Recently, we identified and characterized a novel protein, granuphilin, whose domain structure is similar to that of the Rab3 effector protein rabphilin3 (J. Wang, T. Takeuchi, H. Yokota, and T. Izumi, J. Biol. Chem. 274:28542-28548, 1999). Screening its possible Rab partner by a yeast two-hybrid system revealed that an amino-terminal zinc-finger domain of granuphilin interacts with Rab27a. Granuphilin preferentially bound to the GTP form of Rab27a. Formation of the Rab27a/granuphilin complex was readily detected in the pancreatic beta cell line MIN6. Moreover, the tissue distributions of Rab27a and granuphilin are remarkably similar: both had significant and specific expression in pancreatic islets and in pituitary tissue, but no expression was noted in the brain. Analyses by immunofluorescence, immunoelectron microscopy, and sucrose density gradient subcellular fractionation showed that Rab27a and granuphilin are localized on the membrane of insulin granules. These findings suggest that granuphilin functions as a Rab27a effector protein in beta cells. Overexpression of wild-type Rab27a and its GTPase-deficient mutant significantly enhanced high K ؉ -induced insulin secretion without affecting basal insulin release. Although Rab3a, another exocytotic Rab protein, has some similarities with Rab27a in primary sequence, intracellular distribution, and affinity toward granuphilin, overexpression of Rab3a caused different effects on insulin secretion. These results indicate that Rab27a is involved in the regulated exocytosis of conventional dense-core granules possibly through the interaction with granuphilin, in addition to its recently identified role in lysosome-related organelles.

NMDA receptors are found in neurons both at synapses and in extrasynaptic locations. Extrasynaptic locations are poorly characterized. Here we used preembedding immunoperoxidase and postembedding immunogold electron microscopy and fluorescence light microscopy to characterize extrasynaptic NMDA receptor locations in dissociated hippocampal neurons in vitro and in the adult and postnatal hippocampus in vivo. We found that extrasynaptic NMDA receptors on neurons in vivo and in vitro were usually concentrated at points of contact with adjacent processes, which were mainly axons, axon terminals, or glia. Many of these contacts were shown to contain adhesion factors such as cadherin and catenin. We also found associations of extrasynaptic NMDA receptors with the MAGUKs, PSD-95 and SAP102. Developmental differences were also observed. At postnatal day 2 in vivo, extrasynaptic NMDA receptors could often be found at sites with distinct densities whereas dense material was seen only rarely at sites of extrasynaptic NMDA receptors in the adult hippocampus in vivo. This difference probably indicates that many sites of extrasynaptic NMDA receptors in early postnatal ages represent synapse formation or possibly sites for synapse elimination. At all ages, as suggested in both in vivo and in vitro studies, extrasynaptic NMDA receptors on dendrites or the sides of spines may form complexes with other proteins, in many cases, at stable associations with adjacent cell processes. These associations may facilitate unique functions for extrasynaptic NMDA receptors. KeywordsPSD-95; SAP102; cadherin; catenin; NR2A; NR2B Extrasynaptic NMDA receptors (NMDARs) are common on neurons but are little understood compared to synaptic NMDARs. In hippocampal neuronal cultures, physiological studies showed that ~ 75% of NMDARs are extrasynaptic at about 1 WIV (= week or weeks in vitro; Rosenmund et al., 1995; Westbrook, 1999, 2002) but this decreases with the published values ranging from ~ 20% to 50% at 2 WIV (Ivanov et al., 2006).Correspondence: Ronald S. Petralia 50 South Drive, Rm. 4142 NIDCD/NIH Bethesda, MD 20892-8027 301-496-3804 petralia@nidcd.nih.gov. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2011 April 28. Published in final edited form as:Neuroscience. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptImmunocytochemical studies indicate that about 80-90% of NMDARs are extrasynaptic at 1 WIV. These comprise both NR2A-and NR2B-containing receptors. At 1 WIV, the majority of N...

Rab GTPases regulate the membrane transport pathways by recruiting their specific effector proteins. Melanophilin, a putative Rab effector, has recently been identified as a gene that is mutated in leaden mice, in which peripheral localization of melanosomes is impaired in melanocytes. Genetic studies suggest that three coat-color mutation genes, dilute (MyoVa d ), ashen (Rab27a ash ), and leaden (Mlph ln ), act in the same or overlapping pathways. Here we have cloned and characterized a human melanophilin homolog, which belongs to the rabphilin3/granuphilin-like Rab effector family. Cosedimentation assays using recombinant proteins reveal that melanophilin directly binds to Rab27a and myosin Va through its N-terminal and its first C-terminal coiled-coil region, respectively. Moreover, we show that Rab27a, melanophilin, and myosin Va form a ternary complex in the human melanocyte cell line HMV-II. These findings suggest that melanophilin has a role in bridging Rab27a on melanosomes and myosin Va on actin filaments during melanosome transport. We also propose that the Rab-binding region conserved in a novel rabphilin3/ granuphilin-like Rab effector family constitutes an K K-helixbased coiled-coil structure. ß

The molecular mechanism for the regulated exocytosis of dense-core granules in endocrine cells remains relatively uncharacterized compared to that of synaptic vesicles in neurons. A novel set of Rab and its effector, Rab27a/granuphilin, which is localized on insulin granules in pancreatic beta cells, was recently identified. Here we demonstrate that granuphilin directly binds to syntaxin 1a on the plasma membrane, and this interaction is regulated by Rab27a. Granuphilin shows affinity to syntaxin 1a with a closed conformation but not to mutant syntaxin 1a, which adopts an open conformation constitutively. Overexpression of granuphilin significantly enhances basal insulin secretion but profoundly inhibits high K ؉ -induced insulin secretion. The effect of granuphilin on insulin secretion was impaired by its mutation that disrupts the binding to either Rab27a or syntaxin 1a. Thus, granuphilin is the first regulator in the exocytotic pathway that functions by directly connecting two critical vesicle transport proteins, Rab and SNARE.

Maternal effect genes play critical roles in early embryogenesis of model organisms where they have been intensively investigated. However, their molecular function in mammals remains largely unknown. Recently, we identified a subcortical maternal complex (SCMC) that contains four proteins encoded by maternal effect genes (Mater, Filia, Floped and Tle6). Here we report that TLE6, similar to FLOPED and MATER, stabilizes the SCMC and is necessary for cleavage beyond the two-cell stage of development. We document that the SCMC is required for formation of the cytoplasmic F-actin meshwork that controls the central position of the spindle and ensures symmetric division of mouse zygotes. We further demonstrate that the SCMC controls formation of the actin cytoskeleton specifically via Cofilin, a key regulator of F-actin assembly. Our results provide molecular insight into the physiological function of TLE6, its interaction with the SCMC and their roles in the symmetric division of the zygote in early mouse development.

The molecular pathogenesis of diabetes remains poorly understood because of the genetic complexity of the disease. One possibly effective approach to elucidate the pathogenesis is to study an animal model with a similar phenotype. The TSOD (Tsumura, Suzuki, Obese Diabetes) mouse, a newly developed animal model, exhibits both diabetes and obesity with marked hyperinsulinemia and hypertrophy of the pancreatic islets and might represent a common form of obese type 2 diabetes in humans. Phenotypic characterization revealed that the TSOD mouse had both insulin resistance and impaired glucose-stimulated insulin secretion. A comprehensive genetic dissection of diabetes and obesity has been performed using F1 and F2 progeny between the TSOD and control BALB/cA strains. A genome-wide screen for loci linked to glucose homeostasis and body weight allowed us to map three quantitative trait loci (QTLs) involved in this disorder. The major genetic determinant of blood glucose levels was identified on chromosome 11. Furthermore, two independent QTLs involved in controlling body weight were found on chromosomes 1 and 2. The QTL on chromosome 2 also affected insulin levels significantly. Each QTL has distinct effects on different traits and a different mode of inheritance. Our study indicates that hyperglycemia and obesity are clearly controlled by distinct combinations of genetic loci in this mouse model and provides insights into the genetic basis of common forms of human type 2 diabetes with obesity.

No abstract

Breast cancer amplified sequence 2 (BCAS2) is involved in multiple biological processes, including pre-mRNA splicing. However, the physiological roles of BCAS2 are still largely unclear. Here we report that BCAS2 is specifically enriched in spermatogonia of mouse testes. Conditional disruption of Bcas2 in male germ cells impairs spermatogenesis and leads to male mouse infertility. Although the spermatogonia appear grossly normal, spermatocytes in meiosis prophase I and meiosis events (recombination and synapsis) are rarely observed in the BCAS2-depleted testis. In BCAS2 null testis, 245 genes are altered in alternative splicing forms; at least three spermatogenesis-related genes (Dazl, Ehmt2 and Hmga1) can be verified. In addition, disruption of Bcas2 results in a significant decrease of the full-length form and an increase of the short form (lacking exon 8) of DAZL protein. Altogether, our results suggest that BCAS2 regulates alternative splicing in spermatogonia and the transition to meiosis initiation, and male fertility.