BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATION OF LY-49H AS AN ACTIVATING NATURAL KILLER CELL RECEPTOR

Abstract

Natural Killer (NK) cells are part of the innate immune system and as such serve a critical role in immune surveillance. They are often described as the first line of defense against invading pathogens and aberrant cells. Scientific studies in the last 15 years have led to the discovery of a plethora of cell surface receptors on NK cells and the field of NK biology has exploded. Receptors with similar function can be found in both the human and mouse. The mouse serves as a research tool in studying one particular family of cell surface proteins, the Ly-49 receptors. This receptor family has been characterized as having mostly inhibitory receptors that block the lytic and cytokine secretion functions of the NK cells. However, one family member, Ly-49D, has been described as an activating receptor not only because of its ability to trigger cytolysis and cytokine secretion by NK cells, but because it has been shown to have an associated adaptor protein which contains a canonical immune receptor tyrosine-based activation motif. An additional Ly-49 family member, Ly-49H, has been identified. It has been presumed that Ly-49H is also an activating Ly-49 family member because of its amino acid sequence and a similarity to Ly-49D in the transmembrane domain. In this domain both Ly-49D and Ly-49H have a positively charged amino acid residue. Through this residue, Ly-49D has been shown to bind the adaptor molecule DAP12 which becomes phosphorylated upon stimulation. It is the purpose of this work to demonstrate that Ly-49H is an activating Ly-49 family member. These studies were designed to compare Ly-49H and Ly-49D in biochemical and functional assays. The experiments presented herein use a variety of model systems to accomplish this comparison. Cell lines transfected with Ly-49H or Ly- 49D are used as a model system as well as primary mouse cells to verify the results in a natural environment. Association with an adaptor molecule as well as the functional phosphorylation of the adaptor in transfected cell lines and primary cells is demonstrated. Calcium mobilization in response to cross-linking of the Ly-49H receptor is shown in the cell lines and primary cells, as well. Cytokine production as a measurement of functional activation following stimulation through the Ly-49H receptor is also demonstrated. Finally, an in vivo role for NK cells and perhaps these activating Ly-49 family members with regard to induction of an acquired T cell response is presented.