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Notes: Abstract: Serotonin binding protein (SBP) is present in all neurectodermally derived cells that store serotonin (5-HT). Three forms of SBP have been detected (68, 56, and 45 kDa), and antibodies to SBP that interfere with the binding of 5-HT react with each of these proteins. The current experiments test two hypotheses: (a) that the 56- and 45-kDa forms of SBP are produced by posttranslational cleavage of a 68-kDa precursor molecule; and (b) that 45-kDa SBP is a constituent of serotonergic secretory vesicles. Pulse-chase experiments were carried out using medullary thyroid carcinoma cells as a model. These neurectodermally derived cells produce 5-HT and all three forms of SBP. Following pulse labeling for 20 min with l-[35S]methionine, the cells were incubated in the presence of an excess of unlabeled l-methionine for 0, 30, 60, or 90 min at 37°C. Alternatively, the chase was performed under conditions (20°C, inhibition of ATP generation) that delay or stop transport of newly synthesized proteins from the rough endoplasmic reticulum through the Golgi apparatus. Following incubation, the cells were washed and solubilized, and SBP was immunoprecipitated. Radioactive proteins in the immunoprecipitate were electrophoretically resolved and quantified. Immediately after the pulse, each of the three forms of SBP was found to be labeled with 35S. The relative proportions of 35S-labeled 68-, 56-, and 45-kDa SBP remained the same at each interval of chase. These proportions were not changed when the chase was carried out at 20°C or under conditions that blocked the biosynthesis of ATP. These observations suggest that each form of SBP is a primary product of translation, that the smaller forms of SBP are not produced by cleavage from a larger molecule, and that the size of the primary products of translation is not altered by passage to the Golgi apparatus or a post-Golgi compartment. When secretion was induced, 45-kDa SBP, but not 56- or 68-kDa SBP, was released to the medium. When antibodies to 45-kDa SBP were added to the medium at the time secretion was induced, antibody binding sites appeared as patches on the cell surfaces. Because of these sites, cells were lysed when they were stimulated to secrete in the presence of antibodies to 45-kDa SBP and guinea pig complement. Antibody binding sites disappeared from cell surfaces after 20 min, at which time antibodies to SBP were found inside the cells. It is suggested that 45-kDa SBP is packaged with 5-HT in secretory vesicles. Some 45-kDa SBP is lost during secretion as a result of exocytosis; however, a fraction of the 45-kDa SBP remains bound to the luminal surface of the membrane of secretory vesicles. This protein is exposed to the ambient medium as a consequence of exocytosis, but is reinternalized when the vesicular membrane is recaptured during vesicle recycling.

Notes: Abstract: Several gangliosides, especially GD3 (disialosyllactosyl ceramide) in the presence of another lipid (lecithin) were found to enhance the binding of serotonin to serotonin binding protein (SBP) severalfold. In our conditions, this enhancement was linear to a concentration of 2.7 × 10−6I GD3 and a three- to fivefold increase in binding capacity of SBP was obtained with 8.8 × 10−6 M. The addition of this ganglioside led to an increase of serotonin binding sites, but not to an increase in the affinity of SBP to serotonin. Optimal binding capacity was found with a ratio of lecithin to ganglioside of 6: 1 (w/w). No binding was found in the absence of either SBP or Fe2+ (binding of serotonin to SBP is dependent on Fe2+). Other glycosphingolipids (sulfatide, GD1a, GD1b, GM1) showed lesser effects at low concentration, whereas asialo-GM1, cytolipin H, galactocerebroside and GM3 had insignificant effects. Since earlier studies suggested a storage role for serotonin binding protein, the interaction of gangliosides with this protein may regulate the concentration of the biogenic amine in the synapse.

Notes: Abstract: Serotonin binding protein (SBP) is found in synaptic vesicles of mammalian central and peripheral serotonergic neurons. 5-Hydroxytryptamine (5-HT, serotonin) is physiologically stored as a complex with SBP in vivo. Two forms of SBP have been detected with apparent molecular weights of 45,000 and 56,000 (45K and 56K). To understand the relationship between the two forms more fully, we purified the two proteins to homogeneity and partially characterized them. Purification steps included (NH4)2SO4 fractionation and chromatography on Sepharose 4-B, Affi-Gel-Blue, hydroxylapatite, and phosphocellulose. The 45K form of SBP was obtained pure, whereas the 56K form of SBP was obtained about 90% pure by these methods. To isolate pure 56K SBP for induction of antibodies, the protein was further purified by sodium dodecyl sulfate-gel electrophoresis followed by electroelution. The 56K form of SBP was thus isolated, but in a denatured state; its purity was established by two-dimensional gel electrophoresis. The two forms of SBP (pure 45K and 90% pure undenatured 56K SBP) were similar in their 5-HT binding capacity; the enhancement of 5-HT binding by Fe2+; and inhibition by –SH reagents, chelators, and sodium salts. Antibodies raised against the pure 56K form of SBP cross-reacted with the 45K SBP. The two forms of SBP differed in the following properties: (1) dissociation constants—56K form showed higher affinity for 5-HT (KD1= 0.4 nM; KD2= 32 nM), whereas the 45K form showed lower affinity (KD1= 9.7 nM; KD2= 120 nM); (2) ratio of number of 5-HT binding sites with low affinity to those with high affinity—56K (19:1), 45K (10:1); (3) isoelectric point—the 56K form of SBP is more acidic (5.6 and 5.9) than the 45K form (6.1); (4) binding enhancement by gangliosides and bicarbonate. To establish whether the 45K form of SBP is found in vivo or is produced by proteolysis during isolation, two additional experiments were carried out. (1) We added a mixture of proteolytic enzyme inhibitors to our homogenization buffer; this addition did not change the ratio of the two forms of SBP. (2) We mixed regions of the CNS enriched in the 45K form of SBP (spinal cord) with regions rich in the 56K form of SBP (raphe nuclei) and homogenized them together. Again, this procedure failed to change the ratio of the two forms of SBP as judged by polyacrylamide gel electrophoresis. We suggest that a precursor-product relationship may exist between 45K and 56K SBP and that the two forms may be located within different parts of serotonergic neutrons.

Notes: Abstract: A protein fraction that binds serotonin was obtained from astrocytes in primary cultures. It was found to differ in some of its physical, chemical, and pharmacological properties from neuronal serotonin binding protein.

Notes: Anti-idiotypic antibodies were generated by immunizing rabbits with affinity-purified antibodies to serotonin (5-hydroxytryptamine; 5-HT). Anti-5-HT activity was removed from the resulting antisera by chromatography through a 5-HT affinity column. The anti-idiotypic antibodies were demonstrated by enzyme-linked immunosorbent assay to bind to affinity-purified whole anti-5-HT antibodies and their Fab fragments. Anti-idiotypic antibodies, purified by affinity chromatography on columns to which antibodies to 5-HT were coupled, competed with 5-HT (covalently bound to protein) for the binding sites on anti-5-HT antibodies and serotonin binding protein. The anti-idiotypic antibodies antagonized the binding of [3H]5-HT to membranes isolated from the cerebral cortex, striatum, and raphe area more than to membranes from hippocampus or cerebellum. The anti- idiotypic antibodies also blocked the binding of the 5-HT1B- selective ligand (-)-[125I]iodocyanopindolol (in the presence of 30 μM isoproterenol) to cortical membranes. In contrast, anti-idiotypic antibodies failed to inhibit binding of the 5- HT1A-selective ligand 8-hydroxy-2-(di-n)-[3H]propylamino)- tetralin ([3H]8-OH-DPAT) to raphe area membranes or hippocampal membranes. These observations suggested that the anti-idiotypic antibodies may recognize some 5-HT receptor subtypes but not others. This hypothesis was tested by ascertaining the ability of anti-idiotypic antibodies to immunostain cells transfected in vitro with cDNA encoding the 5- HT1C or 5-HT2 receptor or with a genomic clone encoding the 5-HT1A receptor. Punctate sites of immunofluorescence were found on the surfaces of fibroblasts that expressed 5- HT1C and 5-HT2 receptors, but not on the surfaces of HeLa cells that expressed 5-HT1A receptors. Immunostaining of cells by the anti-idiotypic antibodies was inhibited by appropriate pharmacological agents: immunostaining of cells expressing 5-HT1C receptors was blocked by mesulergine (but not ketanserin, 8-OH-DPAT. or spiperone), whereas that of cells expressing 5-HT2 receptors was blocked by ketanserin or spiperone (but not mesulergine or 8-OH-DPAT). The anti- idiotypic antibodies failed to inhibit the uptake of [3H]5-HT by serotonergic neurons. It is concluded that the anti-idiotypic antibodies generated with anti-5-HT serum recognize the 5- HT1B, 5-HTlC, and 5-HT2 receptor subtypes; however, neither 5-HT1A receptors nor 5-HT uptake sites appear to react with these antibodies.

Notes: Abstract: Serotonin binding protein (SBP) is a vesicular protein found in neurectoderm-derived cells that store 5-hydroxytryptamine (5-HT, serotonin), such as central and peripheral serotonergic neurons and paraneurons (parafollicular cells of the thyroid). 5-HT is stored as a complex with SBP in vivo. Two forms of the protein are found. These differ in molecular mass: one is 45 kDa and the other 56 kDa. It has been suggested that the 56-kDa form of SBP may be the precursor of the 45-kDa form. To study the relationship between these two proteins, we have used a covalently bound radiolabeled probe to analyze their binding domains. A photoaffinity reagent, N-(4-azido-2-nitrophenyl)-5-hydroxytryptamine (NAP-5-HT), was synthesized and characterized by nuclear magnetic resonance spectroscopy, mass spectra, and UV-visible absorption spectra. A 1 M excess of NAP-5-HT inhibited the binding of [3H]5-HT to SBP by 50%. NAP[3H]5-HT was also synthesized and attached to both high- and lowaffinity binding sites on both forms of SBP. The high-affinity binding constants for 45-kDa and 56-kDa proteins were 0.8 nM and 0.02 nM, respectively, whereas the low-affinity constants were 0.3 γM and 0.15 γM. When the high-affinity site of partially purified SBP was photoaffinity-labeled with the reagent, two covalently labeled proteins (45 kDa and 56 kDa) were found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition of the labeling of both proteins by 50% was observed in the presence of a 15-fold molar excess of 5-HT. Drugs and reagents affected to the same degree the binding of [3H]5-HT to SBP (45 kDa and 56 kDa) and the binding of NAP[3H]5-HT to the two proteins. The 45-kDa SBP and 56-kDa SBP covalently labeled with NAP[3H]5-HT were analyzed by partial proteolytic digestion with either Staphylococcus aureus V8 protease or proteinase K. The generated radiolabeled peptides, separated on SDS-PAGE from both forms of the labeled SBP, exhibited a similar pattern, suggesting their close structural similarity. Structure-binding requirements suggest that this probe will be also useful in studying other proteins that bind 5-HT, such as carriers of 5-HT in the plasma membrane and vesicles, as well as some serotonergic receptors (5-HT1p).

Notes: Abstract: The endogenous phosphorylation of serotonin binding protein (SBP), a soluble protein found in central and peripheral serotonergic neurons, inhibits the binding of 5-hydroxytryptamine (5-HT, serotonin). A protein kinase activity that copurifies with SBP (SBP-kinase) was partially characterized and compared with calcium/calmodulin-dependent protein kinase II (CAM-PK II). SBP itself is not the enzyme since heating destroyed the protein kinase activity without affecting the capacity of the protein to bind [3H]5-HT. SBP-kinase and CAM-PK II kinase shared the following characteristics: (1) size of the subunits; (2) autophosphorylation in a Ca2+-dependent manner; and (3) affinity for Ca2+. In addition, both forms of protein kinase phosphorylated microtubule-associated proteins well and did not phosphorylate myosin, phosphorylase b., and casein. Phorbol esters or diacylglycerol had no effect on either of the protein kinases. However, substantial differences between SBP-kinase and CAM-PK II were observed: (1) CAM enhanced CAM-PK II activity, but had no effect on SBP-kinase; (2) synapsin I was an excellent substrate for CAM-PK II, but not for SBP-kinase; (3) 5-HT inhibited both the autophosphorylation of SBP-kinase and the phosphorylation of SBP, but had no effect on CAM-PK II. These data indicate that SBP-kinase is different from CAM-PK II. Phosphopeptide maps of SBP and SBP-kinase generated by digestion with S. aureus V8 protease are consistent with the conclusion that these proteins are distinct molecular entities. It is suggested that phosphorylation of SBP may regulate the transport of 5-HT within neurons.

Notes: Abstract: Rat brain serotonin binding protein (SBP) was found to have essential -S-S and -SH groups. Both reduction of the disulfide bond by dithiothreitol or mercaptoethanol and modification of -SH group(s) by Ellman reagent or alkylating agents caused loss of binding capacity. In contrast, formation of a mixed disulfide bond with sodium metabisulfite did not affect the binding capacity. Serotonin in the presence of Fe2+ and phosphate was found to bind to either an -SH group or to a site in very close proximity. Addition of serotonin protected -SH groups from modification by Ellman reagent and from denaturation of protein upon storage. Lipids that enhance binding of serotonin to SBP also protected -SH groups from modification. Nucleotides were found to be strong inhibitors of the binding of serotonin to SBP. The inhibitory effect of nucleotides was due to their chelating properties and not to their ability to phosphorylate the protein or to bind directly to it. Inhibition by nucleotides and other chelators was reversible. Binding capacity was fully restored after removal of the chelator by molecular sieve chromatography and addition of Fe2+. The ionic environment had a marked effect on the binding: intracellular ions such as K+ were found to enhance the binding, and extracellular ions such as Na+ and Ca2+ inhibited the binding. Based on these data and our previous studies, we suggest that SBP is an intracellular protein that acts as a storage protein. Consistent with our data is formation of a complex of SBP-S-Fe-S that in a hydrophobic surrounding could bind up to four molecules of serotonin in coordination bond with Fe2+ and thereby reduce the osmotic pressure within a storage vesicle. Extracellular ionic conditions that favor the dissociation of the complex would free the amine to interact with its receptor or the presynaptic reuptake carrier.

Notes: To investigate the functional consequences of cross-talk between multiple effectors of serotonin (5-HT) 1A receptor, we employed transfected Chinese hamster ovary cells. Activation of 5-HT1A receptor stimulated extracellular signal-regulated kinase (ERK)1/2, Akt and nuclear transcription factor-κB (NF-κB). Stimulation of cells with 5-HT1A receptor agonist induced a rapid but transient ERK1/2 phosphorylation followed by increased phosphorylation of Akt. Elevated Akt activity in turn suppressed Raf activity and induced a decline in ERK activation. The activation of ERK and Akt downstream of 5-HT1A receptor was sensitive to inhibitors of Ras, Raf and phosphatidylinositol 3-kinase (PI3K). Stimulation of 5-HT1A receptor also resulted in activation of NF-κB through a decrease in inhibitor of nuclear transcription factor-κB. In support of the importance of 5-HT1A receptor signaling for cell survival, inhibition of NF-κB facilitated caspase 3 activation and cleavage of poly (ADP-ribose) polymerase, while treatment of cells with agonist inhibited caspase 3, DNA fragmentation and cell death. Both agonist-dependent NF-κB activation and cell survival were decreased by Akt Inhibitor II or by overexpression of dominant-negative Akt. These findings suggest a role for 5-HT1A receptor signaling in the Ras/Raf-dependent regulation of multiple intracellular signaling pathways that include ERK and PI3K/Akt. Of these, only PI3K/Akt and NF-κB activation were required for 5-HT1A receptor-dependent cell survival, implying that the relative distribution of signals between competing transduction pathways determines the functional outcome of 5-HT1A receptor activation.