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• W1665767818 abstract "Transmembrane proteins of the tetraspanin superfamily are associated with various integrins and modulate their function. We performed mutagenesis analysis to establish structural requirements for the interaction of CD151 with the α3β1 integrin and with other tetraspanins. Using a panel of CD151/CD9 chimeras and CD151 deletion mutants we show that the minimal region, which confers stable (e.g. Triton X-100-resistant) association of the tetraspanin with α3β1, maps within the large extracellular loop (LECL) of CD151 (the amino acid sequence between residues Leu149 and Glu213). Furthermore, the substitution of 11 amino acids (residues 195–205) from this region for a corresponding sequence from CD9 LECL or point mutations of cysteines in the conserved CCG and PXXCC motifs abolish the interaction. The removal of the LECL CD151 does not affect the association of the protein with other tetraspanins (e.g. CD9, CD81, CD63, and wild-type CD151). On the other hand, the mutation of the CCG motif selectively prevents the homotypic CD151·CD151 interaction but does not influence the association of the mutagenized CD151 with other tetraspanins. These results demonstrate the differences in structural requirements for the heterotypic and homotypic tetraspanin·tetraspanin interactions. Various deletions involving the small extracellular loop and the first three transmembrane domains prevent surface expression of the CD151 mutants but do not affect the CD151·α3β1interaction. The CD151 deletion mutants are accumulated in the endoplasmic reticulum and redirected to the lysosomes. The assembly of the CD151·α3β1 complex occurs early during the integrin biosynthesis and precedes the interaction of CD151 with other tetraspanins. Collectively, these data show that the incorporation of CD151 into the “tetraspanin web” can be controlled at various levels by different regions of the protein. Transmembrane proteins of the tetraspanin superfamily are associated with various integrins and modulate their function. We performed mutagenesis analysis to establish structural requirements for the interaction of CD151 with the α3β1 integrin and with other tetraspanins. Using a panel of CD151/CD9 chimeras and CD151 deletion mutants we show that the minimal region, which confers stable (e.g. Triton X-100-resistant) association of the tetraspanin with α3β1, maps within the large extracellular loop (LECL) of CD151 (the amino acid sequence between residues Leu149 and Glu213). Furthermore, the substitution of 11 amino acids (residues 195–205) from this region for a corresponding sequence from CD9 LECL or point mutations of cysteines in the conserved CCG and PXXCC motifs abolish the interaction. The removal of the LECL CD151 does not affect the association of the protein with other tetraspanins (e.g. CD9, CD81, CD63, and wild-type CD151). On the other hand, the mutation of the CCG motif selectively prevents the homotypic CD151·CD151 interaction but does not influence the association of the mutagenized CD151 with other tetraspanins. These results demonstrate the differences in structural requirements for the heterotypic and homotypic tetraspanin·tetraspanin interactions. Various deletions involving the small extracellular loop and the first three transmembrane domains prevent surface expression of the CD151 mutants but do not affect the CD151·α3β1interaction. The CD151 deletion mutants are accumulated in the endoplasmic reticulum and redirected to the lysosomes. The assembly of the CD151·α3β1 complex occurs early during the integrin biosynthesis and precedes the interaction of CD151 with other tetraspanins. Collectively, these data show that the incorporation of CD151 into the “tetraspanin web” can be controlled at various levels by different regions of the protein. large extracellular loop 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid fetal calf serum Dulbecco's modified Eagle's medium Chinese hamster ovary hemagglutinin antibody monoclonal antibody phosphate-buffered saline polyacrylamide gel electrophoresis fluorescein isothiocyanate retinal degeneration slow endoplasmic reticulum tetramethyl rhodamine isothiocyanate The tetraspanin superfamily (TM4SF proteins) is a large group of cell surface transmembrane proteins, which combines more than 25 members (1Hemler M.E. Mannion B.A. Berditchevski F. Biochim. Biophys. Acta. 1996; 1287: 67-71PubMed Google Scholar, 2Maecker H.T. Todd S.C. Levy S. FASEB J. 1997; 11: 428-442Crossref PubMed Scopus (811) Google Scholar). Although the exact biochemical function of tetraspanins is unknown, the proteins were implicated in a variety of biological processes, including egg-sperm fusion (3Le Naour F. Rubinstein E. Jasmin C. Prenant M. Boucheix C. Science. 2000; 287: 319-321Crossref PubMed Scopus (543) Google Scholar, 4Miyado K. Yamada G. Yamada S. Hasuwa H. Nakamura Y. Ryu F. Suzuki K. Kosai K. Inoue K. Ogura A. Okabe M. Mekada E. Science. 2000; 287: 321-324Crossref PubMed Scopus (563) Google Scholar), tissue morphogenesis and differentiation (5Boismenu R. Rhein M. Fischer W.H. Havran W.L. Science. 1996; 271: 198-200Crossref PubMed Scopus (78) Google Scholar, 6Kopczynski C.C. Davis G.W. Goodman C.S. Science. 1996; 271: 1867-1870Crossref PubMed Scopus (155) Google Scholar), and tumor cell growth and metastasis (7Dong J.-T. Lamb P.W. Rinker-Schaeffer C.W. Vukanovic J. Ichikawa T. Isaacs J.T. Barret J.C. Science. 1995; 268: 884-886Crossref PubMed Scopus (764) Google Scholar, 8Ikeyama S. Koyama M. Yamaoko M. Sasada R. Miyake M. J. Exp. Med. 1993; 177: 1231-1237Crossref PubMed Scopus (275) Google Scholar). Tetraspanins are expressed in different combinations on all cell types analyzed, and they are able to associate with one another and with a number of other transmembrane proteins thereby forming large multiprotein clusters on the cell surface (often referred to as a “tetraspanin web”) (9Lagaudriere-Gesbert C. Le Naour F. Lebel-Binay S. Billard M. Lemichez E. Boquet P. Boucheix C. Conjeaud H. Rubinstein E. Cell. Immunol. 1997; 182: 105-112Crossref PubMed Scopus (139) Google Scholar). It has been proposed that tetraspanins may participate in a lateral cross-talk between various types of the receptors within the “web” thereby contributing to their signaling properties (9Lagaudriere-Gesbert C. Le Naour F. Lebel-Binay S. Billard M. Lemichez E. Boquet P. Boucheix C. Conjeaud H. Rubinstein E. Cell. Immunol. 1997; 182: 105-112Crossref PubMed Scopus (139) Google Scholar, 10Sugiura T. Berditchevski F. J. Cell Biol. 1999; 146: 1375-1389Crossref PubMed Scopus (183) Google Scholar). To understand how tetraspanins affect signaling processes one has to examine spatial orientation of the proteins within the tetraspanin web and establish the mechanisms that control its assembly. This issue has been addressed in a number of recent studies, which revealed that both extracellular and cytoplasmic regions could be involved in mediating interactions of tetraspanins with other transmembrane proteins (reviewed in Refs. 1Hemler M.E. Mannion B.A. Berditchevski F. Biochim. Biophys. Acta. 1996; 1287: 67-71PubMed Google Scholar, 2Maecker H.T. Todd S.C. Levy S. FASEB J. 1997; 11: 428-442Crossref PubMed Scopus (811) Google Scholar). Specifically, it has been shown that the large extracellular loop (LECL)1 is required for the association of CD9 with the heparin-binding epidermal growth factor-like growth factor (11Sakuma T. Higashiyama S. Hosoe S. Hayashi S. Taniguchi N. J. Biochem. 1997; 122: 474-480Crossref PubMed Scopus (23) Google Scholar). Similarly, the extracellular portions of CD19 and the α3β1 integrin were critical for the assembly of the CD19·CD81 and α3β1·CD151 complexes thereby implicating the extracellular domains of the tetraspanins in these interactions (12Bradbury L.E. Goldmacher V.S. Tedder T.F. J. Immunol. 1993; 151: 2915-2927PubMed Google Scholar, 13Yauch R.L. Berditchevski F. Harler M.B. Reichner J. Hemler M.E. Mol. Biol. Cell. 1998; 9: 2751-2765Crossref PubMed Scopus (267) Google Scholar). On the other hand, the region critical for the interaction of CD4 with CD81/TAPA-1 was mapped to the juxtamembrane portion of CD4 cytoplasmic tail (14Imai T. Kakizaki M. Nishimura M. Yoshie O. J. Immunol. 1995; 155: 1229-1239PubMed Google Scholar). Finally, both cytoplasmic and extracellular domains of CD4 are required for its association with CD82 (14Imai T. Kakizaki M. Nishimura M. Yoshie O. J. Immunol. 1995; 155: 1229-1239PubMed Google Scholar). The associations of tetraspanins with different integrins have been extensively examined in various cell types (13Yauch R.L. Berditchevski F. Harler M.B. Reichner J. Hemler M.E. Mol. Biol. Cell. 1998; 9: 2751-2765Crossref PubMed Scopus (267) Google Scholar, 15Rubinstein E. Le Naour F. Billard M. Prenant M. Boucheix C. Eur. J. Immunol. 1994; 24: 3005-3013Crossref PubMed Scopus (142) Google Scholar, 16Berditchevski F. Bazzoni G. Hemler M.E. J. Biol. Chem. 1995; 270: 17784-17790Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 17Nakamura K. Iwamoto R. Mekada E. J. Cell Biol. 1995; 129: 1691-1705Crossref PubMed Scopus (230) Google Scholar, 18Berditchevski F. Zutter M.M. Hemler M.E. Mol. Biol. Cell. 1996; 7: 193-207Crossref PubMed Scopus (251) Google Scholar, 19Mannion B.A. Berditchevski F. Kraeft S.-K. Chen L.B. Hemler M.E. J. Immunol. 1996; 157: 2039-2047PubMed Google Scholar, 20Tachibana I. Bodorova J. Berditchevski F. Zutter M.M. Hemler M.E. J. Biol. Chem. 1997; 272: 29181-29189Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 21Claas C. Seiter S. Claas A. Savelyeva L. Schwab M. Zöller M. J. Cell Biol. 1998; 141: 267-280Crossref PubMed Scopus (95) Google Scholar, 22Yáñez-Mó M. Alfranca A. Cabañas C. Marazuela M. Tejedor R. Ursa A. Ashman L.K. De Landázuri M.O. Sanchez-Madrid F. J. Cell Biol. 1998; 141: 791-804Crossref PubMed Scopus (239) Google Scholar, 23Fitter S. Sincock P.M. Jolliffe C.N. Ashman L.K. Biochem. J. 1999; 338: 61-70Crossref PubMed Scopus (105) Google Scholar, 24Yauch R.L. Kazarov A.R. Desai B. Lee R.T. Hemler M.E. J. Biol. Chem. 2000; 275: 9230-9238Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). In most of these studies to prevent dissociation of the integrin·tetraspanin complexes the analysis (e.g.immunoprecipitation) was carried out in the presence of so-called “non-stringent,” less hydrophobic detergents (e.g.CHAPS, various Brij detergents). Although solubilization of the complexes with non-stringent detergents allows assessing the specificity of the association, this approach precludes a detailed mapping analysis of the integrin·tetraspanin interactions. Indeed, under these experimental conditions the immunoprecipitated complexes include a varying number of tetraspanins and some other transmembrane proteins thereby complicating a pair-wise analysis of a particular protein·protein interaction. We have previously shown that the interaction of tetraspanin CD151 with the α3β1 integrin could be observed under the experimental conditions that dissociated all other integrin·tetraspanin complexes (e.g. in the presence of Triton X-100) (13Yauch R.L. Berditchevski F. Harler M.B. Reichner J. Hemler M.E. Mol. Biol. Cell. 1998; 9: 2751-2765Crossref PubMed Scopus (267) Google Scholar, 25Berditchevski F. Chang S. Bodorova J. Hemler M.E. J. Biol. Chem. 1997; 272: 29174-29180Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Not only did these data suggest a close proximity of CD151 and α3β1, but they also pointed to a critical role this tetraspanin might play in the regulation of other α3β1·tetraspanin interactions. Thus, thorough assessment of the α3β1·CD151 association (under both “stringent” and “non-stringent” conditions) may prove to be crucial for understanding the assembly and function of the integrin·tetraspanin web. It has been recently illustrated that the resistance of the α3β1·CD151 complex to Triton X-100 was lost when a C-terminal region of the CD151 LECL (a sequence between residues Lys186 and Gln217) was substituted for the corresponding residues of tetraspanin NAG-2/Tspan-4 (24Yauch R.L. Kazarov A.R. Desai B. Lee R.T. Hemler M.E. J. Biol. Chem. 2000; 275: 9230-9238Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Because a reciprocal chimeric protein has not been examined in this study, it was possible that this “loss-of-function” substitution within CD151 affected overall folding of the LECL rather than specifically removed the sequences that were engaged in interaction with the integrin. In addition, it remains unclear whether other parts of the CD151 LECL can contribute to the incorporation of the protein into the tetraspanin web. In this report we utilized a large panel of the CD151 mutants to carry out a detailed examination of the α3β1·CD151 and tetraspanin·CD151 interactions. We found that the 186–217 region of the CD151 LECL was necessary but not sufficient to confer a stable interaction of the tetraspanin with α3β1. We showed that, although the assembly of the α3β1·CD151 complex is an early step during the integrin biosynthesis, the association with other tetraspanins is likely to occur when CD151 is delivered to the cell surface and it does not involve the CD151 LECL. The MDA-MB-231 human breast cancer cell line was purchased from the ATCC and maintained in L-15 Leibovitz medium (Sigma Chemical Co., Dorset, UK) supplemented with 15% fetal calf serum (FCS). The HT1080, 293T cells, and HT1080- and 293T-derived transfectants were maintained in DMEM supplemented with 10% FCS. The Chinese hamster ovary cells expressing human α3 and β1 integrin subunits, CHO-A3B1 (26Weitzman J.B. Pasqualini R. Takada Y. Hemler M.E. J. Biol. Chem. 1993; 268: 8651-8657Abstract Full Text PDF PubMed Google Scholar,16Berditchevski F. Bazzoni G. Hemler M.E. J. Biol. Chem. 1995; 270: 17784-17790Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) were maintained in α-minus minimum essential media supplemented with 10% FCS. To generate the stable cell lines CHO-A3B1/CD151-HA, CHO-A3B1/CD9, CHO-A3B1/SW1, CHO-A3B1/SW2, CHO-A3B1/SW3, CHO-A3B1/SW4, CHO-A3B1/SW5, CHO-A3B1/SW6, CHO-A3B1/SW7, CHO-A3B1/SW8, CHO-A3B1/SW9, and CHO-A3B1/SW10, the CHO-A3B1 cells were transfected with various pZeoSV-based plasmids containing mutagenized human CD151 cDNA (Fig.1). The transfection experiments were carried out using LipofectAMINE (Life Technologies, Inc.) or FUGENE 6 (Roche Molecular Biochemicals). Pools of zeocin-resistant colonies were selected after 15–20 days, and the expression of the tetraspanins was verified by flow cytometry or by Western blotting. The stable cell lines 293T/CD151mA, 293T/CD151wt, CHO-A3B1/CD151-mA, CHO-A3B1/CD151-mB, CHO-A3B1/CD151/Δ1, CHO-A3B1/CD151/Δ2, CHO-A3B1/CD151/Δ3, and CHO-A3B1/CD151/Δmyc1 were generated as above, and the expression of the mutagenized variants of CD151 in the individual zeocin-resistant clones was verified by Western blotting with rabbit polyclonal anti-CD151 or anti-HA-tag antibodies. The stable cell lines HT1080/CD151/Δmyc1, HT1080/CD151/Δ1, HT1080/CD151/Δ2, and HT1080/CD151/Δ3 were established as a population of pooled zeocin-resistant clones. Anti-integrin mAbs used were: anti-α3, P1B5 (27Carter W.G. Kaur P. Gil S.G. Gahr P.J. Wayner E.A. J. Cell Biol. 1990; 111: 3141-3154Crossref PubMed Scopus (378) Google Scholar) and A3-IIF5 (26Weitzman J.B. Pasqualini R. Takada Y. Hemler M.E. J. Biol. Chem. 1993; 268: 8651-8657Abstract Full Text PDF PubMed Google Scholar). Anti-TM4SF mAbs were: anti-CD151, 5C11 (25Berditchevski F. Chang S. Bodorova J. Hemler M.E. J. Biol. Chem. 1997; 272: 29174-29180Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar), 14A2.1 (28Fitter S. Tetaz T.H. Berndt M. Ashman L.K. Blood. 1995; 86: 1348-1355Crossref PubMed Google Scholar), 11B5 (29Ashman L.K. Fitter S. Sincock P.M. Nguyen L. Cambareri A.C. Kishimoto T. Kikutani H. von dem Borne A.E.G.Kr. Goyert S.M. Mason D. Miyasaka M. Moretta L. Okumure K. Shaw S. Springer T.A. Leukocyte Typing VI White Cell Differentiation Antigens. Garland Publishing, New York1997: 681-683Google Scholar), 14B5 (29Ashman L.K. Fitter S. Sincock P.M. Nguyen L. Cambareri A.C. Kishimoto T. Kikutani H. von dem Borne A.E.G.Kr. Goyert S.M. Mason D. Miyasaka M. Moretta L. Okumure K. Shaw S. Springer T.A. Leukocyte Typing VI White Cell Differentiation Antigens. Garland Publishing, New York1997: 681-683Google Scholar), 11B1G4 (29Ashman L.K. Fitter S. Sincock P.M. Nguyen L. Cambareri A.C. Kishimoto T. Kikutani H. von dem Borne A.E.G.Kr. Goyert S.M. Mason D. Miyasaka M. Moretta L. Okumure K. Shaw S. Springer T.A. Leukocyte Typing VI White Cell Differentiation Antigens. Garland Publishing, New York1997: 681-683Google Scholar); anti-CD9, C9-BB (20Tachibana I. Bodorova J. Berditchevski F. Zutter M.M. Hemler M.E. J. Biol. Chem. 1997; 272: 29181-29189Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Rabbit polyclonal serum was generated against purified human CD151 (23Fitter S. Sincock P.M. Jolliffe C.N. Ashman L.K. Biochem. J. 1999; 338: 61-70Crossref PubMed Scopus (105) Google Scholar). The anti-CD81 mAb, M38, was kindly provided by Dr. O. Yoshie. The anti-CD9 mAbs, ALB-6 and SYB-1, and anti-CD151 mAb, TS151r, were generously provided by Dr. E. Rubinstein. The anti-CD63 hamster mAb, eh1C9b, was provided by Dr. M. Marsh. The anti-CD151 mAbs, LIA1/1 and VJ1/16, were provided by Dr. F. Sanchez-Madrid. The anti-calnexin mAb, AF8, was provided by Dr. M. Brenner. The anti-lamp-1 mAb, BB6, was a generous gift from Dr. S. Carlsson. Rabbit polyclonal (Y-11) and mouse monoclonal (F-7) Ab to HA-tag were purchased from Autogen Bioclear. The pZeoSV-CD151 and pZeoSV-CD9 were described previously (13Yauch R.L. Berditchevski F. Harler M.B. Reichner J. Hemler M.E. Mol. Biol. Cell. 1998; 9: 2751-2765Crossref PubMed Scopus (267) Google Scholar, 18Berditchevski F. Zutter M.M. Hemler M.E. Mol. Biol. Cell. 1996; 7: 193-207Crossref PubMed Scopus (251) Google Scholar). The pZeoSV-CD151-HA construct was kindly provided by Dr. M. Hemler (Dana-Farber Cancer Institute, Boston, MA). The DNA constructs encoding chimeric CD9-CD151 and mutagenized variants of CD151 were generated by overlapping polymerase chain reaction usingPfuI-DNA polymerase (Stratagene) (TableI). The amplified fragments were subcloned into pZeoSV expression vector cut either withHindIII and SpeI (pZeoSV-SW1, pZeoSV-SW2 and pZeoSV-CD151/Δ1, pZeoSV-CD151/Δ2) or with SpeI andEcoRI (pZeoSV-CD151-mA, pZeoSV-CD151-mB, pZeoSV-CD151/Δmyc1, pZeoSV-CD151/Δ3). To generate pZeoSV-SW6, pZeoSV-SW7, pZeoSV-SW8, pZeoSV-SW9, and pZeoSV-SW10, the amplified fragments were subcloned into pZeoSV expression vector cut withHindIII and EcoRI. To generate pZeoSV-CD151/Δ1-HA, pZeoSV-CD151/Δ2-HA, and pZeoSV-CD151/Δ3-HA constructs, the HindIII-BsgI fragments from the CD151Δ1, -2, -3 were subcloned into pZeoSV-CD151-HA. The pZeoSV-SW4 was constructed after subcloning of an amplified fragment into pZeoSV-CD9 cut with SphI (partial cut) and SpeI. The pZeoSV-SW3 was constructed after subcloning of an amplified fragment into pZeoSV-CD9 cut with SacI (partial cut) andEcoRI. The pZeoSV-SW5 was generated after an amplified fragment (cut with PstI and SpeI) was combined with the PstI-EcoRI fragment from CD151 and ligated into pZeoSV (cut with SpeI andEcoRI).Table ISequences of primers used for generation of chimeric and mutant CD151 constructsConstructPrimersDNA templateSW11.5′-gaaggtctcttttagggcatcaggacagga-3′1.pZeoSV-CD95′-gcaagcttctcaccatgccggtcaaaggaggcacc-3′2.5′-cgtctagactagtagtgctccagcttgag-3′2.pZeoSV-CD1515′-gccatcaaagagaccttcatccaggagc-3′SW21.5′-cgtgttcagctcatccttgtgggaatatcc-3′1.pZeoSV-CD95′-gcaagcttctcaccatgccggtcaaaggaggcacc-3′2.5′-aattaaccctcactaaaggg-3′2.pZeoSV-CD151-HA5′-aaggatgagctgaacacggagctcaagg-3′SW31.5′-gaaggtctcttttagggcatcaggacagga-3′1.pZeoSV-CD95′-attaaggagctccaggagttttacaagg-32.5′-tatttaggttgacactatag-3′2.pZeoSV-CD151-HA5′-gccatcaaagagaccttcatccaggagc-3′SW45′-cgtctagactagtagtgctccagcttgag-3′pZeoSV-CD1515′-cagtgcatgctgtacttcatcctgctcc-3′SW55′-ctcctgctgcagggctttcagcgtttcccg-3′pZeoSV-CD95′-gcaagcttctcaccatgccggtcaaaggaggcacc-3′SW61.5′-tatttaggttgacactatag-3′1.pZeoSV-CD95′-accaagttgaaagaggtcttcgacaataaattcc-3′2.5′-aattaaccctcactaaaggg-3′2.pZeoSV-SW25′-gacctctttcaacttggtgatgcagcc-3′SW75′-aattaaccctcactaaaggg-3′pZeoSV-SW35′-ctcctgctgcagggctttcagcgtttcccg-3′SW81.5′-ttccagaagtagtgaggagg-3′1.pZeoSV-CD95′-atggtctcgcttggggcagatgtctgag-3′2.5′-tatttaggttgacactatag-3′2.pZeoSV-SW65′-tgccccaagcgagaccatgcctccaacatc-3′SW91.5′-ttccagaagtagtgaggagg-3′1.pZeoSV-CD95′-actgtctcgctgttccacgcccccagccaaacc-3′2.5′-tatttaggttgacactatag-3′2.pZeoSV-SW65′-gtggaacagcgagacagtgagtggatcc-3′SW101.5′-ttccagaagtagtgaggagg-3′1.pZeoSV-CD1515′-ggtgaaggtttcgagtacgtccttctgtccacaaagagccacc-3′2.5′-tatttaggttgacactatag-3′2.pZeoSV-CD1515′-gtactcgaaaccttcaccgtgaagggcggctgcatcaccaagttgg-3′CD151-Δ11.5′-actcttgagggccagcgtcc-3′1.KS-CD1515′-taatacgactcactataggg-3′2.5′-cgtctagactagtagtgctccagcttgag-3′2.KS-CD1515′-acgctggccctcaagagtcagcagctgaacacggagc-3′CD151-Δ21.5′-cagttgccgacgctccttgaggcaaacggtgccac-3′1.KS-CD1515′-taatacgactcactataggg-3′2.5′-aaggagcgtggcaacctgc-3′2.KS-CD1515′-tatttaggttgacactatag-3′CD151-Δ31.5′-ttcgcgctgggcacccacggagcctccgggctgaacacggagctcaag-3′1.KS-CD1515′-tatttaggttgacactatag-3′2.5′-cgaagcttatcatggcggctgcgctgttcgtgctgctgggattcgcgctgc-3′2.1st RCR product5′-tatttaggttgacactatag-3′CD151-Δmyc11.5′-ctcttctgagatgagtttttgttctgctggtagtaggcgtagg-3′1.pZeoSV-CD151-HA5′-taatacgactcactataggg-3′2.5′-aaactcatctcagaagaggatctgagcacctgagggtcattgg-3′2.pZeoSV-CD151-HA5′-tatttaggttgacactatag-3′CD151-mA1.5′-gctgccagcggcgtggaactcctgctgcag-3′1.pZeoSV-CD151-HA5′-taatacgactcactataggg-3′2.5′-tcccacgaagctggcagcaacaactcacag-3′2.pZeoSV-CD151-HA5′-tatttaggttgacactatag-3′CD151-mB1.5′-cgtcttggcggcgctgtctgggaccacac-3′1.pZeoSV-CD151-HA5′-taatacgactcactataggg-3′2.5′-gacagcgccgccaagacggtggtggctc-3′2.pZeoSV-CD151-HA5′-tatttaggttgacactatag-3′ Open table in a new tab Cellular distribution of tetraspanins in the CHOA3B1 and HT1080 transfectants was analyzed by indirect immunofluorescence staining. The cells were plated in serum-free L-15 media on glass coverslips coated with the laminin-5-containing extracellular matrix (prepared as described previously (31Berditchevski F. Odintsova E. J. Cell Biol. 1999; 146: 477-492Crossref PubMed Scopus (257) Google Scholar)). In other experiments cells were grown on glass coverslips in complete media for 24–36 h. Spread cells were fixed with 2% paraformaldehyde/PBS for 10–15 min. After subsequent washes with PBS, the cells were permeabilized with 1% Brij 98/PBS for 1.5 min (or left untreated). In some experiments fixed cells were permeabilized with 0.1%Triton X-100/PBS for 30 s. The staining with primary and fluorochrome-conjugated secondary Abs was carried out as previously described (32Golberg A.F.X. Molday R.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13726-13730Crossref PubMed Scopus (105) Google Scholar). The staining was analyzed using the Nikon Eclipse E600 microscope. Images were acquired using the Leica DC200 digital camera and subsequently processed using the DC200 image processing program. The proteins were solubilized into the immunoprecipitation buffer containing 1% Brij 98/PBS (or 1% Triton X-100/PBS), 2 mm phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin for 2–16 h at 4 °C. The insoluble material was pelleted at 12,000 rpm for 10 min, and the cell lysates were precleared by incubation for 30 min at 4 °C with agarose beads conjugated with goat anti-mouse antibodies (Sigma). Immune complexes were collected on the agarose beads that were prebound with mAbs, followed by four washes with the immunoprecipitation buffer. Immune complexes were eluted from the beads with Laemmli sample buffer. Proteins were resolved in 11% SDS-PAGE, transferred to the nitrocellulose membrane, and developed with the appropriate Ab. Protein bands were visualized using horseradish peroxidase-conjugated secondary antibodies and Enhanced Chemiluminescence reagent (Amersham Pharmacia Biotech, Buckinghamshire, UK). Cells were incubated with saturating concentrations of primary mouse mAbs for 45 min at 4 °C, washed twice, and then labeled with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin. Stained cells were analyzed on a FACScan (Becton Dickinson, UK). The cells were labeled with 1 mCi of EXPRE35S35S (PerkinElmer Life Sciences) for 15 min in the serum-, methionine-, and cysteine-free MEM and subsequently chased for various time intervals with the complete DMEM containing 10% FCS. The immunoprecipitation was carried out as described in the above section. The gel pretreated with the Amplifier (Amersham Pharmacia Biotech) was dried and exposed to the x-ray Kodak film at −70 °C. In this study our goal was to identify the regions within CD151 that are critical for the interactions of the protein with the α3β1 integrin and with other tetraspanins. Previous studies have shown that stability of the CD9·α3β1 complex is compromised in the presence of Triton X-100 (18Berditchevski F. Zutter M.M. Hemler M.E. Mol. Biol. Cell. 1996; 7: 193-207Crossref PubMed Scopus (251) Google Scholar). Hence, to delineate the sequences required for mediating tight (e.g. Triton X-100-resistant) association of CD151 with the α3β1integrin, a series of the chimeric CD9-CD151 cDNA constructs were generated (Fig. 1). To avoid antibody counter-reactivity toward the endogenous CD9 and CD151 (both tetraspanins are widely expressed on cultured human cells), the association of the chimeric proteins with α3β1 was analyzed in the CHOA3B1 hamster cells (a variant of CHO cells that expresses human α3β1 integrin). The immunoprecipitation experiments have shown that in CHOA3B1 cells wild-type human CD151 is associated with the α3β1 in the presence of both Triton X-100 and Brij 98 (Fig.2A, lanes 1 and2). On the other hand, the association of human CD9 with the integrin can be observed only when the immunoprecipitation is carried out in Brij 98 (Fig. 2A, lanes 3 and4). These results established that the detergent sensitivity of the CD151·α3β1 and CD9·α3β1 complexes purified from the hamster cells was similar to that observed in human cells. Hence, the CHOA3B1 cells represent a suitable model system to carry out the biochemical analysis of the CD9/CD151 chimeras. The association of the chimeric proteins (SW-series) with the α3β1integrin was examined by the immunoprecipitation→Western blotting protocol using both Brij 98 and Triton X-100. As illustrated in Fig. 2B, in the presence of Triton X-100 the α3β1 integrin was co-immunoprecipitated only when the chimeric proteins included the LECL of the CD151 (e.g. chimeras SW2, SW4, and SW6) (lanes 4,8, and 10). In contrast, when this region of the protein was substituted for a corresponding part of CD9 (chimeras SW1 and SW3) the association was detected only in the presence of Brij 98 (Fig. 2B, lanes 1 and 5). In the control experiments we showed that comparable amounts of the SW1 and SW3 proteins were immunoprecipitated from both Brij 98 and Triton X-100 cellular lysates (results not shown). In addition, we examined the association of α3β1 with the truncated variants of CD151 (mutants CD151/Δ1, CD151/Δ2, and CD151/Δ3 (Fig.1)). In the Δ mutants various parts of CD151 that preceded the LECL were deleted. Fig. 2C shows that all three truncated proteins could be co-immunoprecipitated with α3β1 from both Brij 98 and Triton X-100 lysates. Collectively, these results demonstrate that the presence of the CD151 LECL (the amino acid sequence between residues Leu118 and Glu213) is necessary and sufficient for mediating stable (e.g. Triton-resistant) association of the tetraspanin with the integrin. A computer-based analysis of the large extracellular loops of all tetraspanins in the GenBank™ data base (BLAST algorithm) indicate that there are three unique regions within LECL of CD151 that show no similarity to the corresponding sequences of any other member of the family (sequences between the amino acids 119–150, 166–181, and 194–206). We therefore hypothesized that one of these regions may be important for mediating tight association of CD151 with α3β1. To investigate this idea we generated mutants of CD151 in which two of these regions (120–148 and 195–205) were swapped for the corresponding CD9 sequences (chimeras SW7 and SW10, respectively (Fig. 1)). The immunoprecipitation experiments have shown that the α3β1·SW7 complex was resistant to the presence of Triton X-100 (Fig.3, lane 2). Notably, due to poor extractability of SW7 protein we were unable to detect the signal when cells were lysed in Brij 98 (Fig. 3, lane 1). These results showed that, although the 120–148 region of the CD151 LECL was not required for mediating stable association with α3β1, it might regulate other properties of the protein. In agreement with this conclusion we found that the protein, in which the first 148 amino acids of CD151 were replaced for the CD9 sequence (SW5 chimera (Fig. 1)), also formed a stable complex with the integrin and was poorly extracted from the cells by Brij 98 (Fig. 3, lanes 3 and 4). In contrast, the α3β1·SW10 complex was sensitive to Triton X-100 (Fig. 3, compare lanes 5 and 6) thereby suggesting that the 195–205 region of CD151 was important. To substantiate this conclusion further we generated a reciprocal chimera, in which this sequence was embedded into the LECL of CD9 (SW8 chimera (Fig. 1)). Surprisingly, the SW8 protein also failed to form a stable complex with α3β1 (Fig. 3, comparelanes 7 and 8). Hence, by itself the CD151195–205 sequence does not confer the stable association of the chimeric protein with α3β1. Finally, we tested the interaction of α3β1 integrin with the SW9 chimera. This chimeric protein contains the CD151 sequence that includes the second and third unique regions" @default.
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• W1665767818 title "Analysis of the CD151·α3β1 Integrin and CD151·Tetraspanin Interactions by Mutagenesis" @default.
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• W1665767818 doi "https://doi.org/10.1074/jbc.m104041200" @default.
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