. 1989; 341: 619-624Crossref PubMed Scopus (1288) Google Scholar). Upon binding to the extracellular matrix, integrins induce signals required for the reorganization of actin cytoskeleton and the formation of focal adhesion complexes (4Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2816) Google Scholar, 5Yamada K.M. Miyamoto S. Curr. Opin. Cell Biol. 1995; 7: 681-689Crossref PubMed Scopus (588) Google Scholar, 6Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-519Crossref PubMed Scopus (1659) Google Scholar). The adhesion-dependent clustering of integrins leads to the activation of nonreceptor tyrosine kinase focal adhesion kinase and Ras/mitogen-activated protein kinase pathway, the stimulation of inositol lipid metabolism, an increase in intracellular Ca2+ and pH, and the activation of PKC (4Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2816) Google Scholar, 7Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1469) Google Scholar, 8Lafrenie R.M. Yamada K.M. J. Cell. Biochem. 1996; 61: 543-553Crossref PubMed Scopus (127) Google Scholar). Each subunit of integrins consists of a large extracellular ligand-binding domain, a transmembrane domain, and a short cytoplasmic domain that lacks any enzymatic activity. Although the cytoplasmic domains of α subunits are variable in size and sequence, the cytoplasmic domains of β subunits are more conserved in size and sequence. In particular, three conserved regions, termed cyto-1, cyto-2, and cyto-3, found in β integrin cytoplasmic domains have been implicated in the recruitment of integrins to the focal adhesion plaques and the regulation of adhesive functions of integrins (9Reszka A.A. Hayashi Y. Horwitz A.F. J. Cell Biol. 1992; 117: 1321-1330Crossref PubMed Scopus (240) Google Scholar, 10O'Toole T.E. Ylanne J. Culley B.M. J. Biol. Chem. 1995; 270: 8553-8558Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 11Dedhar S. Hannigan G.E. Curr. Opin. Cell Biol. 1996; 8: 657-669Crossref PubMed Scopus (346) Google Scholar). Although both integrin subunits are required for the ligand binding, the interaction between intracellular proteins and integrin cytoplasmic domain can occur in the absence of subunit association. Studies have shown that chimeric molecules composed of the β integrin cytoplasmic domains and the extracellular domain of the interleukin-2 receptor can be directed to the focal adhesion plaques and activate focal adhesion kinase in the absence of ligand binding (12LaFlamme S.E. Thomas L.A. Yamada S.S. Yamada K.M. J. Cell Biol. 1994; 126: 1287-1298Crossref PubMed Scopus (207) Google Scholar). Based on these findings, it has been hypothesized that the integrin β subunit cytoplasmic domain provides binding sites for proteins involved in the regulation of integrin functions. Proteins that have been shown to directly interact with β subunit cytoplasmic domains include known cytoskeletal proteins α-actinin (13Otey C.A. Vasquez G.B. Burridge K. Erickson B.W. J. Biol. Chem. 1993; 268: 21193-21197Abstract Full Text PDF PubMed Google Scholar), paxillin (14Schaller M.D. Otey C.A. Hildebrand J.D. Parsons J.T. J. Cell Biol. 1995; 130: 1181-1187Crossref PubMed Scopus (556) Google Scholar), talin (11Dedhar S. Hannigan G.E. Curr. Opin. Cell Biol. 1996; 8: 657-669Crossref PubMed Scopus (346) Google Scholar, 15Horwitz A. Duggan K. Buck C. Beckerle M.C. Burridge K. Nature. 1986; 320: 531-533Crossref PubMed Scopus (825) Google Scholar), and filamin (16Sharma C.P. Ezzell R.M. Arnaout M.A. J. Immunol. 1995; 154: 3461-3470PubMed Google Scholar); protein kinases focal adhesion kinase (14Schaller M.D. Otey C.A. Hildebrand J.D. Parsons J.T. J. Cell Biol. 1995; 130: 1181-1187Crossref PubMed Scopus (556) Google Scholar) and integrin-linked kinase-1 (ILK-1) (17Hannigan G.E. Leung-Hagesteijn C. Fitz-Gibbon L. Coppolino M.G. Radeva G. Filmus J. Bell J.C. Dedhar S. Nature. 1996; 379: 91-96Crossref PubMed Scopus (967) Google Scholar); and potential regulatory proteins such as β3-endonexin (18Shattil S.J. O'Toole T. Eigenthaler M. Thon V. Williams M. Babior B.M. Ginsberg M.H. J. Cell Biol. 1995; 131: 807-816Crossref PubMed Scopus (167) Google Scholar), cytohesin-1 (19Kolanus W. Nagel W. Schiller B. Zeitlmann L. Godar S. Stockinger H. Seed B. Cell. 1996; 86: 233-242Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar), and integrin cytoplasmic domain-associated protein-1 (Icap-1) (20Chang D.D. Wong C. Smith H. Liu J. J. Cell Biol. 1997; 138: 1149-1157Crossref PubMed Scopus (150) Google Scholar). In this study, we report a direct association of receptor for activated protein kinase C (Rack1) to the integrin β subunit cytoplasmic domain. Rack1 consists of seven repeating units of WD motifs presumed to be involved in a protein-protein interaction (2Springer T.A. Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6400) Google Scholar, 21Ron D. Chen C.H. Caldwell J. Jamieson L. Orr E. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 839-843Crossref PubMed Scopus (646) Google Scholar). Deletion studies indicate that the C-terminal three WD motifs (WD-5, WD-6, and WD-7) of Rack1 interact with the conserved membrane-proximal region of β subunit cytoplasmic domain. Interestingly, interaction of Rack1 with integrins in vivo requires a treatment with PMA, which promotes cell spreading and integrin-dependent cell adhesion (3Dustin M.L. Springer T.A. Nature. 1989; 341: 619-624Crossref PubMed Scopus (1288) Google Scholar, 22Stewart M.P. Cabanas C. Hogg N. J. Immunol. 1996; 156: 1810-1817PubMed Google Scholar). Our finding suggests a direct linkage between integrins and PKC through Rack1 and further implicates PKC in integrin-mediated cell signaling. The anti-β1integrin antibody producing hybridoma cell line, TS2/16, was generously provided by Dr. M. Hemler (Dana Farber Cancer Institute, Boston, MA). The mouse hybridoma cell line TS1/18 producing anti-β2integrin antibody was obtained from the ATCC (Rockville, MD). The mAb TS2/16 and TS1/18 recognize the extracellular domains of β1 and β2 integrins, respectively. Anti-Rack1 antibody was purchased from Transduction Labs. The anti-αL antibody, directed against the integrin αL cytoplasmic domain, was described previously (20Chang D.D. Wong C. Smith H. Liu J. J. Cell Biol. 1997; 138: 1149-1157Crossref PubMed Scopus (150) Google Scholar). 293T cells, a derivative of human kidney embryonal fibroblast containing SV40 T antigen, were obtained from Dr. K. Shuai (University of California at Los Angeles) and cultured in Dulbecco's modified Eagle's medium + 5% fetal calf serum (BioWhittaker). JY cells, an Epstein-Barr virus-transformed human lymphoid cell line, were obtained from Dr. T. Springer (Harvard Medical School, Boston, MA) and cultured in RPMI + 10% fetal calf serum. The yeast genetic screening for the isolation of proteins interacting with the cytoplasmic domain of β2 integrin was carried out essentially as described previously (20Chang D.D. Wong C. Smith H. Liu J. J. Cell Biol. 1997; 138: 1149-1157Crossref PubMed Scopus (150) Google Scholar, 23Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1322) Google Scholar). The C-terminal 46 amino acids (724–769) of the β2 integrin were cloned in frame into LexA coding sequence to generate a “bait” plasmid, pNlex-β2cyto. pNlex-β2cyto and a JY cDNA library in the pJG45 yeast expression vector (23Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1322) Google Scholar) were introduced into a yeast strain containing a chromosomal copy of the Leu2 gene (EGY48 (Matα trp1 ura3–52 leu2::pLeu2-lexAop6(ΔUAS leu2)) and an episomal β-galactosidase gene (as a JK103 plasmid) under the control of a synthetic promoter with LexA-binding sites (23Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1322) Google Scholar). Yeast transformants were selected for the formation of blue colonies on 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside indicator plates with leucine. The interacting cDNA clones were rescued from the selected yeast transformants as described previously (23Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1322) Google Scholar). Rack1-ΔBH1 was constructed by cloning the C-terminal BamHI fragment of Rack1 (amino acids 204–317) into the pJG45 vector (23Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1322) Google Scholar). The β1 and β5 integrin cytoplasmic domain and the β2 integrin cytoplasmic domain mutants were generated by polymerase chain reaction and cloned into the pNLex vector. All constructs used in this study were verified by direct DNA sequence analysis. For in vitro GST interaction assays, the β2 integrin cytoplasmic domain (amino acids 724–769), a truncation mutant β2(E16) (amino acids 750–769), and the αL integrin subunit cytoplasmic domain were individually expressed as GST fusion proteins using the bacterial expression vector pGEX4T1 (Pharmacia Biotech Inc.) (24Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1994: 911-919Google Scholar). Rack1-WD5/7 was synthesized in vitro using a co-transcription/translation system (Promega) and added to approximately 2 μg of GST fusion proteins and incubated overnight at 4 °C in NET (25 mm Tris-HCl, pH 7.6, 100 mmNaCl, 3 mm EDTA) containing 1 mmdithiothreitol, 1% bovine serum albumin, and 0.1% Triton X-100. The GST fusion proteins were purified using glutathione-Sepharose beads (Pharmacia). The beads were washed twice in the binding buffer and twice in 0.05% Triton X-100 in NET. The bound proteins were eluted by boiling in SDS sample buffer, subjected to SDS/polyacrylamide gel electrophoresis, and then transferred to a nitrocellulose membrane (Bio-Rad). The filter membrane was probed with an anti-Rack1 antibody (0.06 μg/ml) and then developed using an enhanced chemiluminescence method (Amersham Corp.). A full-length Rack1 cDNA was obtained from a JY cDNA library using a polymerase chain reaction and cloned into the eukaryotic expression vectors pcDNA3 (Invitrogen) and pEBG (20Chang D.D. Wong C. Smith H. Liu J. J. Cell Biol. 1997; 138: 1149-1157Crossref PubMed Scopus (150) Google Scholar). 5–10 μg of plasmid DNA was transfected into 293T cells by using a calcium phosphate precipitation method (24Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1994: 911-919Google Scholar). 48 h post-transfection, cells were lysed in TBSM (25 mm Tris-HCl, pH 7.6, 150 mm NaCl, and 2 mm MgCl2) containing 0.5% Nonidet P-40, leupeptin, aprotinin, and phenylmethylsulfonyl fluoride. When indicated, the cells were treated with PMA (100 ng/ml, stock solution 1 mg/ml in Me2SO) for 30 min at 37 °C prior to lysis. Detergent insoluble were by at for μg of the were with an of TBSM and incubated with glutathione-Sepharose beads for 3 h at 4 were washed with TBSM + Nonidet and TBSM three of β1 integrins with the bound was by with the mAb TS2/16 of β1 integrins was carried out by the with 4 of the mAb TS2/16 for 3 h at 4 in the of protein beads JY cell were as and LFA1 was using the mAb of Rack1 with the integrins was by using an anti-Rack1 The yeast two-hybrid were used to proteins that interact with the cytoplasmic domain of A which the cytoplasmic domain (amino acids of β2 subunit was introduced into a yeast strain Leu2 and β-galactosidase under the control of a synthetic promoter with LexA-binding sites (23Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1322) Google Scholar). The strain was used as a for with a human cell cDNA library in the pJG45 yeast expression vector. were found to interact with the cytoplasmic domain of with the control containing that clones of the C-terminal of the Rack1 is composed of repeating units of WD D. Chen C.H. Caldwell J. Jamieson L. Orr E. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 839-843Crossref PubMed Scopus (646) Google Scholar). Rack1 is to a where WD repeat composed of β a of the R. Smith Nature. 1994; PubMed Scopus Google Scholar, J. A. Nature. 1996; 379: PubMed Scopus Google Scholar, M.A. E. J.A. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). The Rack1 cDNA clones in yeast two-hybrid screening through these three C-terminal WD repeats are for the binding to the β2 integrin cytoplasmic domain A of amino acids to the of the we that is for the integrin The amino acids in Rack1-ΔBH1 the β and the that are on the of the D. Chen C.H. Caldwell J. Jamieson L. Orr E. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 839-843Crossref PubMed Scopus (646) Google J. A. Nature. 1996; 379: PubMed Scopus Google Scholar, M.A. E. J.A. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). of the Rack1 clones the WD motifs to The full-length Rack1 to interact with the containing the β integrin cytoplasmic domains in a that the interaction between the full-length Rack1 and the β integrins may be In a of we the Rack1-binding site on the β2 integrin cytoplasmic domain by the interaction between Rack1-WD5/7 (amino acids and β2 integrin cytoplasmic domain mutants A C-terminal of the β2 integrin cytoplasmic domain to amino which the sites at and three at affect the binding of a of 4 amino acids in the membrane-proximal of cytoplasmic domain or a of the to the the Rack1-binding site resides within a region (amino acids of the β2 integrin cytoplasmic domain. The membrane-proximal region of the β integrin cytoplasmic domains is conserved, and Rack1-WD5/7 was to interact with two integrins, β1 and the interaction between integrin β subunit and Rack1 in Rack1-WD5/7 was synthesized in vitro and incubated with GST fusion proteins containing the β integrin cytoplasmic domains Rack1-WD5/7 with a GST fusion protein the cytoplasmic domain of integrin β2 amino acids with GST fusion proteins of integrin domain amino acids and amino acids 1 and or a GST fusion protein the cytoplasmic domain of integrin αL The of in vitro binding were to the findings in the yeast two-hybrid interaction the interaction of Rack1 and integrinsin Rack1-WD5/7 or a full-length Rack1 were expressed as GST fusion proteins in 293T cells. The transfected cells were lysed in a buffer containing and incubated with glutathione-Sepharose beads to fusion The binding of β1 integrins to the fusion proteins were by with the mAb TS2/16 The β1 integrins were with with GST used as the 4 of of the GST fusion proteins in 293T cells was by with antibody In two-hybrid Rack1 of which the WD motifs to the were We that the β2 integrin cytoplasmic domain interact with the full-length in yeast These indicate that the of Rack1 to may with the integrin In of the report that Rack1 binds PKC in the of Ca2+ and lipid to activate PKC D. Chen C.H. Caldwell J. Jamieson L. Orr E. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 839-843Crossref PubMed Scopus (646) Google Scholar), we that the binding of full-length Rack1 to integrins in vivo is and may activation of in we to association of integrins with the transfected in 293T cells stimulation with PMA, a formation between the β1 integrins and 4 In a of 293T cells were transfected with and the formation between Rack1 and β1 integrins was by with the mAb TS2/16 by a using anti-Rack1 antibody 4 in GST interaction assays, Rack1 and β1 integrins a a PMA treatment 4 In the was association between the two 4 The of Rack1 by control PMA stimulation 4 3 and focal adhesion kinase or two proteins known to with integrins to the focal adhesion were in the by antibody that Rack1 can with different β as in the yeast two-hybrid assay, we the in JY cells that LFA1 The association of Rack1 with LFA1 can be PMA treatment 1 and of of LFA1 was by the with anti-αL antibody These a specific association of Rack1 with different β integrins in to a that is known to integrin-mediated cell adhesion. Integrins have been as transmembrane proteins cytoskeleton to the extracellular studies have that integrin-dependent cell adhesion is in to extracellular In stimulation in the extracellular ligand-binding domain of the integrin to an M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1469) Google Scholar). A has been for integrin-dependent cell adhesion of cell is enhanced cell spreading is by a PMA treatment (3Dustin M.L. Springer T.A. Nature. 1989; 341: 619-624Crossref PubMed Scopus (1288) Google Scholar, 22Stewart M.P. Cabanas C. Hogg N. J. Immunol. 1996; 156: 1810-1817PubMed Google Scholar, Ginsberg M.H. J. 1994; PubMed Scopus Google Scholar). of these integrin cytoplasmic that the interaction between integrins and proteins an important in the regulation of cell adhesion. into cell adhesion we to proteins that directly with integrin β subunit cytoplasmic In the study, we report isolation of Rack1 as an Our for the interaction of Rack1 with integrins is on the interaction of the two proteins in a yeast two-hybrid assay, the binding vitro C-terminal of Rack1 to expressed GST fusion protein containing the β2 integrin cytoplasmic domain, and the of a between Rack1 and integrins in Rack1 is a of composed of seven WD repeats an β M.A. E. J.A. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). WD repeats motifs by β and have been implicated in protein-protein interaction R. Smith Nature. 1994; PubMed Scopus Google Scholar). Although the function of Rack1 is Rack1 binds to PKC in the of Ca2+ and lipid in vitro and has been as an intracellular receptor for activated protein kinase C for of PKC D. Chen C.H. Caldwell J. Jamieson L. Orr E. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 839-843Crossref PubMed Scopus (646) Google Scholar). cDNA from yeast screening that the WD repeats are for interaction with β a of WD repeat to bind β2 The region in Rack1-ΔBH1 to the of the β that be to with The region of the integrin β2 cytoplasmic domain for the Rack1 binding within the membrane-proximal region This region is conserved different β and Rack1 with at least two β β1 and The of three a at the to be for the Rack1 a in this region or a of to the Rack1 of β1 integrins to the focal adhesion plaques (9Reszka A.A. Hayashi Y. Horwitz A.F. J. Cell Biol. 1992; 117: 1321-1330Crossref PubMed Scopus (240) Google Scholar) and regulation of integrins O'Toole T.E. Ylanne J. S.J. Ginsberg M.H. J. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus (171) Google Scholar), have been to the membrane-proximal region. be of to these known integrin functions The finding of study is that the association between Rack1 and integrins in vivo is and requires a PMA β subunits are known to on and PMA treatment M.L. S. Springer T.A. J. PubMed Scopus Google Scholar). the that the of β cytoplasmic domain is for the recruitment of Rack1 Rack1-WD5/7 binds integrins both in vitro and the of sites on β cytoplasmic domain affect this Based on a previously function of Rack1 as a cytoplasmic receptor of activated PKC D. Chen C.H. Caldwell J. Jamieson L. Orr E. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 839-843Crossref PubMed Scopus (646) Google Scholar), it is that a PMA activated PKC binds and Rack1, recruitment of Rack1 to the integrin cytoplasmic domain. Cell stimulation with PMA or receptor activation integrin and promotes cell spreading M.P. Cabanas C. Hogg N. J. Immunol. 1996; 156: 1810-1817PubMed Google Scholar, Ginsberg M.H. J. 1994; PubMed Scopus Google Scholar). The of association to be In to Rack1, two α-actinin F.M. J. Immunol. 1993; Google Scholar) and M. Leung-Hagesteijn C. Dedhar S. J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), have been shown to bind integrin cytoplasmic domain in an through integrins to the actin cytoskeleton may function to integrin Rack1 or which bind to β subunit cytoplasmic with α subunits through a conserved short M. V. Dedhar S. PubMed Scopus Google Scholar). The function of to be of by antibody into cells or gene with integrin-dependent cell adhesion M. Leung-Hagesteijn C. Dedhar S. J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, N. S. R. Dedhar S. Nature. 1997; PubMed Scopus Google Scholar). The of protein-protein interaction WD domains may Rack1 to function as a protein to PKC proteins to the membrane at the site of by to bind integrin cytoplasmic domain. Rack1 may be an in this association and participate in regulation of cell adhesion. We are to R. Brent for the yeast and for the yeast genetic We are to M. Hemler Cancer T. Springer and K. Shuai (University of California at Los Angeles) for and C. for on the