Rab GTPases are key to membrane-trafficking events in eukaryotic cells, and human cells contain more than 60 Rab proteins that are localized to distinct compartments. The recent determination of the structure of a monoprenylated Rab GTPase bound to GDP-dissociation inhibitor provides new molecular details that are relevant to models of Rab delivery. The further discovery of an integral membrane protein that can dissociate prenylated Rab proteins from GDP-dissociation inhibitor gives new insights into the mechanisms of Rab localization.

Human cells contain more than 60 small G proteins of the Rab family, which are localized to the surfaces of distinct membrane compartments and regulate transport vesicle formation, motility, docking and fusion. Prenylated Rabs also occur in the cytosol bound to GDI (guanine nucleotide dissociation inhibitor), which binds to Rabs in their inactive state. Prenyl Rab-GDI complexes contain all of the information necessary to direct Rab delivery onto distinct membrane compartments. The late endosomal, prenyl Rab9 binds GDI with very high affinity, which led us to propose that there might be a 'GDI-displacement factor' to catalyse dissociation of Rab-GDI complexes and to enable transfer of Rabs from GDI onto membranes. Indeed, we have previously shown that endosomal membranes contain a proteinaceous factor that can act in this manner. Here we show that the integral membrane protein, Yip3, acts catalytically to dissociate complexes of endosomal Rabs bound to GDI, and to deliver them onto membranes. We propose that the conserved Yip proteins serve as GDI-displacement factors for the targeting of Rab GTPases in eukaryotic cells.

Antigen receptors on T cells, B cells, mast cells, and basophils all have cytoplasmic domains containing one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor engagement in an early and obligatory event in the signaling cascade. How clustering of receptor extracellular domains leads to phosphorylation of cytoplasmic domain ITAMs is not known, and little structural or biochemical information is available for the ITAM-containing cytoplasmic domains. Here we investigate the conformation and oligomeric state of several immune receptor cytoplasmic domains, using purified recombinant proteins and a variety of biophysical and biochemical techniques. We show that all of the cytoplasmic domains of ITAM-containing signaling subunits studied are oligomeric in solution, namely, T cell antigen receptor zeta, CD3epsilon, CD3delta, and CD3gamma, B cell antigen receptor Igalpha and Igbeta, and Fc receptor FcepsilonRIgamma. For zeta(cyt), the oligomerization behavior is best described by a two-step monomer-dimer-tetramer fast dynamic equilibrium with dissociation constants in the order of approximately 10 microM (monomer-dimer) and approximately 1 mM (dimer-tetramer). In contrast to the other ITAM-containing proteins, Igalpha(cyt) forms stable dimers and tetramers even below 10 microM. Circular dichroic analysis reveals the lack of stable ordered structure of the cytoplasmic domains studied, and oligomerization does not change the random-coil-like conformation observed. The random-coil nature of zeta(cyt) was also confirmed by heteronuclear NMR. Phosphorylation of zeta(cyt) and FcepsilonRIgamma(cyt) does not significantly alter their oligomerization behavior. The implications of these results for transmembrane signaling and cellular activation by immune receptors are discussed.

Multichain immune recognition receptors (MIRRs) found on the surface of T cells, B cells, mast cells, natural killer cells, basophils, and other immune cells, are formed by the association of several single-pass transmembrane proteins, with immunoglobulin-like ligand recognition domains and signal-transducing domains present on separate subunits. The MIRR signaling subunits all have cytoplasmic domains containing one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor engagement in an early and obligatory event in the signaling cascade. Despite the proximity to the cell membrane and crucial role in transmembrane signal transduction, little is known about the structure and lipid-binding activity of the ITAM-containing cytoplasmic domains. Here we investigate the conformation and lipid-binding activity of several MIRR cytoplasmic domains, namely T cell receptor ζ cyt , CD3ε cyt , CD3δ cyt , and CD3γ cyt , B cell receptor Igα cyt and Igβ cyt , and Fc receptor FcεRIγ cyt , using purified recombinant proteins. Secondary structure prediction analysis and experimental circular dichroism spectra identify each of these cytoplasmic domains as natively unfolded proteins. We also report that ζ cyt , CD3ε cyt , and FcεRIγ cyt bind to acidic and mixed phospholipid vesicles and that the binding strength correlates with the protein net charge and the presence of clustered basic amino acid residues. Circular dichroism analysis reveals the lack of secondary structure for these domains in lipid-bound form. Phosphorylation of ζ cyt and FcεRIγ cyt does not alter their random-coil conformation but weakens binding to membranes. The implications of these results for transmembrane signal transduction by immune receptors are discussed.Immune cells respond to the presence of foreign antigens with a wide range of responses. Antigen recognition by immune cells resulting in the initiation of these responses is mediated by the interaction of membrane-bound receptors with soluble, particulate, and cellular antigens. † This study was supported in part by the National Science Foundation (NSF; project MCB-0091072, to A.B.S., D.A.A., and L.J.S.) and by the NIH/NIAID, University of Massachusetts Center for AIDS Research (project P30 AI42845-08, to A.B.S.). The Programs of the Russian Academy of Sciences for the "Molecular and cellular biology" and "Fundamental science for medicine" provided partial support to V.N.U. * Corresponding author. Address: Department of Pathology, S2-302 The family of antigen receptors named multichain immune recognition receptors (MIRRs) (1) shares common structural and functional features, including multiple subunits with extracellular ligand-binding domains and intracellular signaling domains carried on separate protein chains. Members of the MIRR family include the T cell receptor (TCR), the B cell receptor (BCR), and the high-affinity IgE receptor (FcεRI) expressed by mast cells and basophils. A common feature of the members of...

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2′, 3′ -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2′-5′ and 3′-5′ phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

The human genome encodes ∼70 Rab GTPases that localize to the surfaces of distinct membrane compartments. To investigate the mechanism of Rab localization, chimeras containing heterologous Rab hypervariable domains were generated, and their ability to bind seven Rab effectors was quantified. Two chimeras could bind effectors for two distinctly localized Rabs; a Rab5/9 hybrid bound both Rab5 and Rab9 effectors, and a Rab1/9 hybrid bound to certain Rab1 and Rab9 effectors. These unusual chimeras permitted a test of the importance of effector binding for Rab localization. In both cases, changing the cellular concentration of a key Rab9 effector, which is called tail-interacting protein of 47 kD, moved a fraction of the proteins from their parental Rab localization to that of Rab9. Thus, relative concentrations of certain competing effectors could determine a chimera's localization. These data confirm the importance of effector interactions for Rab9 localization, and support a model in which effector proteins rely on Rabs as much as Rabs rely on effectors to achieve their correct steady state localizations.

The cytoplasmic domain of the T cell receptor zeta subunit (zeta(cyt)) is sufficient to couple receptor ligation to intracellular signaling cascades, but little is known about its structure or mechanism of signaling. In aqueous solution, zeta(cyt) is unstructured. Here we report that in the presence of lipid vesicles zeta(cyt) assumes a folded structure. The folding transition is reversible and dependent on the presence of acidic phospholipids. In the lipid-bound conformation, zeta(cyt) is refractory to phosphorylation by src family tyrosine kinases, which are believed to play a key role in signal initiation in vivo. In the lipid-free, unstructured form, zeta(cyt) is readily phosphorylated, and phospho-zeta cyt exhibits neither membrane association nor structure induction. The conformational change may provide a mechanism for coupling receptor clustering to cytoplasmic signaling events.

(2011) A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity, mAbs, 3:3, 273-288,