A presynaptic role for the cytomatrix protein GIT in synaptic vesicle recycling.

Neurotransmission involves the exo-endocytic cycling of synaptic vesicles (SVs) within nerve terminals. Exocytosis is facilitated by a cytomatrix assembled at the active zone (AZ). The precise spatial and functional relationship between exocytic fusion of SVs at AZ membranes and endocytic SV retrieval is unknown. Here, we identify the scaffold G protein coupled receptor kinase 2 interacting (GIT) protein as a component of the AZ-associated cytomatrix and as a regulator of SV endocytosis. GIT1 and its D. melanogaster ortholog, dGIT, are shown to directly associate with the endocytic adaptor stonin 2/stoned B. In Drosophila dgit mutants, stoned B and synaptotagmin levels are reduced and stoned B is partially mislocalized. Moreover, dgit mutants show morphological and functional defects in SV recycling. These data establish a presynaptic role for GIT in SV recycling and suggest a connection between the AZ cytomatrix and the endocytic machinery.

Structure of the Eps15-stonin2 complex provides a molecular explanation for EH-domain ligand specificity.

Eps15 homology (EH) domain-containing proteins play a key regulatory role in intracellular membrane trafficking and cell signalling. EH domains serve as interaction platforms for short peptide motifs comprising the residues NPF within natively unstructured regions of accessory proteins. The EH-NPF interactions described thus far are of very low affinity and specificity. Here, we identify the presynaptic endocytic sorting adaptor stonin2 as a high-affinity ligand for the second EH domain (EH2) of the clathrin accessory protein Eps15. Calorimetric data indicate that both NPF motifs within stonin2 interact with EH2 simultaneously and with sub-micromolar affinity. The solution structure of this complex reveals that the first NPF motif binds to the conserved site on the EH domain, whereas the second motif inserts into a novel hydrophobic pocket. Our data show how combination of two EH-attachment sites provides a means for modulating specificity and allows discrimination from a large pool of potential binding partners containing NPF motifs.

Clathrin-mediated endocytosis at synapses.

Neurons are communication specialists that convert electrical into chemical signals at specialized cell-cell junctions termed synapses. Arrival of an action potential triggers calcium-regulated exocytosis of neurotransmitter (NT) from small synaptic vesicles (SVs), which then diffuses across the synaptic cleft and binds to postsynaptic receptors to elicit specific changes within the postsynaptic cell. Endocytosis of pre- and postsynaptic membrane proteins including SV components and postsynaptic NT receptors is essential for the proper functioning of the synapse. During the past several years, we have witnessed enormous progress in our understanding of the mechanics of clathrin-mediated endocytosis (CME) and its role in regulating exo-endocytic vesicle cycling at synapses. Here we summarize the molecular machinery used for recognition of synaptic membrane protein cargo and its clathrin-dependent internalization, and describe the inventory of tools that can be used to monitor vesicle cycling at synapses or to inhibit CME in a stage-specific manner.

Molecular basis of synaptic vesicle cargo recognition by the endocytic sorting adaptor stonin 2.

Synaptic transmission depends on clathrin-mediated recycling of synaptic vesicles (SVs). How select SV proteins are targeted for internalization has remained elusive. Stonins are evolutionarily conserved adaptors dedicated to endocytic sorting of the SV protein synaptotagmin. Our data identify the molecular determinants for recognition of synaptotagmin by stonin 2 or its Caenorhabditis elegans orthologue UNC-41B. The interaction involves the direct association of clusters of basic residues on the surface of the cytoplasmic domain of synaptotagmin 1 and a beta strand within the mu-homology domain of stonin 2. Mutation of K783, Y784, and E785 to alanine within this stonin 2 beta strand results in failure of the mutant stonin protein to associate with synaptotagmin, to accumulate at synapses, and to facilitate synaptotagmin internalization. Synaptotagmin-binding-defective UNC-41B is unable to rescue paralysis in C. elegans stonin mutant animals, suggesting that the mechanism of stonin-mediated SV cargo recognition is conserved from worms to mammals.

The Aspergillus nidulans F-box protein GrrA links SCF activity to meiosis.

Cellular differentiation relies on precise and controlled means of gene expression that act on several levels to ensure a flexible and defined spatio-temporal expression of a given gene product. In our aim to identify transcripts enriched during fruiting body formation of the homothallic ascomycete Aspergillus (Emericella) nidulans, the grrA gene could be identified in a negative subtraction hybridization screening procedure. It encodes a protein similar to fungal F-box proteins, which function as substrate receptors for ubiquitin ligases, and that is highly related to the Saccharomyces cerevisiae regulatory protein Grr1p. Expression studies confirmed induction of grrA transcription and expression of its gene product during cleistothecial development of A. nidulans. Functional complementation of a yeast grr1Delta mutant was achieved by overexpression of the grrA coding sequence. A grrADelta deletion mutant resembles the wild-type in hyphal growth, asexual sporulation, Hülle cell formation or development of asci-containing cleistothecia, but is unable to produce mature ascospores due to a block in meiosis as demonstrated by cytological staining of cleistothecial contents. Our results specify a particular involvement of the E3 ubiquitin ligase SCFGrrA in meiosis and sexual spore formation of an ascomyceteous fungus and shed light on the diverse functions of ubiquitin-proteasome-mediated protein degradation in eukaryotic development.

Stonin 2 is an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling.

Clathrin-mediated endocytosis is involved in the internalization, recycling, and degradation of cycling membrane receptors as well as in the biogenesis of synaptic vesicle proteins. While many constitutively internalized cargo proteins are recognized directly by the clathrin adaptor complex AP-2, stimulation-dependent endocytosis of membrane proteins is often facilitated by specialized sorting adaptors. Although clathrin-mediated endocytosis appears to be a major pathway for presynaptic vesicle cycling, no sorting adaptor dedicated to synaptic vesicle membrane protein endocytosis has been indentified in mammals. Here, we show that stonin 2, a mammalian ortholog of Drosophila stoned B, facilitates clathrin/AP-2-dependent internalization of synaptotagmin and targets it to a recycling vesicle pool in living neurons. The ability of stonin 2 to facilitate endocytosis of synaptotagmin is dependent on its association with AP-2, an intact mu-homology domain, and functional AP-2 heterotetramers. Our data identify stonin 2 as an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling.

Functional dissection of the interactions of stonin 2 with the adaptor complex AP-2 and synaptotagmin.

Synaptic vesicle recycling is in part mediated by clathrin-mediated endocytosis. This process involves the coordinated assembly of clathrin and adaptor proteins and the concomitant selection of cargo proteins. Here, we demonstrate that the endocytotic protein stonin 2 localizes to axonal vesicle clusters through its micro-homology domain. Interaction of this domain with synaptotagmin I is sufficient to recruit stonin 2 to the plasmalemma. The N-terminal domain of stonin 2 harbors multiple AP-2-interaction motifs that bind to the clathrin adaptor complex AP-2. These motifs with the consensus sequence WVxF are capable of binding to the alpha-adaptin ear domain and to micro2. Mutation of the tyrosine motif-binding pocket of micro2 abolishes recognition of the WVxF peptide, suggesting that association with stonin 2 renders AP-2 incompetent to sort tyrosine motif-containing cargo proteins. We hypothesize that stonin 2 may function as an AP-2-dependent sorting adaptor for synaptic vesicle recycling.

The MHC class I-like IgG receptor controls perinatal IgG transport, IgG homeostasis, and fate of IgG-Fc-coupled drugs.

Abs of the IgG isotype are efficiently transported from mother to neonate and have an extended serum t(1/2) compared with Abs of other isotypes. Circumstantial evidence suggests that the MHC class I-related protein, the neonatal FcR (FcRn), is the FcR responsible for both in vivo functions. To understand the phenotypes imposed by FcRn, we produced and analyzed mice with a defective FcRn gene. The results provide direct evidence that perinatal IgG transport and protection of IgG from catabolism are mediated by FcRn, and that the latter function is key to IgG homeostasis, essential for generating a potent IgG response to foreign Ags, and the basis of enhanced efficacy of Fc-IgG-based therapeutics. FcRn is therefore a promising therapeutic target for enhancing protective humoral immunity, treating autoimmune disease, and improving drug efficacy.

Immunodominance of H60 is caused by an abnormally high precursor T cell pool directed against its unique minor histocompatibility antigen peptide.

The H60 minor histocompatibility (H) antigen peptide is derived from a glycoprotein that serves as a ligand for the stimulatory NKG2D receptor. We show that this peptide is remarkably immunodominant in that it competes effectively with MHC alloantigens, is efficiently crosspresented by host antigen-presenting cells (APCs), and readily elicits naive CD8 T cell responses in vitro. H60 immunodominance is neither a consequence of NKG2D engagement nor competition among minor H antigens on APCs. Instead, H60 immunodominance is a consequence of an abnormally high naive precursor frequency of H60 peptide reactive CD8 T cells. Understanding why the H60 peptide is so immunogenic has important implications in tissue transplantation and vaccine design.

Real-time T-cell profiling identifies H60 as a major minor histocompatibility antigen in murine graft-versus-host disease.

Although CD8 T cells are thought to be a principal effector population of graft-versus-host disease (GVHD), their dynamics and specificity remain a mystery. Using a mouse model in which donor and recipient were incompatible at many minor histocompatibility antigens (minor H Ags), the CD8 T-cell response was tracked temporally and spatially through the course of GVHD. Donor CD8 T cells in the circulation, spleen, lung, and liver demonstrated virtually identical kinetics: rapid expansion and then decline prior to morbidity. Remarkably, up to one fourth of the CD8 T cells were directed against a single minor antigen, H60. Extreme H60 immunodominance occurred regardless of sampling time, site, and genetic background. This study is the first to analyze the T cells participating in GVHD in "real-time," demonstrates the exceptional degree to which immunodominance of H60 can occur, and suggests that such superdominant minor H Ags could be risk factors for GVHD.