Jay C Unkeless, PhD

Our group studies the structure and function of receptors for IgG (Fc[gamma]R) that are found on neutrophils, macrophages and NK cells. Fc[gamma]R's link the effector cells of the immune system to the production of IgG following an immune response. Fc[gamma]Rs on effector cells, following crosslinking by multivalent immune complexes, can trigger the secretion of superoxide, release of hydrolytic enzymes, synthesis of cytokines, and killing of sensitized target cells.\r\n\r\n

A major aim of the laboratory is to analyze signaling pathways leading from Fc[gamma]R activation (by crosslinking) to cell effector activity. In particular, we are interested in the signaling mechanisms of glycan-phosphatidyl inositol-anchored Fc[gamma]R IIIb, and with the architecture of Fc[gamma]RIIIb in the plasma membrane. We have recently observed that crosslinking Fc[gamma]RIIIb results in the co-capping of Fc[gamma]RIIa and in the rapid partition into the Fc[gamma]RIIIb cap of a lipid probe, dI-I, that is initially diffusely distributed in the plasma membrane. It is thus likely that GPI-anchored protein capping results in a lipid membrane domain with significantly different physical properties from the bulk membrane. The properties of these domains, and the proteins that associate with the capped Fc[gamma]RIIIb caps are under investigation.

\r\n\r\n

Following our interest in phagocytosis, we have derivsed a screen for proteins that bind inositol hexaphosphate (IP6) and have probed a recently constructed macrophage expression library with a multivalent complex consisting of biotinylated IP6 conjugated to 125I-BSA. We have identified a serine/threonine kinase reported to be expressed strongly in spermatids, where it associates with microtubule associated proteins. This kinase is likely to be involved with exocytic events. The proteins that interact with the kinase are being studied biochemically, and by yeast two-hybrid interaction analysis. We are also mapping the domain(s) of the protein that bind IP6, and have mapped 5' genomic clones for use in isolating a knockout mutant.

\r\n\r\n

We have developed a system for obtaining synchronous and maximal stimulation of macrophages by Fc[gamma]R ligation to glass, and are screening for transcripts activated by this stimulation. Initial efforts in this direction used differential display PCR (ddPCR), and resulted in the isolation of a protein that may play a role in control of deubiquitination. However, ddPCR is fraught with technical difficulties, and we are now revisiting this problem utilizing a recently developed subtractive hybridization/PCR protocol.

\r\n