Structure Function Analysis of SIRPα-CD47 Interactions Público
Lee, Winston Yuhsien (2009)
Abstract
Abstract Structure Function Analysis of SIRPα-CD47 Interactions By Winston Y. Lee
Signal regulatory proteins (SIRPs) are important regulators of innate immune functions, such as, leukocyte migration and phagocytosis. SIRPs, as a paired receptor family, include an inhibitory member (SIRPα) and an activating member (SIRPβ). They are highly homologous in their ectodomains, which consist of a membrane distal IgV fold (D1) and two membrane proximal IgC folds (D2D3). Despite their similarities, only SIRPα but not SIRPβ binds CD47. Furthermore, SIRPαD1 alone is sufficient to mediate CD47 binding. Therefore, we hypothesized that the unique residues in SIRPαD1 account for its specificity to recognize CD47. Using site directed mutagenesis and in vitro binding assay, we identified residues in SIRPαD1 that were critical in binding CD47. Mapping these critical residues onto the crystal structure of SIRPαD1 revealed a novel region that is required for CD47 binding that is distinct from the known CD47 binding site. On the other hand, the high degree of homology between the non-ligand binding domains (D2D3) of SIRPα and SIRPβ signifies a conserved theme in structure. SIRPβ is present on cell surface as a dimer linked covalently through a disulfide bond at D3. Although lacking the necessary cysteine in D3, SIRPα may still dimerize through noncovalent interactions. Our biochemical analysis revealed that SIRPα on cell surface can form homodimers in cis through noncovalent interactions at each of the three extracellular Ig folds. Notably, in tunicamycin treated cells, SIRPα dimerization but not CD47 binding was inhibited, suggesting that a SIRPα dimer may be bivalent and capable of binding CD47 with increased avidity. Lastly, in adherent neutrophils stimulated with bacterial derived products, dimerization of SIRPα was enhanced. Collectively, these data suggest that cis dimerization of SIRPα plays an important role in regulating CD47 binding and function in leukocytes. A better understanding of the structural basis of SIRPα/CD47 interactions may provide insights into therapeutics targeting pathologic inflammation.
Table of Contents
Table of Contents
Chapter 1: Introduction...1
Figures...19 Figure Legends...26
Chapter 2...29
Introduction...31 Methods...34
Results...40
The most membrane-distal Ig loop (D1) of SIRPα binds to CD47...40 Comparative analysis of protein sequences of SIRPα.D1 and SIRPβ.D1...41 Analyses of the role of disparate residues between SIRPα.D1 and SIRPβ.D1 in mediating binding to CD47...42 Homology modeling of SIPRa.D1 reveals the topological relationships of critical residues and identifies a region critical for CD47 binding...44 Identification of critical residues that are common to SIRPα and SIRPβ...45 The lateral surface of SIRPα.D1 is important in binding CD47...46 A monoclonal antibody that binds to a residue adjacent to the lateral surface of SIRPα.D1 blocks binding of CD47...47
Discussion...49 Figures...56
Figure Legends...64
Chapter 3: The role of Cis dimerization of SIRPα in binding to CD47...68
Introduction...69 Methods...72
Results...81
SIRPα forms noncovalent dimers on cell surfaces...81 CD47 is not a component of crosslinked SIRPα complexes...82 Extracellular domains contribute to the dimerization of SIRPα on cell surfaces...85 SIRPα dimerizes in cis on cell surface...85 N glycans facilitate SIRPα dimerization...87 SIRPα dimerization is not necessary for binding to soluble CD47...89 SIRPα dimerization is enhanced in stimulated adherent human PMN...91
Discussion...92
Figures...99
Figure Legends...109
Chapter 4: Discussion and Future Directions...114
Figures...124
Figure Legends...129
References...132
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