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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: 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: 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: Several analogues of 5-hydroxytryptophan were tested for their ability to inhibit the binding of serotonin to serotonin-binding protein (SBP), a protein found within serotonergic neurons which has a high affinity for serotonin. An N-substituted dipeptide, N-acetyl-5-hydroxytryptophan-5-hydroxytryptophan amide, was found to be an inhibitor of this binding. The inhibition (50% at 1.0 μM) was specific, since it did not affect other known sites of serotonin binding. The binding of serotonin to its membrane receptor was not affected by the dipeptide (up to 10 μM). Uptake of serotonin by synaptosomes was only slightly affected (9% at 10 μM), and aromatic-L-amino-acid carboxy-lyase(EC 4.1.1.28) and amine: oxygen oxidoreductase (deaminating) (flavin-containing) (EC 1.4.3.4) were not inhibited (10 μM and 5 mM respectively), The peptide was not hydrolyzed by honiogenates of brain or myenteric plexus. The 14C-labelled dipeptide was shown to be taken up by synaptosomes. However, the uptake of the peptide was not affected either by drugs that inhibit serotonin uptake or by serotonin itself although the uptake was abolished by excess 5-hydroxytryptophan. Intraventricular injection of N-acetyl dipeptide caused a biphasic effect depending on dose. Lower doses (10nmol) induced a decrease in serotonin brain levels (40%). Higher doses (300 nmol) caused a 95% increase in serotonin levels.It is suggested that 5-hydroxytryptophyl peptides may be used as potent specific inhibitors of SBP, a storage compartment of serotonin.

Notes: We have used the newly introduced method of DeLorenzo & Freedman (1978) for isolating synaptic vesicles to determine if such vesicles contain both serotonin (5-HT) and serotonin binding protein (SBP). Two fractions were obtained. A 55, 000 g fraction was morphologically heterogeneous and contained coated vesicles. A 135, 0000 vesicle (dia. 51.3 nm) fraction was homogeneous in ultra-structure and contained no coated vesicles. The specific activity of SBP in this fraction was much higher than that in the supernatant. Unlike SBP, very little lactic dehydrogenase activity appeared in the 135, 000 g fraction. Qualitative and quantitative differences were observed between the polypeptide profiles of soluble proteins extracted from the vesicles and supernatant proteins on SDS gels. Therefore, entrapment of cytosol in the vesicles of the 135, 000 g fraction was minimal. The 5-HT concentration of the 135, 000 g vesicles was 5.5 ng/mg protein and in the supernatant, 11.3 ng/mg protein. The ATP concentration in the 135, 000 g vesicle fraction was only 0.8 ng/mg Pr. Rabbit spinal cords were transected in order to determine if SBP is moved proximo-distally in axons by rapid axonal transport as would be predicted for a constituent of synaptic vesicles. SBP accumulated above the cut at a rate consistent with fast transport (78 mm/day). SBP activity fell caudal to the point of transection and there was no evidence, such as an accumulation below the lesion, that might indicate retrograde transport of SBP. These experiments indicate that SBP is probably synthesized in the cell bodies of serotonergic neurons and some is rapidly transported down axons to be stored in terminals in vesicles.

Notes: The binding of serotonin to a soluble, high affinity binding protein, present in synaptosomes and associated with serotonergic tracts, has now been studied for the effects of metallic ions and various drugs. At optimal concentration (10-4 M) of Fe2+ the enhancement of binding was close to 20-fold. A much smaller effect was noted with Cu2+. With other ions (Fe3+, Mn2+, Co2+, Ni2+, Cr3+, Mg2+, Ca2+) little or no effect was seen. For the effect with Fe2+. preincubation was required (10 min, 25°C) and concentrations higher than 10-4M were inhibitory. Studies based on equilibrium dialysis show that the effect of Fe2+ was on the affinity of the binding of serotonin to the protein, rather than on the binding capacity. In polydcrylamide gels at pH 8.6 the migratory properties of thc serotonin-protein complex formed in the presence of Fe2+ differ from those of the complex formed without Fe2+. Nucleotides (ATP, GTP, ADP, AMP) inhibited thc binding. The effects of several classes of drugs (inhibitors of biogenic amine storage and uptake, psychotomimetics, MAO) inhibitors and drugs binding to contractile proteins) were also studied. The only effective inhibitors of serotonin binding were reserpine, vinblastine and CZ-74, which caused 50% inhibition at 2 × 10-6 M, 7.5 × 10-6 M and 0.2 × 10-6M respectively.

Notes: Mice lacking dopamine D2 receptors exhibit a significantly decreased agonist-promoted forebrain neocortical D1 receptor activation that occurs without changes in D1 receptor expression levels. This raises the possibility that, in brains of D2 mutants, a substantial portion of D1 receptors are uncoupled from their G protein, a phenomenon known as receptor desensitization. To test this, we examined D1-agonist-stimulated [35S]GTPγS binding (in the presence and absence of protein phosphatase inhibitors) and cAMP production (in the presence and absence of pertussis toxin) in forebrain neocortical tissues of wild-type mice and D2-receptor mutants. These studies revealed a decreased agonist-stimulated G-protein activation in D2 mutants. Moreover, whereas protein phosphatase 1/2A (PP1/2A) and 2B (PP2B) inhibitors decrease [35S]GTPγS binding in a concentration-dependent manner in wild type, they have either no (PP2B) or only partial (PP1/2A) effects in D2 mutants. Furthermore, for D2 mutants, immunoprecipitation experiments revealed increased basal andD1-agonist-stimulated phosphorylation of D1-receptor proteins at serine residues. Finally, D1 immunoprecipitates of both wild type and D2 mutants also contain protein kinase A (PKA) and PP2B immunoreactivities. In D2 mutants, however, the catalytic activity of the immunoprecipitated PP2B is abolished. These data indicate that neocortical D1 receptors are physically linked to PKA and PP2B and that the increased phosphorylation of D1 receptors in brains of D2 mutants is due to defective dephosphorylation of the receptor rather than increasedkinase-mediated phosphorylation.

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: 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.