Inhibition of exocytosis by intracellularly applied antibodies against a chromaffin granule-binding protein

Schiweizer, Felix E.; Schäfer, Theo; Tapparelli, Carlo; Grob, Marianne; Karli, Urs O.; Heumann, Rolf; Thoenen, H.; Bookman, Richard J.; Burger, Max M.

doi:10.1038/339709a0

Cited by 49 publications

(11 citation statements)

References 21 publications

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“…In this context it was of interest to check the effect of anti-JAK2 antibody on K Ca channel activity. The effectiveness of using antibodies in patch-clamp experiments was previously shown by Schweizer et al (45). When anti-JAK2 antibody was introduced into the patch pipette in whole cell experiments, the K Ca conductance was almost completely inhibited within an average of 15 min, and PRL was no longer able to stimulate K Ca conductance.…”

Section: Prl Receptor and Kmentioning

confidence: 82%

Role of Tyrosine Phosphorylation in Potassium Channel Activation

Prevarskaya

Skryma

Vacher

et al. 1995

Journal of Biological Chemistry

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Chinese hamster ovary (CHO) cells, stably transfected with the long form of the prolactin (PRL) receptor (PRL-R) cDNA, were used for PRL-R signal transduction studies. Patch-clamp technique in whole cell and cellfree configurations were employed. Exposure of transfected CHO cells to 5 nM PRL led to the increase of Ca 2؉ -and voltage-dependent K ؉ channel (K Ca ) activity. The effect was direct as it was observed also in excised patch experiments. A series of tyrosine kinase inhibitors was studied to investigate the possible involvement of protein tyrosine kinases in K Ca functioning and its stimulation by PRL. Genistein, lavendustin A, and herbimycin A decreased in a concentration and time-dependent manner the amplitude of the K Ca current in whole cell and the open probability of K Ca channels in cell-free experiments. The subsequent application of PRL was ineffective. The protein tyrosine phosphatase inhibitor orthovanadate (1 mM) stimulated K Ca channel activity in excised patches, indicating that channels can be modulated in opposite directions by protein tyrosine kinase and protein tyrosine phosphatase. Moreover, in whole cell experiments as well as in excised patch recordings, anti-JAK2 tyrosine kinase antibody decreased the K Ca conductance and the open probability of the K Ca channels. Subsequent application of PRL was no longer able to stimulate K Ca conductance. Immunoblotting studies using the same anti-JAK2 antibody, revealed the constitutive association of JAK2 kinase with PRL-R. Preincubation of anti-JAK2 antibody with the JAK2 Immunizing Peptide abolished the effects observed using anti-JAK2 antibody alone in both electrophysiological and immunoblotting studies.We conclude from these findings that these K Ca channels are regulated through tyrosine phosphorylation/ dephosphorylation; JAK2 tyrosine kinase, constitutively associated with PRL-R, is implicated in PRL stimulation of K Ca channels.Prolactin (PRL) 1 is a multifunctional pituitary hormone involved in the control of a wide variety of physiological processes in vertebrates, including lactation, reproduction, immune responses, and osmoregulation, as well as cell proliferation (1-3). The PRL receptor (PRL-R) belongs to the cytokine-growth factor receptor superfamily that includes receptors for growth hormone, erythropoietin, numerous hematopoietic interleukins (IL)-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-9, granulocyte colonystimulating factor, granulomacrophage colony-stimulating factor, and ciliary neurotrophic factor (4, 5). This family of receptors possesses common structural motifs, both external (two disulfide loops and the WSXWS homology box) and internal (proline-rich homology box 1). Recent studies have been marked by considerable progress in understanding the mechanisms of intracellular signaling for the different members of this family, particularly for PRL-R. Most of the data were obtained in the PRL-dependent rat T lymphoma cell line Nb2. It has been shown by several groups that, following binding of PRL to the PRL-R in these cells, di...

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Section: Prl Receptor and Kmentioning

confidence: 82%

Role of Tyrosine Phosphorylation in Potassium Channel Activation

Prevarskaya

Skryma

Vacher

et al. 1995

Journal of Biological Chemistry

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“…Antibodies have been widely used as probes of regulated secretion (Alder et al 1992;Ali et al 1989;Elferink et al 1993;Kenigsberg and Trifaro 1985;Mikoshiba et al 1995;Mochida et al 1995;Perrin et al 1987;Pieribone et al 1995;Schweizer et al 1989;Skehel et al 1995;Walent et al 1992). We address three important considerations that are relevant to the design and interpretation of such experiments (also see comments in the preceding cited papers and Augustine et al 1996).…”

Section: Discussionmentioning

confidence: 99%

Antibodies Against Cysteine String Proteins Inhibit Evoked Neurotransmitter Release at Xenopus Neuromuscular Junctions

Poage

Meriney

Gundersen³

et al. 1999

Journal of Neurophysiology

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Cysteine string proteins (CSPs) are evolutionarily conserved proteins that are associated with synaptic vesicles and other regulated secretory organelles. To investigate the role of CSPs in vertebrate neuromuscular transmission, we introduced anti-CSP antibodies into the cell bodies of Xenopus spinal motor neurons that form synapses with embryonic muscle cells in culture. These antibodies produced a rapid (within 3-6 min), and in most cases complete, inhibition of stimulus-dependent neurotransmitter secretion. However, spontaneous neurotransmitter release was stable (both in frequency and amplitude) throughout the period of antibody exposure. Several control experiments validated the specificity of the anti-CSP antibody effects. First, the anti-CSP antibody actions were not mimicked either by antibodies against another synaptic vesicle protein SV2, or by nonspecific immunoglobins. Second, heat treatment of the anti-CSP antibodies eliminated their effect on evoked secretion. Third, immunoblot experiments showed that the anti-CSP and anti-SV2 antibodies were highly selective for their respective antigens in these Xenopus cultures. We conclude from these results that CSPs are vital constituents of the pathway for regulated neurotransmitter release in vertebrates. Moreover, the selective inhibition of evoked, but not spontaneous transmitter release by anti-CSP antibodies indicates that there is a fundamental difference in the machinery that mediates these secretory processes.

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“…However, this issue requires a more detailed investigation to resolve which membrane compartments harbor csp in adrenal. Then, given the relative accessibility of adrenal cells to studies of secretion employing either permeabilized (Knight and Baker 1982;Holz, 1983, Wilson andKirshner 1983) or intact cells (Neher and Marty, 1982;Schweizer et al, 1989), this system may offer useful clues to the function of csps.…”

Section: Csp Immunoreactivity In the Adrenalmentioning

confidence: 99%

Cysteine string protein immunoreactivity in the nervous system and adrenal gland of rat

et al. 1995

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Cysteine string proteins (csps) are a recently discovered class of cysteine-rich proteins. They have been shown to associate preferentially with synaptic vesicle fractions of Torpedo electric organ or rat brain where they have been implicated in events associated with transmitter secretion. However, to date there has been no information concerning the distribution of csps in rat tissues. We investigated the localization of csps in the rat retina and CNS using immunohistochemistry with affinity purified anti-csp antibodies. Specific csp immunoreactivity having a punctate appearance is present throughout the neuraxis. Csp immunoreactivity is particularly abundant in synapse-rich regions including those of the retina, main olfactory bulb, hippocampal formation, and cerebellum. White matter tracts are devoid of csp immunoreactivity. Neuromuscular junctions show strong csp immunoreactivity. This localization of csp immunoreactivity is compatible with a role for csps in presynaptic events at a wide variety of synapses. Immunohistochemical analysis of a non-neuronal, secretory tissue, the adrenal gland, reveals prominent csp immunoreactivity in the chromaffin cells of the adrenal medulla. However, csp immunoreactivity is not detected in adrenal cortical regions. These findings are confirmed and extended by immunoblot and Northern analyses which identify a 35 kDa and a 5 kb product, respectively, in extracts of adrenal. The presence of csps in the adrenal medulla suggests that these proteins may also participate in secretion-related events in certain non-neuronal cells.

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Inhibition of exocytosis by intracellularly applied antibodies against a chromaffin granule-binding protein

Cited by 49 publications

References 21 publications

Role of Tyrosine Phosphorylation in Potassium Channel Activation

Role of Tyrosine Phosphorylation in Potassium Channel Activation

Antibodies Against Cysteine String Proteins Inhibit Evoked Neurotransmitter Release at Xenopus Neuromuscular Junctions

Cysteine string protein immunoreactivity in the nervous system and adrenal gland of rat

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