Ly49 RECEPTORS: REQUIREMENTS FOR LIGAND RECOGNITION AND STRUCTURAL ASSEMBLY

• Author / Creator

  Craveiro Salvado, Carla M

• Natural killer (NK) cells in the mouse and rat rely on homodimeric Ly49 receptors to detect virally infected or transformed cells. The identification of altered cells requires the engagement of Ly49 receptors with MHC class I molecules. The engagement requires contact at three specific sites located below the floor of the peptide binding groove of MHC class I by three loop regions located within the ligand-interacting domain of Ly49s, the natural killer domain (NKD). Any alteration within either the MHC class I molecule or the Ly49 receptor could inhibit or disrupt the interaction, affecting the recognition of the ligand by the receptor, ultimately reflected in the functions of the NK cell. This relationship has not been as extensively studied in the rat
  as compared to the mouse. Given the sequence variability between the mouse and rat Ly49 loop regions, the significance of the loops in rat Ly49 receptors should be explored. Utilizing surface plasmon resonance (SPR), I was able to quantify the binding affinity of the rat Ly49i2 receptor for the MHC class I molecule RT1-A1c. Furthermore, I also
  showed that at least two of the three loop regions are required for RT1-A1c recognition
  by the Ly49i2 receptor. While complete binding between RT1-A1c and Ly49i2 was lost with mutated NKD L3 loop regions, only partial binding was lost with a mutated L6 loop, implying that the L6 loop may be required for MHC class I recognition, while the L3 loop may be required for MHC specificity. To assess the practical significance of the
  Ly49 loop regions, a functional assay is required. The RNK-16 rat leukemic cell line is
  heavily relied upon for NK cell functional studies. Expression of exogenous genes in
  RNK-16 cells, however, is difficult and extremely time consuming. I outlined a
  lentivirus-mediated transduction method that will allow RNK-16 cells to be transfected rapidly resulting in high transfection efficiency rate. The functional output of NK cells
  is a result of the signaling induced by the engagement of the MHC class I molecule with
  the Ly49 homodimer receptor. Utilizing biochemical assays, I demonstrate that Ly49
  receptors belonging to the same group can associate as heterodimers. The ability of these receptors to form heterodimers may allow for manipulation of the receptors, altering the function of the NK cell.

• Subjects / Keywords

  • Ly49 Receptors
  • NK cells

• Graduation date

  Fall 2015

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R38K75257

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.