Haruko Kumanogoh

Characterization of a Novel Rat Brain Glycosylphosphatidylinositol-anchored Protein (Kilon), a Member of the IgLON Cell Adhesion Molecule Family

In the central nervous system, many cell adhesion molecules are known to participate in the establishment and remodeling of the neural circuit. Some of the cell adhesion molecules are known to be anchored to the membrane by the glycosylphosphatidylinositol (GPI) inserted to their C termini, and many GPI-anchored proteins are known to be localized in a Triton-insoluble membrane fraction of low density or so-called “raft.” In this study, we surveyed the GPI-anchored proteins in the Triton-insoluble low density fraction from 2-week-old rat brain by solubilization with phosphatidylinositol-specific phospholipase C. By Western blotting and partial peptide sequencing after the deglycosylation with peptide N-glycosidase F, the presence of Thy-1, F3/contactin, and T-cadherin was shown. In addition, one of the major proteins, having an apparent molecular mass of 36 kDa after the peptide N-glycosidase F digestion, was found to be a novel protein. The result of cDNA cloning showed that the protein is an immunoglobulin superfamily member with three C2 domains and has six putative glycosylation sites. Since this protein shows high sequence similarity to IgLON family members including LAMP, OBCAM, neurotrimin, CEPU-1, AvGP50, and GP55, we termed this protein Kilon (akindred of IgLON). Kilon-specific monoclonal antibodies were produced, and Western blotting analysis showed that expression of Kilon is restricted to brain, and Kilon has an apparent molecular mass of 46 kDa in SDS-polyacrylamide gel electrophoresis in its expressed form. In brain, the expression of Kilon is already detected in E16 stage, and its level gradually increases during development. Kilon immunostaining was observed in the cerebral cortex and hippocampus, in which the strongly stained puncta were observed on dendrites and soma of pyramidal neurons.

Cholesterol-dependent Localization of NAP-22 on a Neuronal Membrane Microdomain (Raft)

A membrane microdomain called raft has been under extensive study since the assembly of various signal-transducing molecules into this region has been envisaged. This domain is isolated as a low buoyant membrane fraction after the extraction with a nonionic detergent such as Triton X-100. The characteristic low density of this fraction is ascribed to the enrichment of several lipids including cholesterol. To clear the molecular mechanism of raft formation, several extraction methods were applied to solubilize raft components. Cholesterol extraction using methyl-β-cyclodextrin was found to be effective to solubilize NAP-22, a neuron-enriched Ca2+-dependent calmodulin-binding protein as well as one of the main protein components of brain raft. Purified NAP-22 bound to the liposomes that were made from phosphatidylcholine and cholesterol. This binding was dependent on the amount of cholesterol in liposomes. Calmodulin inhibited this binding in a dose-dependent manner. These results suggest that the presence of a calcium-dependent regulatory mechanism works on the assembly of raft within the neuron.

A Truncated Tropo-Myosine-Related Kinase B Receptor, T1, Regulates Glial Cell Morphology via Rho GDP Dissociation Inhibitor 1

Through tropo-myosine-related kinase B (TrkB) receptors, brain-derived neurotrophic factor (BDNF) performs many biological functions such as neural survival, differentiation, and plasticity. T1, an isoform of TrkB receptors that lacks a tyrosine kinase, predominates in the adult mammalian CNS, yet its role remains controversial. In this study, to examine whether T1 transduces a signal and to determine its function, we first performed an affinity purification of T1-binding protein with the T1-specific C-terminal peptide and identified Rho GDP dissociation inhibitor 1 (GDI1), a GDP dissociation inhibitor of Rho small G-proteins, as a signaling protein directly associated with T1. The binding of BDNF to T1 caused Rho GDI1 to dissociate from the C-terminal tail of T1. Astrocytes cultured for 30 d expressed only endogenous T1 among the BDNF receptors. In 30 d cultured astrocytes, Rho GDI1, when dissociated in a BDNF-dependent manner, controlled the activities of the Rho GTPases, which resulted in rapid changes in astrocytic morphology. Furthermore, using 2 d cultured astrocytes that were transfected with T1, a T1 deletion mutant, or cyan fluorescent protein fusion protein of the T1-specific C-terminal sequence, we demonstrated that T1-Rho GDI1 signaling was indispensable for regulating the activities of Rho GTPases and for the subsequent morphological changes among astrocytes. Therefore, these findings indicate that the T1 signaling cascade can alter astrocytic morphology via regulation of Rho GTPase activity.

Calcium‐dependent association of annexin VI, protein kinase Cα, and neurocalcinα on the raft fraction derived from the synaptic plasma membrane of rat brain

A membrane microdomain enriched in cholesterol and sphingolipids or so called “raft” region was found to contain many signal transducing proteins such as GPI‐anchored proteins, trimeric G proteins and protein tyrosine kinases. Because brain‐derived raft contains two calmodulin‐binding proteins, GAP‐43 and NAP‐22 as the major protein components, the raft domain is assumed to be important in the Ca2+‐signaling. In this study, we analyzed protein components showing Ca2+‐dependent binding to the raft of synaptic plasma membrane from rat brain. SDS‐PAGE analysis of the protein components in the EGTA eluate from the raft prepared in the presence of Ca2+‐ions showed the elution of 80 kDa, 68 kDa, 22 kDa, and 21 kDa proteins. These proteins were identified as protein kinase Cα (80 kDa) and annexin VI (68 kDa) from the partial amino‐acid sequencing, and neurocalcinα (22 kDa) and calmodulin (21 kDa) with western blotting and electrophoretic mobilities in the presence or absence of Ca2+ ions. Further immunoblotting experiments showed the Ca2+‐dependent association of conventional, but not non‐conventional, subtypes of PKC to the raft. J. Neurosci. Res. 64:235–241, 2001.

Identification of NAP-22 and GAP-43 (neuromodulin) as major protein components in a Triton insoluble low density fraction of rat brain

NAP-22 is a membrane-localized brain enriched acidic protein having a Ca(2+)-dependent calmodulin binding activity. Further fractionation of the NAP-22 containing membrane showed the localization of NAP-22 in a Triton insoluble fraction of low density. Besides NAP-22, this fraction was found to contain GAP-43 (neuromodulin), trimeric G proteins, and some GPI-anchored proteins such as Thy-1 and N-CAM-120. Presence of some protein tyrosine kinases, such as src and fyn, was also shown.

Identification of V-ATPase as a major component in the raft fraction prepared from the synaptic plasma membrane and the synaptic vesicle of rat brain.

Cholesterol is important in the maintenance and remodeling of the synapse. Since membrane cholesterol participates in the formation of the membrane microdomain (raft), the characterization of raft components within membrane structures in the synaptic region could be a good approach to understand the role of cholesterol in the synaptic function. In this study, protein complexes in the raft prepared from synaptic plasma membrane and the synaptic vesicle were analyzed with blue-native polyacrylamide gel electrophoresis and vacuolar H(+)-pump (V-ATPase) was identified as a major raft component using mass spectrometry. The ATPase activity was reduced through cholesterol deprivation with methyl-beta-cyclodextrin. Since the H(+) -gradient is used to transport synaptic transmitters or their precursors into the vesicle, this result suggests the essential role of cholesterol and raft in the synaptic function.

Ouabain-induced isoform-specific localization change of the Na+, K+-ATPase α subunit in the synaptic plasma membrane of rat brain

Na+, K+-ATPase is one of major membrane proteins that has two subunits, alpha and beta. The alpha subunit has the ATPase activity and the ouabain binding site. Among four isoforms of the alpha subunit, expression of alpha1, alpha2, and alpha3, but not alpha4, is observed in matured rat brain. Ouabain is one of cardiac glycosides, and endogenous ouabain-like compounds have been recognized as a new class of steroid hormone. The alpha subunit is considered as their endogenous receptor. Recent studies envisaged the importance of membrane microdomains (MDs) as signaling platforms, which are recovered as a detergent-resistant membrane microdomain fraction (DRM). Although this ATPase has been considered as a non-DRM protein, some amount of the alpha subunit was found to be a component of the DRM prepared from the synaptic plasma membrane fraction (SPM) of rat brain. Ouabain treatment increased the amount of alpha3 isoform, but not alpha1, in the DRM derived from synaptosome fraction and SPM. These results suggest that the localization of the alpha subunit of Na+, K+-ATPase is regulated with isoform-specific mechanisms and the physiological importance of DRM in the signal transduction of the endogenous ouabain-like steroid hormone in neurons.

Changes in the localization of NAP-22, a calmodulin binding membrane protein, during the development of neuronal polarity

NAP-22, a neuronal tissue-enriched acidic membrane protein, is a Ca(2+)-dependent calmodulin binding protein and has similar biochemical characteristics to GAP-43 (neuromodulin). Recent biochemical studies have demonstrated that NAP-22 localizes in the membrane raft domain with a cholesterol-dependent manner. Since the raft domain is assumed to be important to establish and/or to maintain the cell polarity, we have investigated the changes in the localization of NAP-22 during the development of the neuronal polarity in vitro and in vivo, using cultured hippocampal neurons and developing cerebellum neurons, respectively. Cultured hippocampal neurons initially extended several short processes, and at this stage NAP-22 was distributed more or less evenly among them. During the maturation of neuronal cells, NAP-22 was sorted preferentially into the axon. Throughout the developmental stages of hippocampal neurons, the localization change of NAP-22 was quite similar to that of tau, an axonal marker protein, but not to that of microtubule-associated protein-2 (MAP-2), a dendritic marker protein. Further confocal microscopic observation demonstrated the colocalization of NAP-22 and either tau or vesicle-associated protein-2 (VAMP-2). A comparison of the time course of the axonal localization of NAP-22 and GAP-43 showed that NAP-22 localization was much later than that of GAP-43. The correlation between the expression of NAP-22 and synaptogenesis in the cerebellar granular layer, particularly in the synaptic glomeruli, was also investigated. There existed many VAMP-2 positive synapses but no NAP-22 positive ones in 1-week-old cerebellum. On sections of 2-week-old cerebellum, accumulation of NAP-22 to the synaptic glomeruli was clearly observed and this accumulation became clearer during the maturation of the synaptic structure. The present results suggest the possibility that NAP-22 plays an important role in the maturation and/or the maintenance of synapses rather than in the process of the axonal outgrowth, by controlling cholesterol-dependent membrane dynamics.

Biochemical interaction of an actin-capping protein, CapZ, with NAP-22.

NAP‐22 is a neuronal protein localized in the presynaptic membrane and synaptic vesicles and recovered in a Triton‐insoluble low‐density microdomain fraction after biochemical fractionation of the synaptic plasma membrane. NAP‐22 organizes membrane microdomains through binding to membrane lipids such as cholesterol, phosphatidylethanolamine, and phosphatidylinositol 4,5‐bisphosphate. In this study, NAP‐22‐binding proteins were screened through the pull‐down assay using brain‐derived NAP‐22 bound to Sepharose 4B. An actin‐capping protein, CapZ, was identified in the precipitate through mass spectrometry and Western blotting. CapZ was then expressed in E. coli and the purified protein‐bound NAP‐22 directly. Because bacterially expressed NAP‐22 bound CapZ, it was determined that the N‐terminal myristoyl moiety of NAP‐22 is not necessary for the binding. The binding of NAP‐22 showed no effect on the actin nucleation activity of CapZ measured with centrifugation and viscometric assays. Hence, the CapZ–NAP‐22 complex could work as the nucleation site of actin polymerization or as the actin filament‐anchoring site on the membrane microdomain.

Localization of phospholipase Cβ1 on the detergent-resistant membrane microdomain prepared from the synaptic plasma membrane fraction of rat brain

The membrane microdomain (MD), such as detergent‐resistant low‐density membrane microdomain fraction (DRM), has been paid much attention because many signal‐transducing molecules are recovered in this fraction, although precise localization and interactions of these molecules are largely unclear. To identify neuronal MD‐localized proteins, monoclonal antibodies (mAbs) against the DRM‐components of synaptic plasma membrane fraction (SPM) were produced and the antigens were characterized. One of the antigens reacted with two closely positioned bands of about 140 kDa in SDS‐PAGE and the antigen showed age‐dependent localization on DRM. The antigen was immunoprecipitated with the mAb after partial solubilization with 0.6 M NaCl from SPM‐derived DRM and identified as phospholipase Cβ1 through mass analysis. The identity was further confirmed with Western blotting using a specific polyclonal antibody. The enzyme purified from the DRM was activated by the α subunit of trimeric G protein, Gq, expressed in HEK293 cells. The lipid composition of the liposomes affected the enzymatic activity and the addition of NAP‐22, a neuronal DRM‐localized protein, inhibited the activity. These results suggest that there exists a signal‐transducing MD that performs important roles in neuronal functions through PIP2 signaling and Ca2+ mobilization.