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Extracellular Isoforms of CD6 Generated by Alternative Splicing Regulate Targeting of CD6 to the Immunological Synapse¹

Mónica A. A. Castro^2,*, Marta I. Oliveira^2,*,, Raquel J. Nunes^*,, Stéphanie Fabre, Rita Barbosa^*, António Peixoto, Marion H. Brown^¶, Jane R. Parnes^||, Georges Bismuth, Alexandra Moreira^*, Benedita Rocha and Alexandre M. Carmo^3,*,

^* Group of Cell Activation and Gene Expression, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal; Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal; Institut National de la Santé et de la Recherche Médicale Unité 567, Département de Biologie Cellulaire, Institut Cochin, Paris, France; Institut National de la Santé et de la Recherche Médicale Unité 345, Institut Necker, Paris, France; ^¶ Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom; and ^|| Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, CA 94305

The great majority of mammalian genes yield multiple transcripts arising from differential mRNA processing, but in very few instances have alternative forms been assigned distinct functional properties. We have cloned and characterized a new isoform of the accessory molecule CD6 that lacks the CD166 binding domain and is expressed in rat and human primary cells. The novel isoform, CD6d3, results from exon 5 skipping and consequently lacks the third scavenger receptor cysteine-rich (SRCR) domain of CD6. Differential expression of the SRCR domain 3 resulted in a remarkable functional difference: whereas full-length CD6 targeted to the immunological synapse, CD6d3 was unable to localize at the T cell:APC interface during Ag presentation. Analysis of expression of CD6 variants showed that, while being more frequent in coexpression with full-length CD6, the CD6d3 isoform constituted the sole species in a small percentage of T cells. In the rat thymus, CD6d3 is less represented in double-positive thymocytes but is detectable in nearly 50% of single-positive CD4 or CD8 thymocytes, suggesting that CD6 switching between full-length and d3 isoforms may be involved in thymic selection. Strikingly, CD6d3 is markedly up-regulated upon activation of T lymphocytes, partially substituting full-length CD6, as evaluated by RT-PCR analysis at the single-cell level, by immunoblotting, and by flow cytometry using Abs recognizing SRCR domains 1 and 3 of human CD6. This elegant mechanism controlling the expression of the CD166 binding domain may help regulate signaling delivered by CD6, through different types of extracellular engagement.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Programa Operacional Ciência, Tecnologia, Inovação (POCTI), and Programa Operacional Ciência e Inovação 2010 (POCI), cofunded by the European Regional Development Fund (FEDER). M.A.A.C. is supported by a postdoctoral fellowship from Fundação para a Ciência e a Tecnologia (FCT)-Programa Operacional Sociedade da Informação; M.I.O. and R.J.N. are recipients of studentships from FCT-POCTI; S.F. is supported by the Ministère de l’Education Nationale et de La Recherche; J.R.P. was supported by Grant CA68675 from the National Institutes of Health; and A.P. was funded by FCT, Ligue Contre le Cancer, and Association pour la Recherche sur le Cancer. Additional support was obtained from Programa Pessoa, a Gabinete de Relações Internacionais da Ciência e do Ensino Superior (Portugal)/Egide (France) cooperation.

² M.A.A.C. and M.I.O. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Alexandre M. Carmo, Institute for Molecular and Cellular Biology, Rua do Campo Alegre, Porto, Portugal. E-mail address: acarmo{at}ibmc.up.pt

⁴ Abbreviations used in this paper: IS, immunological synapse; cSMAC, central supramolecular activation cluster; pSMAC, peripheral supramolecular activation cluster; SH, Src homology; SRCR, scavenger receptor cysteine rich; YFP, yellow fluorescent protein; SEA, staphylococcal enterotoxin A; DP, double positive; SP, single positive.