Skip to main content
NCBI home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1993 Aug 1;178(2):537–547. doi: 10.1084/jem.178.2.537

Molecular cloning, expression, and chromosomal localization of the human earliest lymphocyte activation antigen AIM/CD69, a new member of the C-type animal lectin superfamily of signal-transmitting receptors

PMCID: PMC2191117  PMID: 8340758

Abstract

The activation of T lymphocytes, both in vivo and in vitro, induces the expression of CD69. This molecule, which appears to be the earliest inducible cell surface glycoprotein acquired during lymphoid activation, is involved in lymphocyte proliferation and functions as a signal transmitting receptor in lymphocytes, natural killer (NK) cells, and platelets. To determine the structural basis for CD69 function, the cDNA coding for CD69 was isolated by a polymerase chain reaction-based strategy using oligonucleotides deduced from peptide sequences of the purified protein. The isolated cDNA exhibited a single open reading frame of 597 bp coding for CD69, and predicted a 199-amino acid protein of type II membrane topology, with extracellular (COOH-terminal), transmembrane, and intracellular domains. The CD69 clone hybridized to a 1.7-kb mRNA species, which was rapidly induced and degraded after lymphocyte stimulation, consistent with the presence of rapid degradation signals at the 3' untranslated region. Transient expression of the polypeptide encoded by CD69 cDNA in COS-7 cells demonstrated that it presented properties comparable to native CD69 protein. The CD69 gene was regionally mapped to chromosome 12 p13-p12 by both somatic cell hybrid DNA analysis and fluorescence in situ hybridization coupled with GTG banding (G bands by trypsin using Giemsa). Protein sequence homology search revealed that CD69 is a new member of the Ca(2+)-dependent (C-type) lectin superfamily of type II transmembrane receptors, which includes the human NKG2, the rat NKR-P1, and the mouse NKR-P1 families of NK cell-specific genes. CD69 also has a structural homology with other type II lectin cell surface receptors, such as the T cell antigen Ly49, the low avidity immunoglobulin E receptor (CD23), and the hepatic asialoglycoprotein receptors. The CD69 protein also shares functional characteristics with most members of this superfamily, which act as transmembrane signaling receptors in early phases of cellular activation.

Full Text

The Full Text of this article is available as a PDF (1.7 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aubry J. P., Pochon S., Graber P., Jansen K. U., Bonnefoy J. Y. CD21 is a ligand for CD23 and regulates IgE production. Nature. 1992 Aug 6;358(6386):505–507. doi: 10.1038/358505a0. [DOI] [PubMed] [Google Scholar]
  2. Bettler B., Hofstetter H., Rao M., Yokoyama W. M., Kilchherr F., Conrad D. H. Molecular structure and expression of the murine lymphocyte low-affinity receptor for IgE (Fc epsilon RII). Proc Natl Acad Sci U S A. 1989 Oct;86(19):7566–7570. doi: 10.1073/pnas.86.19.7566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bettler B., Maier R., Rüegg D., Hofstetter H. Binding site for IgE of the human lymphocyte low-affinity Fc epsilon receptor (Fc epsilon RII/CD23) is confined to the domain homologous with animal lectins. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7118–7122. doi: 10.1073/pnas.86.18.7118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bieber T., Rieger A., Stingl G., Sander E., Wanek P., Strobel I. CD69, an early activation antigen on lymphocytes, is constitutively expressed by human epidermal Langerhans cells. J Invest Dermatol. 1992 May;98(5):771–776. doi: 10.1111/1523-1747.ep12499948. [DOI] [PubMed] [Google Scholar]
  5. Bjorndahl J. M., Nakamura S., Hara T., Jung L. K., Fu S. M. The 28-kDa/32-kDa activation antigen EA 1. Further characterization and signal requirements for its expression. J Immunol. 1988 Dec 15;141(12):4094–4100. [PubMed] [Google Scholar]
  6. Cambiaggi C., Scupoli M. T., Cestari T., Gerosa F., Carra G., Tridente G., Accolla R. S. Constitutive expression of CD69 in interspecies T-cell hybrids and locus assignment to human chromosome 12. Immunogenetics. 1992;36(2):117–120. doi: 10.1007/BF00215288. [DOI] [PubMed] [Google Scholar]
  7. Caput D., Beutler B., Hartog K., Thayer R., Brown-Shimer S., Cerami A. Identification of a common nucleotide sequence in the 3'-untranslated region of mRNA molecules specifying inflammatory mediators. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1670–1674. doi: 10.1073/pnas.83.6.1670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cebrián M., Redondo J. M., López-Rivas A., Rodríguez-Tarduchy G., De Landázuri M. O., Sánchez-Madrid F. Expression and function of AIM, an activation inducer molecule of human lymphocytes, is dependent on the activation of protein kinase C. Eur J Immunol. 1989 May;19(5):809–815. doi: 10.1002/eji.1830190505. [DOI] [PubMed] [Google Scholar]
  9. Cebrián M., Yagüe E., Rincón M., López-Botet M., de Landázuri M. O., Sánchez-Madrid F. Triggering of T cell proliferation through AIM, an activation inducer molecule expressed on activated human lymphocytes. J Exp Med. 1988 Nov 1;168(5):1621–1637. doi: 10.1084/jem.168.5.1621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chambers W. H., Vujanovic N. L., DeLeo A. B., Olszowy M. W., Herberman R. B., Hiserodt J. C. Monoclonal antibody to a triggering structure expressed on rat natural killer cells and adherent lymphokine-activated killer cells. J Exp Med. 1989 Apr 1;169(4):1373–1389. doi: 10.1084/jem.169.4.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chan P. Y., Takei F. Molecular cloning and characterization of a novel murine T cell surface antigen, YE1/48. J Immunol. 1989 Mar 1;142(5):1727–1736. [PubMed] [Google Scholar]
  12. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  13. Chrétien I., Helm B. A., Marsh P. J., Padlan E. A., Wijdenes J., Banchereau J. A monoclonal anti-IgE antibody against an epitope (amino acids 367-376) in the CH3 domain inhibits IgE binding to the low affinity IgE receptor (CD23). J Immunol. 1988 Nov 1;141(9):3128–3134. [PubMed] [Google Scholar]
  14. Conrad D. H. Fc epsilon RII/CD23: the low affinity receptor for IgE. Annu Rev Immunol. 1990;8:623–645. doi: 10.1146/annurev.iy.08.040190.003203. [DOI] [PubMed] [Google Scholar]
  15. Cosulich M. E., Rubartelli A., Risso A., Cozzolino F., Bargellesi A. Functional characterization of an antigen involved in an early step of T-cell activation. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4205–4209. doi: 10.1073/pnas.84.12.4205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Crabtree G. R. Contingent genetic regulatory events in T lymphocyte activation. Science. 1989 Jan 20;243(4889):355–361. doi: 10.1126/science.2783497. [DOI] [PubMed] [Google Scholar]
  17. Drickamer K. Two distinct classes of carbohydrate-recognition domains in animal lectins. J Biol Chem. 1988 Jul 15;263(20):9557–9560. [PubMed] [Google Scholar]
  18. Fernández-Ruiz E., Pardo-Manuel de Villena F., Rubio M. A., Corbí A. L., Rodriguez de Córdoba S., Sánchez-Madrid F. Mapping of the human VLA-alpha 4 gene to chromosome 2q31-q32. Eur J Immunol. 1992 Feb;22(2):587–590. doi: 10.1002/eji.1830220243. [DOI] [PubMed] [Google Scholar]
  19. Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gaugitsch H. W., Prieschl E. E., Kalthoff F., Huber N. E., Baumruker T. A novel transiently expressed, integral membrane protein linked to cell activation. Molecular cloning via the rapid degradation signal AUUUA. J Biol Chem. 1992 Jun 5;267(16):11267–11273. [PubMed] [Google Scholar]
  21. Giorda R., Rudert W. A., Vavassori C., Chambers W. H., Hiserodt J. C., Trucco M. NKR-P1, a signal transduction molecule on natural killer cells. Science. 1990 Sep 14;249(4974):1298–1300. doi: 10.1126/science.2399464. [DOI] [PubMed] [Google Scholar]
  22. Giorda R., Trucco M. Mouse NKR-P1. A family of genes selectively coexpressed in adherent lymphokine-activated killer cells. J Immunol. 1991 Sep 1;147(5):1701–1708. [PubMed] [Google Scholar]
  23. Hara T., Jung L. K., Bjorndahl J. M., Fu S. M. Human T cell activation. III. Rapid induction of a phosphorylated 28 kD/32 kD disulfide-linked early activation antigen (EA 1) by 12-o-tetradecanoyl phorbol-13-acetate, mitogens, and antigens. J Exp Med. 1986 Dec 1;164(6):1988–2005. doi: 10.1084/jem.164.6.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Harvey R. J., Darlison M. G. Random-primed cDNA synthesis facilitates the isolation of multiple 5'-cDNA ends by RACE. Nucleic Acids Res. 1991 Jul 25;19(14):4002–4002. doi: 10.1093/nar/19.14.4002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Houchins J. P., Yabe T., McSherry C., Bach F. H. DNA sequence analysis of NKG2, a family of related cDNA clones encoding type II integral membrane proteins on human natural killer cells. J Exp Med. 1991 Apr 1;173(4):1017–1020. doi: 10.1084/jem.173.4.1017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ikuta K., Takami M., Kim C. W., Honjo T., Miyoshi T., Tagaya Y., Kawabe T., Yodoi J. Human lymphocyte Fc receptor for IgE: sequence homology of its cloned cDNA with animal lectins. Proc Natl Acad Sci U S A. 1987 Feb;84(3):819–823. doi: 10.1073/pnas.84.3.819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jung L. K., Haynes B. F., Nakamura S., Pahwa S., Fu S. M. Expression of early activation antigen (CD69) during human thymic development. Clin Exp Immunol. 1990 Sep;81(3):466–474. doi: 10.1111/j.1365-2249.1990.tb05357.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Karlhofer F. M., Yokoyama W. M. Stimulation of murine natural killer (NK) cells by a monoclonal antibody specific for the NK1.1 antigen. IL-2-activated NK cells possess additional specific stimulation pathways. J Immunol. 1991 May 15;146(10):3662–3673. [PubMed] [Google Scholar]
  29. Kikutani H., Inui S., Sato R., Barsumian E. L., Owaki H., Yamasaki K., Kaisho T., Uchibayashi N., Hardy R. R., Hirano T. Molecular structure of human lymphocyte receptor for immunoglobulin E. Cell. 1986 Dec 5;47(5):657–665. doi: 10.1016/0092-8674(86)90508-8. [DOI] [PubMed] [Google Scholar]
  30. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  31. Laffón A., García-Vicuña R., Humbría A., Postigo A. A., Corbí A. L., de Landázuri M. O., Sánchez-Madrid F. Upregulated expression and function of VLA-4 fibronectin receptors on human activated T cells in rheumatoid arthritis. J Clin Invest. 1991 Aug;88(2):546–552. doi: 10.1172/JCI115338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lanier L. L., Buck D. W., Rhodes L., Ding A., Evans E., Barney C., Phillips J. H. Interleukin 2 activation of natural killer cells rapidly induces the expression and phosphorylation of the Leu-23 activation antigen. J Exp Med. 1988 May 1;167(5):1572–1585. doi: 10.1084/jem.167.5.1572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lüdin C., Hofstetter H., Sarfati M., Levy C. A., Suter U., Alaimo D., Kilchherr E., Frost H., Delespesse G. Cloning and expression of the cDNA coding for a human lymphocyte IgE receptor. EMBO J. 1987 Jan;6(1):109–114. doi: 10.1002/j.1460-2075.1987.tb04726.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. McCoy J. P., Jr, Chambers W. H. Carbohydrates in the functions of natural killer cells. Glycobiology. 1991 Sep;1(4):321–328. doi: 10.1093/glycob/1.4.321. [DOI] [PubMed] [Google Scholar]
  35. Moretta A., Poggi A., Pende D., Tripodi G., Orengo A. M., Pella N., Augugliaro R., Bottino C., Ciccone E., Moretta L. CD69-mediated pathway of lymphocyte activation: anti-CD69 monoclonal antibodies trigger the cytolytic activity of different lymphoid effector cells with the exception of cytolytic T lymphocytes expressing T cell receptor alpha/beta. J Exp Med. 1991 Dec 1;174(6):1393–1398. doi: 10.1084/jem.174.6.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nakamura S., Sung S. S., Bjorndahl J. M., Fu S. M. Human T cell activation. IV. T cell activation and proliferation via the early activation antigen EA 1. J Exp Med. 1989 Mar 1;169(3):677–689. doi: 10.1084/jem.169.3.677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Reed J. C., Alpers J. D., Nowell P. C., Hoover R. G. Sequential expression of protooncogenes during lectin-stimulated mitogenesis of normal human lymphocytes. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3982–3986. doi: 10.1073/pnas.83.11.3982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Risso A., Cosulich M. E., Rubartelli A., Mazza M. R., Bargellesi A. MLR3 molecule is an activation antigen shared by human B, T lymphocytes and T cell precursors. Eur J Immunol. 1989 Feb;19(2):323–328. doi: 10.1002/eji.1830190216. [DOI] [PubMed] [Google Scholar]
  40. Risso A., Smilovich D., Capra M. C., Baldissarro I., Yan G., Bargellesi A., Cosulich M. E. CD69 in resting and activated T lymphocytes. Its association with a GTP binding protein and biochemical requirements for its expression. J Immunol. 1991 Jun 15;146(12):4105–4114. [PubMed] [Google Scholar]
  41. Ryan J. C., Niemi E. C., Goldfien R. D., Hiserodt J. C., Seaman W. E. NKR-P1, an activating molecule on rat natural killer cells, stimulates phosphoinositide turnover and a rise in intracellular calcium. J Immunol. 1991 Nov 1;147(9):3244–3250. [PubMed] [Google Scholar]
  42. Ryan J. C., Turck J., Niemi E. C., Yokoyama W. M., Seaman W. E. Molecular cloning of the NK1.1 antigen, a member of the NKR-P1 family of natural killer cell activation molecules. J Immunol. 1992 Sep 1;149(5):1631–1635. [PubMed] [Google Scholar]
  43. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  44. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Santis A. G., Campanero M. R., Alonso J. L., Tugores A., Alonso M. A., Yagüe E., Pivel J. P., Sánchez-Madrid F. Tumor necrosis factor-alpha production induced in T lymphocytes through the AIM/CD69 activation pathway. Eur J Immunol. 1992 May;22(5):1253–1259. doi: 10.1002/eji.1830220521. [DOI] [PubMed] [Google Scholar]
  46. Seed B., Aruffo A. Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure. Proc Natl Acad Sci U S A. 1987 May;84(10):3365–3369. doi: 10.1073/pnas.84.10.3365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Shaw G., Kamen R. A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell. 1986 Aug 29;46(5):659–667. doi: 10.1016/0092-8674(86)90341-7. [DOI] [PubMed] [Google Scholar]
  48. Sánchez-Mateos P., Sánchez-Madrid F. Structure-function relationship and immunochemical mapping of external and intracellular antigenic sites on the lymphocyte activation inducer molecule, AIM/CD69. Eur J Immunol. 1991 Oct;21(10):2317–2325. doi: 10.1002/eji.1830211005. [DOI] [PubMed] [Google Scholar]
  49. Takahashi H., Komano H., Kawaguchi N., Kitamura N., Nakanishi S., Natori S. Cloning and sequencing of cDNA of Sarcophaga peregrina humoral lectin induced on injury of the body wall. J Biol Chem. 1985 Oct 5;260(22):12228–12233. [PubMed] [Google Scholar]
  50. Testi R., Phillips J. H., Lanier L. L. Constitutive expression of a phosphorylated activation antigen (Leu 23) by CD3bright human thymocytes. J Immunol. 1988 Oct 15;141(8):2557–2563. [PubMed] [Google Scholar]
  51. Testi R., Phillips J. H., Lanier L. L. T cell activation via Leu-23 (CD69). J Immunol. 1989 Aug 15;143(4):1123–1128. [PubMed] [Google Scholar]
  52. Testi R., Pulcinelli F., Frati L., Gazzaniga P. P., Santoni A. CD69 is expressed on platelets and mediates platelet activation and aggregation. J Exp Med. 1990 Sep 1;172(3):701–707. doi: 10.1084/jem.172.3.701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tugores A., Alonso M. A., Sánchez-Madrid F., de Landázuri M. O. Human T cell activation through the activation-inducer molecule/CD69 enhances the activity of transcription factor AP-1. J Immunol. 1992 Apr 1;148(7):2300–2306. [PubMed] [Google Scholar]
  54. Ullman K. S., Northrop J. P., Verweij C. L., Crabtree G. R. Transmission of signals from the T lymphocyte antigen receptor to the genes responsible for cell proliferation and immune function: the missing link. Annu Rev Immunol. 1990;8:421–452. doi: 10.1146/annurev.iy.08.040190.002225. [DOI] [PubMed] [Google Scholar]
  55. Yokoyama W. M., Jacobs L. B., Kanagawa O., Shevach E. M., Cohen D. I. A murine T lymphocyte antigen belongs to a supergene family of type II integral membrane proteins. J Immunol. 1989 Aug 15;143(4):1379–1386. [PubMed] [Google Scholar]
  56. Yokoyama W. M. Recognition structures on natural killer cells. Curr Opin Immunol. 1993 Feb;5(1):67–73. doi: 10.1016/0952-7915(93)90083-5. [DOI] [PubMed] [Google Scholar]
  57. Yokoyama W. M., Ryan J. C., Hunter J. J., Smith H. R., Stark M., Seaman W. E. cDNA cloning of mouse NKR-P1 and genetic linkage with LY-49. Identification of a natural killer cell gene complex on mouse chromosome 6. J Immunol. 1991 Nov 1;147(9):3229–3236. [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES