Structure/Function Studies of LILRs Enabled by a Bacterially-Derived Ligand

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PROJECT ABSTRACT The Leukocyte Immunoglobulin-Like Receptors (hereafter “LILRs”) are a family of type-I glycoprotein receptors expressed exclusively by hematopoietic lineage cells. While the extracellular regions of the 11 human LILRs share a similar architecture of repeating Ig-domains, their intracellular regions can be classified according to their signaling capacities: LILRs A1-A6 generally associate with FcγR and utilize its signal-activating ITAM motif, whereas LILRs B1-B5 contain their own signal-inhibitory ITIM motif. LILRs themselves are primate- specific, but studies on their murine paralogs (known as PIR-A and PIR-B) or isolated human leukocytes have shown that ligand binding triggers signaling events that either activate or inhibit various immune cells, primarily those of myeloid origin. Known LILR ligands include both cell-surface (e.g. HLA Class-I) and oligomeric proteins (e.g. β-amyloid polymers), yet the ligand(s) of 5 of the 11 human LILRs (i.e. LILR-A2, -A5, -A6, -B3, and -B4) remain unidentified. For those that are known, there is little detailed biochemical information as to how LILRs recognize these ligands. Moreover, no co-crystal structures of a ligand-bound LILR been reported thus far. These shortcomings represent a critical gap in knowledge of basic LILR biology. They have also prevented a more thorough understanding of LILR signaling and its contributions to leukocyte function in health and disease. Our laboratory has worked for many years toward understanding the mechanisms that underlie bacterial evasion of the complement/neutrophil axis. In the course of these studies, we recently made the unexpected discovery that Group B streptococcal cells bind to the extracellular regions of LILR-A6 and LILR-B3. These two LILRs are expressed by phagocytes, including neutrophils and monocytes, which serve critical roles in innate immunity to bacterial pathogens. We have since identified the β-Antigen C Protein (βAC) as the bacterial cell- surface molecule responsible for LILR-A6 and LILR-B3 binding to Group B streptococcal cells. Since βAC is the first known ligand for either of these LILRs, we believe the βAC protein represents a powerful tool for obtaining new insights into how LILRs recognize their ligands and how ligand-binding induces signaling. We believe these principles are likely broadly relevant to LILRs as a family, rather than pertaining to only this system specifically. In this project, we will use the βAC protein as a paradigm for defining the fundamental structure/function relationships of LILRs. We will begin by defining the molecular basis for the interaction of βAC with the extracellular regions of human LILR-A6 and LILR-B3. We will then use a chimeric T-cell based reporter system to determine how βAC influences signaling by these LILRs. Finally, we will define the molecular basis for interaction of βAC with human complement Factor H as a prelude for examining how this endogenous complement regulator might affect LILR signaling in response to βAC. Together, the studies we propose will shed new mechanistic light on this as yet poorly understood family of immune cell surface receptors.