• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of biochemjBJ Latest papers and much more!
Biochem J. Jan 15, 2004; 377(Pt 2): 407–417.
PMCID: PMC1223880

Evidence for specific tetraspanin homodimers: inhibition of palmitoylation makes cysteine residues available for cross-linking.

Abstract

It is a well-established fact that tetraspanin proteins, a large family of integral membrane proteins involved in cell motility, fusion and signalling, associate extensively with one another and with other transmembrane and membrane-proximal proteins. In this study, we present results strongly suggesting that tetraspanin homodimers are fundamental units within larger tetraspanin complexes. Evidence for constitutive CD9 homodimers was obtained using several cell lines, utilizing the following four methods: (1) spontaneous cross-linking via intermolecular disulphide bonds, (2) use of a cysteine-reactive covalent cross-linking agent, (3) use of an amino-reactive covalent cross-linking agent, and (4) covalent cross-linking via direct intermolecular disulphide bridging between unpalmitoylated membrane-proximal cysteine residues. In the last case, incubation of cells with the palmitoylation inhibitor 2-bromopalmitate exposed membrane-proximal cysteine residues, thus effectively promoting 'zero-length' cross-linking to stabilize homodimers. Similar to CD9, other tetraspanins (CD81 and CD151) also showed a tendency to homodimerize. Tetraspanin homodimers were assembled from newly synthesized proteins in the Golgi, as evidenced by cycloheximide and Brefeldin A inhibition studies. Importantly, tetraspanin homodimers appeared on the cell surface and participated in typical 'tetraspanin web' interactions with other proteins. Whereas homodimers were the predominant cross-linked species, we also observed some higher-order complexes (trimers, tetramers or higher) and a much lower level of cross-linking between different tetraspanins (CD81-CD9, CD9-CD151, CD81-CD151). In conclusion, our results strongly suggest that tetraspanin homodimers, formed in the Golgi and present at the cell surface, serve as building blocks for the assembly of larger, multicomponent tetraspanin protein complexes.

Full Text

The Full Text of this article is available as a PDF (314K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Boucheix C, Rubinstein E. Tetraspanins. Cell Mol Life Sci. 2001 Aug;58(9):1189–1205. [PubMed]
  • Seigneuret M, Delaguillaumie A, Lagaudrière-Gesbert C, Conjeaud H. Structure of the tetraspanin main extracellular domain. A partially conserved fold with a structurally variable domain insertion. J Biol Chem. 2001 Oct 26;276(43):40055–40064. [PubMed]
  • Escola JM, Kleijmeer MJ, Stoorvogel W, Griffith JM, Yoshie O, Geuze HJ. Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes. J Biol Chem. 1998 Aug 7;273(32):20121–20127. [PubMed]
  • Hammond C, Denzin LK, Pan M, Griffith JM, Geuze HJ, Cresswell P. The tetraspan protein CD82 is a resident of MHC class II compartments where it associates with HLA-DR, -DM, and -DO molecules. J Immunol. 1998 Oct 1;161(7):3282–3291. [PubMed]
  • Kobayashi T, Vischer UM, Rosnoblet C, Lebrand C, Lindsay M, Parton RG, Kruithof EK, Gruenberg J. The tetraspanin CD63/lamp3 cycles between endocytic and secretory compartments in human endothelial cells. Mol Biol Cell. 2000 May;11(5):1829–1843. [PMC free article] [PubMed]
  • Berditchevski F. Complexes of tetraspanins with integrins: more than meets the eye. J Cell Sci. 2001 Dec;114(Pt 23):4143–4151. [PubMed]
  • Maecker HT, Todd SC, Levy S. The tetraspanin superfamily: molecular facilitators. FASEB J. 1997 May;11(6):428–442. [PubMed]
  • Hemler ME. Specific tetraspanin functions. J Cell Biol. 2001 Dec 24;155(7):1103–1107. [PMC free article] [PubMed]
  • Stipp Christopher S, Kolesnikova Tatiana V, Hemler Martin E. Functional domains in tetraspanin proteins. Trends Biochem Sci. 2003 Feb;28(2):106–112. [PubMed]
  • Zhang Xin A, Kazarov Alexander R, Yang Xiuwei, Bontrager Alexa L, Stipp Christopher S, Hemler Martin E. Function of the tetraspanin CD151-alpha6beta1 integrin complex during cellular morphogenesis. Mol Biol Cell. 2002 Jan;13(1):1–11. [PMC free article] [PubMed]
  • Charrin Stéphanie, Manié Serge, Oualid Michael, Billard Martine, Boucheix Claude, Rubinstein Eric. Differential stability of tetraspanin/tetraspanin interactions: role of palmitoylation. FEBS Lett. 2002 Apr 10;516(1-3):139–144. [PubMed]
  • Berditchevski Fedor, Odintsova Elena, Sawada Shigeaki, Gilbert Elizabeth. Expression of the palmitoylation-deficient CD151 weakens the association of alpha 3 beta 1 integrin with the tetraspanin-enriched microdomains and affects integrin-dependent signaling. J Biol Chem. 2002 Oct 4;277(40):36991–37000. [PubMed]
  • Seehafer JG, Tang SC, Slupsky JR, Shaw AR. The functional glycoprotein CD9 is variably acylated: localization of the variably acylated region to a membrane-associated peptide containing the binding site for the agonistic monoclonal antibody 50H.19. Biochim Biophys Acta. 1988 Dec 2;957(3):399–410. [PubMed]
  • Yang Xiuwei, Claas Christoph, Kraeft Stine-Kathrein, Chen Lan Bo, Wang Zemin, Kreidberg Jordan A, Hemler Martin E. Palmitoylation of tetraspanin proteins: modulation of CD151 lateral interactions, subcellular distribution, and integrin-dependent cell morphology. Mol Biol Cell. 2002 Mar;13(3):767–781. [PMC free article] [PubMed]
  • Yauch RL, Kazarov AR, Desai B, Lee RT, Hemler ME. Direct extracellular contact between integrin alpha(3)beta(1) and TM4SF protein CD151. J Biol Chem. 2000 Mar 31;275(13):9230–9238. [PubMed]
  • Kazarov Alexander R, Yang Xiuwei, Stipp Christopher S, Sehgal Bantoo, Hemler Martin E. An extracellular site on tetraspanin CD151 determines alpha 3 and alpha 6 integrin-dependent cellular morphology. J Cell Biol. 2002 Sep 30;158(7):1299–1309. [PMC free article] [PubMed]
  • Higginbottom A, Quinn ER, Kuo CC, Flint M, Wilson LH, Bianchi E, Nicosia A, Monk PN, McKeating JA, Levy S. Identification of amino acid residues in CD81 critical for interaction with hepatitis C virus envelope glycoprotein E2. J Virol. 2000 Apr;74(8):3642–3649. [PMC free article] [PubMed]
  • Nakamura K, Mitamura T, Takahashi T, Kobayashi T, Mekada E. Importance of the major extracellular domain of CD9 and the epidermal growth factor (EGF)-like domain of heparin-binding EGF-like growth factor for up-regulation of binding and activity. J Biol Chem. 2000 Jun 16;275(24):18284–18290. [PubMed]
  • Kitadokoro K, Bordo D, Galli G, Petracca R, Falugi F, Abrignani S, Grandi G, Bolognesi M. CD81 extracellular domain 3D structure: insight into the tetraspanin superfamily structural motifs. EMBO J. 2001 Jan 15;20(1-2):12–18. [PMC free article] [PubMed]
  • Kitadokoro Kengo, Ponassi Marco, Galli Giuliano, Petracca Roberto, Falugi Fabiana, Grandi Guido, Bolognesi Martino. Subunit association and conformational flexibility in the head subdomain of human CD81 large extracellular loop. Biol Chem. 2002 Sep;383(9):1447–1452. [PubMed]
  • Goldberg AF, Moritz OL, Molday RS. Heterologous expression of photoreceptor peripherin/rds and Rom-1 in COS-1 cells: assembly, interactions, and localization of multisubunit complexes. Biochemistry. 1995 Oct 31;34(43):14213–14219. [PubMed]
  • Goldberg AF, Loewen CJ, Molday RS. Cysteine residues of photoreceptor peripherin/rds: role in subunit assembly and autosomal dominant retinitis pigmentosa. Biochemistry. 1998 Jan 13;37(2):680–685. [PubMed]
  • Wu XR, Medina JJ, Sun TT. Selective interactions of UPIa and UPIb, two members of the transmembrane 4 superfamily, with distinct single transmembrane-domained proteins in differentiated urothelial cells. J Biol Chem. 1995 Dec 15;270(50):29752–29759. [PubMed]
  • Berditchevski F, Zutter MM, Hemler ME. Characterization of novel complexes on the cell surface between integrins and proteins with 4 transmembrane domains (TM4 proteins). Mol Biol Cell. 1996 Feb;7(2):193–207. [PMC free article] [PubMed]
  • Rubinstein E, Le Naour F, Lagaudrière-Gesbert C, Billard M, Conjeaud H, Boucheix C. CD9, CD63, CD81, and CD82 are components of a surface tetraspan network connected to HLA-DR and VLA integrins. Eur J Immunol. 1996 Nov;26(11):2657–2665. [PubMed]
  • Zhang XA, Bontrager AL, Hemler ME. Transmembrane-4 superfamily proteins associate with activated protein kinase C (PKC) and link PKC to specific beta(1) integrins. J Biol Chem. 2001 Jul 6;276(27):25005–25013. [PubMed]
  • Berditchevski F, Tolias KF, Wong K, Carpenter CL, Hemler ME. A novel link between integrins, transmembrane-4 superfamily proteins (CD63 and CD81), and phosphatidylinositol 4-kinase. J Biol Chem. 1997 Jan 31;272(5):2595–2598. [PubMed]
  • Yauch RL, Berditchevski F, Harler MB, Reichner J, Hemler ME. Highly stoichiometric, stable, and specific association of integrin alpha3beta1 with CD151 provides a major link to phosphatidylinositol 4-kinase, and may regulate cell migration. Mol Biol Cell. 1998 Oct;9(10):2751–2765. [PMC free article] [PubMed]
  • Hemler Martin E. Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain. Annu Rev Cell Dev Biol. 2003;19:397–422. [PubMed]
  • Webb Y, Hermida-Matsumoto L, Resh MD. Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids. J Biol Chem. 2000 Jan 7;275(1):261–270. [PubMed]
  • Fukudome K, Furuse M, Imai T, Nishimura M, Takagi S, Hinuma Y, Yoshie O. Identification of membrane antigen C33 recognized by monoclonal antibodies inhibitory to human T-cell leukemia virus type 1 (HTLV-1)-induced syncytium formation: altered glycosylation of C33 antigen in HTLV-1-positive T cells. J Virol. 1992 Mar;66(3):1394–1401. [PMC free article] [PubMed]
  • Weitzman JB, Pasqualini R, Takada Y, Hemler ME. The function and distinctive regulation of the integrin VLA-3 in cell adhesion, spreading, and homotypic cell aggregation. J Biol Chem. 1993 Apr 25;268(12):8651–8657. [PubMed]
  • Kolesnikova TV, Mannion BA, Berditchevski F, Hemler ME. Beta1 integrins show specific association with CD98 protein in low density membranes. BMC Biochem. 2001;2:10–10. [PMC free article] [PubMed]
  • Careaga CL, Falke JJ. Thermal motions of surface alpha-helices in the D-galactose chemosensory receptor. Detection by disulfide trapping. J Mol Biol. 1992 Aug 20;226(4):1219–1235. [PMC free article] [PubMed]
  • Mannion BA, Berditchevski F, Kraeft SK, Chen LB, Hemler ME. Transmembrane-4 superfamily proteins CD81 (TAPA-1), CD82, CD63, and CD53 specifically associated with integrin alpha 4 beta 1 (CD49d/CD29). J Immunol. 1996 Sep 1;157(5):2039–2047. [PubMed]
  • Taylor AM, Zhu Q, Casey JR. Cysteine-directed cross-linking localizes regions of the human erythrocyte anion-exchange protein (AE1) relative to the dimeric interface. Biochem J. 2001 Nov 1;359(Pt 3):661–668. [PMC free article] [PubMed]
  • Jones Larry R, Cornea Razvan L, Chen Zhenhui. Close proximity between residue 30 of phospholamban and cysteine 318 of the cardiac Ca2+ pump revealed by intermolecular thiol cross-linking. J Biol Chem. 2002 Aug 2;277(31):28319–28329. [PubMed]
  • Barkalow FJ, Barkalow KL, Mayadas TN. Dimerization of P-selectin in platelets and endothelial cells. Blood. 2000 Nov 1;96(9):3070–3077. [PubMed]
  • Hamdan Fadi F, Ward Stuart D C, Siddiqui Nasir A, Bloodworth Lanh M, Wess Jürgen. Use of an in situ disulfide cross-linking strategy to map proximities between amino acid residues in transmembrane domains I and VII of the M3 muscarinic acetylcholine receptor. Biochemistry. 2002 Jun 18;41(24):7647–7658. [PubMed]
  • Resh MD. Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim Biophys Acta. 1999 Aug 12;1451(1):1–16. [PubMed]
  • Lomant AJ, Fairbanks G. Chemical probes of extended biological structures: synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate). J Mol Biol. 1976 Jun 14;104(1):243–261. [PubMed]
  • McDermott AM, Haslam RJ. Chemical cross-linking of pleckstrin in human platelets: evidence for oligomerization of the protein and its dissociation by protein kinase C. Biochem J. 1996 Jul 1;317(Pt 1):119–124. [PMC free article] [PubMed]
  • Kürzinger K, Springer TA. Purification and structural characterization of LFA-1, a lymphocyte function-associated antigen, and Mac-1, a related macrophage differentiation antigen associated with the type three complement receptor. J Biol Chem. 1982 Oct 25;257(20):12412–12418. [PubMed]
  • Fukuda M, Kanno E, Ogata Y, Mikoshiba K. Mechanism of the SDS-resistant synaptotagmin clustering mediated by the cysteine cluster at the interface between the transmembrane and spacer domains. J Biol Chem. 2001 Oct 26;276(43):40319–40325. [PubMed]
  • Loewen CJ, Molday RS. Disulfide-mediated oligomerization of Peripherin/Rds and Rom-1 in photoreceptor disk membranes. Implications for photoreceptor outer segment morphogenesis and degeneration. J Biol Chem. 2000 Feb 25;275(8):5370–5378. [PubMed]
  • Musil LS, Goodenough DA. Multisubunit assembly of an integral plasma membrane channel protein, gap junction connexin43, occurs after exit from the ER. Cell. 1993 Sep 24;74(6):1065–1077. [PubMed]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...