DARPP - 32 : Regulator of the Efficacy of Dopaminergic Neurotransmission

Immunol. 157, 207 (1996). 10. D. L. DiGiusto and E. Palmer, Mol. Immunol. 31, 693 (1994). 11. The a wild-type, b wild-type, aIII, and bIII constructs have been previously described (8). The a wild-type amino acid sequence from the interchain Cys to the COOH-terminus is CDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (30), with the a-CPM indicated in bold. The b wild-type amino acid sequence from the interchain Cys to the COOH-terminus is CGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAM VKRKNS. Similarly, the corresponding aIII amino acid sequence is CDATLTEKSFETVTVHTEKVNMMSLTVLGLRLLFAKTIAINFLLTVKLFF. The underlined sequences are derived from murine Cd and consequently, the distal five amino acids (DMNLN) of the a-CPM have been replaced. To permit surface expression of this chimeric a-chain, the aIII construct was paired with the bIII cDNA (8), which encodes the corresponding amino acid sequence CGITSASYQQGVLSATILYLLLLLKSVIYLAIISFSLLRRTSVCGNEKKS. The underlined sequences are derived from murine Cg1. Although this TCR is encoded by chimeric a and b chains, the functional defects associated with the aIII/bIII TCR are due to the absence of an intact a-CPM (8). cDNAs were excised with Eco RI and Bam and individually cloned into the Sal I and Bam sites of the expression vector pHSE39 (31). 12. B. Hausmann and E. Palmer, data not shown. 13. P. Borgulya, H. Kishi, Y. Uematsu, H. von Boehmer, Cell 69, 529 (1992). 14. In contrast to irradiated B6 mice, irradiated B6 athymic nude mice reconstituted with T cell–depleted bone marrow from a-CPM mutant animals failed to generate significant numbers of transgenic T cells. Therefore, the appearance of mutant T cells in the periphery is dependent on the presence of a thymus (12). 15. H. Suzuki, J. A. Punt, L. G. Granger, A. Singer, Immunity 2, 413 (1995). 16. W. Swat, L. Ignatowicz, H. von Boehmer, P. Kisielow, Nature 351, 150 (1991). 17. J. A. Punt, B. A. Osborne, Y. Takahama, S. O. Sharrow, A. Singer, J. Exp. Med. 179, 709 (1994). 18. D. M. Page, L. P. Kane, J. P. Allison, S. M. Hedrick, J. Immunol. 151, 1868 (1993). 19. S. J. Curnow, M. Barad, N. Brun-Roubereau, A. M. Schmitt-Verhulst, Cytometry 16, 41 (1994). 20. B. T. Bäckström and E. Palmer, unpublished observations. 21. V. P. Dave et al., EMBO J. 16, 1360 (1997). 22. M. Cohn and R. Langman, Behring Inst. Mitt. 70, 219 (1982). 23. K. A. Swan et al., EMBO J. 14, 276 (1995). 24. J. Alberola-Ila, K. A. Hogquist, K. A. Swan, M. J. Bevan, R. M. Perlmutter, J. Exp. Med. 184, 9 (1996). 25. C. C. O’Shea, T. Crompton, I. R. Rosewell, A. C. Hayday, M. J. Owen, Eur. J. Immunol. 26, 2350 (1996). 26. R. Amakawa et al., Cell 84, 551 (1996). 27. I. Correa et al., Proc. Natl. Acad. Sci. U.S.A. 89, 653 (1992). 28. M. Bigby et al., J. Immunol. 151, 4465 (1993). 29. E. Schweighoffer and B. J. Fowlkes, J. Exp. Med. 183, 2033 (1996). 30. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. 31. H. Pircher et al., EMBO J. 8, 719 (1989). 32. M. M. Rozdzial, R. T. Kubo, S. L. Turner, T. H. Finkel, J. Immunol. 153, 1563 (1994). 33. T. Shiohara et al., ibid. 138, 1979 (1987). 34. The monoclonal antibodies (mAbs) to Va2.1 (B20.1), Vb8 (MR5-2), CD3« (145-2c11), CD4 (H129.19), and CD8 (53-6.7) were purchased from PharMingen (San Diego, CA). The z chain mAb H146-968 (32) and the I-Abm12 mAb 3JP (33) were purified from culture supernatants using protein G Sepharose beads (Pharmacia). Cells were analyzed on a FACScan or a FACStar Plus (Becton Dickinson) using the CellQuest software (Becton Dickinson). 35. B. T. Bäckström, B. Rubin, A. Peter, G. Tiefenthaler, E. Palmer, Eur. J. Immunol. 27, 1433 (1997). 36. The stimulation of DP thymocytes and Hoechst staining to detect apoptotic cells was carried out as previously described (16–19). Briefly, stimulator splenocytes were prepared from B6 (I-Ab) or B6.C.H2-bm12 (I-Abm12) mice. We cultured 2 3 106 stimulators (in 2 ml) for 12 to 16 hours in the presence or absence of titrated amounts of the I-Abm12 blocking mAb 3JP (34) in 24-well plates with 1 3 106 thymocytes from B6.Rag-2–/– mice expressing the wild-type or the a-CPM mutant TCR. Cells were then harvested and stained with Hoechst 33342 (1 mg/ml) followed by staining with CD4 mAb, CD8 mAb, and propidium iodide (2.5 mg/ml) and analyzed by flow cytometry. 37. We thank A. Peter and S. Stotz for generating the aIII and bIII constructs; J. Bluestone, L. Bolliger, R. Langman, and M. Cohn for discussions; S. Bahram, C. T. Baldari, T. Hünig, J. Howard, H. Jacobs, R. Leibnitz, M. Record, A. Rolink, C. Steinberg, S. Stotz, and R. Torres for comments; M. Dessing for flow cytometric analysis; and E. Wagner, W. Metzger, U. Schneider, E. Singer, and W. Hänggi for animal husbandry. Rag-2–/– (F. Alt) and B6.Rag-2–/– (A. Rolink) mice and the pHSE39 (H. Pircher) vector are gratefully acknowledged. Care of animals was carried out in accordance with the cantonal and federal laws of Switzerland. The Basel Institute for Immunology was founded and is supported by F. Hoffmann–La Roche Ltd., Basel, Switzerland.