Gene. 109 O991) 255-258 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/91/$03.50 GENE 06219 255 cDNA encoding murine FK506-binding protein (FKBP): nucleotide and deduced amino acid sequences* (Recombinant DNA; immunoblotting; immunophilin; immunosuppression; rapamycin) Patricia A. Nelson, Judith A. Lippke, Mark A. Murcko, Sandra L. Rosborough and Debra A. Peattie Immunology, Molecular Biology and Molecular Modeling. Vertex Pharmaceuticals Incorporated. Cambridge, MA 02139-4211 (U.S.A.) Received by S.T. Case: 23 July 1991 Accepted: 24 August 1991 Received at publishers: 30 September 1991 suMMARY A FKBP cDNA encoding murine FK506 binding protein (FKBP) has been cloned, and its complete nucleotide sequence has been determined. The open reading frame within the 1556-bp cDNA segment encodes an 108 amino acid (aa) protein that differs from the human FKBP by three aa and from the bovine FKBP by five aa. Molecular modeling of the protein places the aa substitutions at positions not directly involved in drug binding or interaction with the potential drug target protein, calcineurin A. INTRODUCTION The immunosuppressive compounds Cyclosporin A (CSA) and FK506 (FK) have been shown to be potent immunosuppressive drugs both in vitro and in vivo. They bind to and inhibit the pcptidyl-prolyl cis-trans isomerases cyclophilin (Handschumacher et al., 1984; Harding et al., 1986) and FK506 binding protein (FKBP) (Harding et al., 1989; Siekierka et al., 1989), respectively. CSA and FK are Correspondence to: Dr. P.A. Nelson, Vertex Pharmaceuticals Incor- porated, 40 Ailston St., Cambridge, MA 02139-4211 (U.S.A.) Tel. (617)576-311 !; Fax (617)499-2437; E-mail: nelson@vpharm.eom. * On request, authors will supply detailed experimental evidence for conclusions reached in this short communication Abbreviations: aa, amino acid(s); b, bovine; bp, base pair(s); eDNA, complementary DNA; CSA, immunosuppressant compound Cyclo- sporin A; h, human; EK, immunosuppressantcompoundFK506; FKBP, FK-bindirr ~ protein; FKBP, gene(DNA) encodingFKBP; m, muririe;nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open rel:.di::~ fi ame;PVDF, immobiion-P polyvinylidene difluoride; Rap, immunosup- pressant compound Rapamycin; TBST, 20raM Tri~.HCI/500mM NaCI/0.3°o Tween20 pH 7.5; UTR, untranslated region. inhibitory in T lymphocyte mitogenesis assays (Lin et al., 1991). In addition, inhibition of mRNA transcription for IL-2 is a downs,ream activation event known to be inhibited by CSA and FK, presumably after the drugs complex with their respective cytosolic binding proteins. The drugs appear to inhibit completely transcription activated by NF-AT, NF-IL2 A, and NF-IL2 B, and to inhibit partially transcription activated by NFrB (Mattila et al., 1990; Banerji et ai., 1991). These findings suggest that CSA and FK may interfere with the activity of a novel Ca 2 + depend- ent step that regulates several transcription factors. Rapamycin (Rap) is another immunosuppressant that binds to FKBP, but it does not prevent transcription of IL-2 message, even at concentrations as high as 1/~M (Bierer et ai., 1990; Nelson et al,, 1991). Several other proteins that bind FK and Rap have been described, and their role in immunosuppression is being investigated (Fretz et al., 1991). It has been proposed recently that the FK-FKBP complex inhibits activation of T lymphocytes through specific binding to calcineurin A (Liu et al., 1991), a cal- modulin regulated protein phosphatase whose role in T cell activation has yet to be elucidated. These new, compelling results have recently been revic~ved (McKeon, 1991) and