Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) is a minor component of the lipid bilayer but plays an important role in various cellular functions, including exocytosis and endocytosis. Recently, PI(4,5)P 2 was shown to form microdomains in the plasma membrane. In this study, we investigated the relationship between the spatial organization of PI(4,5)P 2 microdomains and exocytotic machineries in clonal rat pheochromocytoma PC12 cells. Both PI(4,5)P 2 and syntaxin, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein essential for exocytosis, exhibited punctate clusters in isolated plasma membranes. The number of PI(4,5)P 2 microdomains colocalizing with syntaxin clusters and large dense core vesicles (LDCVs) was decreased after catecholamine release. Alternatively, the expression of type I phosphatidylinositol-4-phosphate 5-kinase (PIP5KI) increased the number of PI(4,5)P 2 microdomains at syntaxin clusters with docked LDCVs and enhanced exocytotic activity, possibly by increasing the number of release sites. About half of the PI(4,5)P 2 microdomains were not colocalized with Thy-1, a specific marker of lipid rafts, and the colocalization of transfected PIP5KI with syntaxin clusters was observed. These results suggest that the formation of PI(4,5)P 2 microdomains at syntaxin clusters with docked LDCVs is essential for Ca 2؉ -dependent exocytosis.

Astrocytes release various bioactive substances via Ca(2+) - and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent exocytosis; however the regulatory mechanisms of glial exocytosis are still poorly understood. In the present study, we investigated the effect of protein kinase C (PKC) on exocytosis in glial cells using primary cultured astrocytes and clonal rat glioma C6 cells. Mass spectrometry and Western blot analysis using phospho-specific antibodies revealed that phorbol 12-myristate 13-acetate (PMA) treatment induced the phosphorylation of synaptosomal-associated protein of 23 kDa (SNAP-23) on Ser(95), Ser(120), and Ser(160) in cultured astrocytes and C6 cells. Phosphorylation at these sites was suppressed by treatment with the PKC inhibitor, bisindolylmaleimide I (BIS). In contrast, Ser(110) of SNAP-23 was constitutively phosphorylated in these cells and was dephosphorylated in a PKC-dependent manner. Exogenously expressed human growth hormone (hGH) accumulated in cytoplasmic granular structures in cultured astrocytes, and its release after ATP-treatment was Ca(2+) - and SNARE-dependent. PMA treatment suppressed the ATP-induced hGH release from astrocytes and this inhibition was reversed by BIS. We also observed PMA-dependent suppression and an attenuation of that suppression by BIS in ionomycin-induced hGH release from C6 cells. These results suggest that intracellular activation of PKC suppresses Ca(2+) - and SNARE-dependent exocytosis in astroglial cells.

Mucin-type O-glycans (O-glycans) are one of the most common glycans attached to proteins. To develop an optimized glycomic analysis protocol, O-glycans were released from glycoproteins using hydrazine, ammonia, or sodium hydroxide treatment, followed by hydrophilic interaction liquid chromatography to evaluate O-glycan release. We found that porcine gastric mucin or bovine fetuin treated at 60 °C for 6 h with hydrazine gas in the presence of malonic acid yielded O-glycans with only a small amount of degraded, so-called "peeled" products. Ammonia treatment also yielded intact O-glycans but with additional peeled products containing GlcNAc at the reducing end. In contrast, sodium hydroxide treatment yielded mainly peeled glycans, including those containing GlcNAc at the reducing end. Importantly, O-glycans obtained from rat gastric mucin treated with hydrazine and labeled with anthranilic acid had a nearly identical profile following hydrophilic interaction liquid chromatography as permethylated O-glycan alditols analyzed by mass spectroscopy. Taken together, the data suggest that glycan release using hydrazine treatment, followed by high-performance liquid chromatography after fluorescent labeling, is a suitable method for glycomic analysis of mucin-type O-glycans.

Whole mucins and reduced subunits were extracted from the corpus of the rat stomach. After purification by Sepharose CL-4B chromatography followed by cesium trifluoroacetate equilibrium centrifugation, they were analyzed by Sepharose CL-2B chromatography, rate-zonal sedimentation centrifugation, and Q-Sepharose chromatography. Monoclonal antibodies RGM21 and HIK1083, which histochemically stained mucins in the surface and glandular mucosa of the rat stomach, respectively, were used to detect the site-specific mucins. Although RGM21- and HIK1083-reactive mucins both had a multimerized structure, the density and size of both the whole mucins and reduced subunits differed, thus indicating the presence of distinct mucin species in the surface and glandular mucosa. The mucin subunits were separated into four fractions, UB, B1, B2a, and B2b, by Q-Sepharose chromatography. HIK1083 reacted mainly with UB, while RGM21 reacted with B1, B2a, and B2b. These results, combined with dot-blot, amino acid, and carbohydrate composition analyses, showed that the surface mucins may consist of three kinds of subunits. In contrast, the glandular mucins may consist of one kind of subunit which differs from that of surface mucins.

Transmembrane a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor regulatory proteins (TARPs) are auxiliary subunits that regulate AMPA receptor trafficking to the plasma membrane and localization to postsynaptic sites. The classical TARP family consists of four members: stargazin/c-2, c-3, c-4 and c-8. The TARP c-8 isoform, which is highly expressed in the hippocampus, has a unique, long C-terminal domain with five distinct regions: two glycine-rich regions, a serine/arginine-rich region, a proline/alanine (P/A) rich region, and a PSD-95/ Dlg/ZO-1 (PDZ) binding motif. We performed mass spectrometry and immunoprecipitation assays to identify specific binding partners for the c-8 C-terminal tail and found that c-8, but not stargazin/c-2, co-immunoprecipitated with calcineurin/PP2B, a Ca 2+ /calmodulin-dependent Ser/Thr phosphatase. Co-immunoprecipitation and immunoblot analyses of lysates from COS-7 cells co-transfected with calcineurin and either wild-type or chimeric c-8 revealed that a section of the C-terminal tail (residues 356-421) can bind calcineurin. Futhermore, c-8 lacking the P/A-rich region (residues 383-399) did not bind to calcineurin. In addition, the GST-c-8 C-terminal tail (residues 353-414) fusion protein containing the P/A-rich region bound to purified calcineurin in a Ca 2+ /calmodulin-dependent manner, whereas GST-c-8 with a deletion of the P/A-rich region did not. Peptide competition assays demonstrated that c-8 may interact with the hydrophobic pocket defined by b-sheet 14 and/or adjacent regions of the catalytic A subunit of calcineurin. These results indicate that the c-8 P/A-rich region is essential for binding calcineurin, suggesting that the c-8/calcineurin complex may regulate AMPA receptor phosphorylation and trafficking. Structured digital abstractgamma-8 physically interacts with Cn A by anti bait coimmunoprecipitation (View interaction) gamma-8 binds to Cn A by pull down (View interaction) gamma-2 physically interacts with PSD-95 by anti bait coimmunoprecipitation (View interaction) gamma-2 physically interacts with GluA1 and GluA2 by anti bait coimmunoprecipitation (View interaction) gamma-8 physically interacts with beta -tubulin, Cn A and alpha-tubulin by anti bait coimmunoprecipitation (View interaction) gamma-8 physically interacts with GluA1 and GluA2 by anti bait coimmunoprecipitation (View interaction)Abbreviations AKAP, A-kinase anchoring protein; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionate; Cn, calcineurin; GST, glutathione S-transferase; LTD, long-term depression; LTP, long-term potentiation; NFAT, nuclear factor of activated T cells; NMDA, N-methyl-D-aspartate; P/A, proline/ alanine; PDZ, PSD-95/Dlg/ZO-1; S/R, serine/arginine; TARP, transmembrane AMPA receptor regulatory protein.