scholarly journals PI(4,5)P2-dependent regulation of exocytosis by amisyn, the vertebrate-specific competitor of synaptobrevin 2

2020 ◽  
Vol 117 (24) ◽  
pp. 13468-13479
Author(s):  
Ilona Kondratiuk ◽  
Shrutee Jakhanwal ◽  
Jialin Jin ◽  
Udhayabhaskar Sathyanarayanan ◽  
Benjamin Kroppen ◽  
...  
Keyword(s):  
Specific Protein ◽  
Neurosecretory Cells ◽  
Snare Complex ◽  
Pleckstrin Homology Domain ◽  
Secretory Vesicles ◽  
Pleckstrin Homology ◽  
Inhibitory Function ◽  
Snare Motif ◽  
Key Aspects ◽  
Functional Analyses

The functions of nervous and neuroendocrine systems rely on fast and tightly regulated release of neurotransmitters stored in secretory vesicles through SNARE-mediated exocytosis. Few proteins, including tomosyn (STXBP5) and amisyn (STXBP6), were proposed to negatively regulate exocytosis. Little is known about amisyn, a 24-kDa brain-enriched protein with a SNARE motif. We report here that full-length amisyn forms a stable SNARE complex with syntaxin-1 and SNAP-25 through its C-terminal SNARE motif and competes with synaptobrevin-2/VAMP2 for the SNARE-complex assembly. Furthermore, amisyn contains an N-terminal pleckstrin homology domain that mediates its transient association with the plasma membrane of neurosecretory cells by binding to phospholipid PI(4,5)P2. However, unlike synaptrobrevin-2, the SNARE motif of amisyn is not sufficient to account for the role of amisyn in exocytosis: Both the pleckstrin homology domain and the SNARE motif are needed for its inhibitory function. Mechanistically, amisyn interferes with the priming of secretory vesicles and the sizes of releasable vesicle pools, but not vesicle fusion properties. Our biochemical and functional analyses of this vertebrate-specific protein unveil key aspects of negative regulation of exocytosis.

scholarly journals The Munc18-1 domain 3a hinge-loop controls syntaxin-1A nanodomain assembly and engagement with the SNARE complex during secretory vesicle priming

2016 ◽  
Vol 214 (7) ◽  
pp. 847-858 ◽  
Author(s):  
Ravikiran Kasula ◽  
Ye Jin Chai ◽  
Adekunle T. Bademosi ◽  
Callista B. Harper ◽  
Rachel S. Gormal ◽  
...  
Keyword(s):  
Single Molecule ◽  
Botulinum Neurotoxin ◽  
Secretory Vesicle ◽  
Neurosecretory Cells ◽  
Snare Complex ◽  
Secretory Vesicles ◽  
Deficient Mutant ◽  
Syntaxin 1A ◽  
Single Molecule Analysis ◽  
Snare Complex Formation

Munc18-1 and syntaxin-1A control SNARE-dependent neuroexocytosis and are organized in nanodomains on the plasma membrane of neurons and neurosecretory cells. Deciphering the intra- and intermolecular steps via which they prepare secretory vesicles (SVs) for fusion is key to understanding neuronal and hormonal communication. Here, we demonstrate that expression of a priming-deficient mutant lacking 17 residues of the domain 3a hinge-loop (Munc18-1Δ317-333) in PC12 cells engineered to knockdown Munc18-1/2 markedly prolonged SV docking. Single-molecule analysis revealed nonhomogeneous diffusion of Munc18-1 and syntaxin-1A in and out of partially overlapping nanodomains. Whereas Munc18-1WT mobility increased in response to stimulation, syntaxin-1A became less mobile. These Munc18-1 and syntaxin-1A diffusional switches were blocked by the expression of Munc18-1Δ317-333, suggesting that a conformational change in the Munc18-1 hinge-loop controls syntaxin-1A and subsequent SNARE complex assembly. Accordingly, syntaxin-1A confinement was prevented by expression of botulinum neurotoxin type E. The Munc18-1 domain 3a hinge-loop therefore controls syntaxin-1A engagement into SNARE complex formation during priming.

scholarly journals GαqDirectly Activates p63RhoGEF and Trio via a Conserved Extension of the Dbl Homology-associated Pleckstrin Homology Domain

2007 ◽  
Vol 282 (40) ◽  
pp. 29201-29210 ◽  
Author(s):  
Rafael J. Rojas ◽  
Marielle E. Yohe ◽  
Svetlana Gershburg ◽  
Takeharu Kawano ◽  
Tohru Kozasa ◽  
...  
Keyword(s):  
Pleckstrin Homology Domain ◽  
Pleckstrin Homology

Structural Characterization of the Interaction between a Pleckstrin Homology Domain and Phosphatidylinositol 4,5-Bisphosphate

Biochemistry ◽  
1995 ◽  
Vol 34 (31) ◽  
pp. 9859-9864 ◽  
Author(s):  
John E. Harlan ◽  
Ho Sup Yoon ◽  
Philip J. Hajduk ◽  
Stephen W. Fesik
Keyword(s):  
Structural Characterization ◽  
Pleckstrin Homology Domain ◽  
Pleckstrin Homology

scholarly journals Critical Role for Kalirin in Nerve Growth Factor Signaling through TrkA

2005 ◽  
Vol 25 (12) ◽  
pp. 5106-5118 ◽  
Author(s):  
Kausik Chakrabarti ◽  
Rong Lin ◽  
Noraisha I. Schiller ◽  
Yanping Wang ◽  
David Koubi ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Pc12 Cells ◽  
Critical Role ◽  
Neuronal Morphology ◽  
Neurotrophin Receptor ◽  
General Mechanism ◽  
Pleckstrin Homology Domain ◽  
Pleckstrin Homology ◽  
Nerve Growth

ABSTRACT Kalirin is a multidomain guanine nucleotide exchange factor (GEF) that activates Rho proteins, inducing cytoskeletal rearrangement in neurons. Although much is known about the effects of Kalirin on Rho GTPases and neuronal morphology, little is known about the association of Kalirin with the receptor/signaling systems that affect neuronal morphology. Our experiments demonstrate that Kalirin binds to and colocalizes with the TrkA neurotrophin receptor in neurons. In PC12 cells, inhibition of Kalirin expression using antisense RNA decreased nerve growth factor (NGF)-induced TrkA autophosphorylation and process extension. Kalirin overexpression potentiated neurotrophin-stimulated TrkA autophosphorylation and neurite outgrowth in PC12 cells at a low concentration of NGF. Furthermore, elevated Kalirin expression resulted in catalytic activation of TrkA, as demonstrated by in vitro kinase assays and increased NGF-stimulated cellular activation of Rac, Mek, and CREB. Domain mapping demonstrated that the N-terminal Kalirin pleckstrin homology domain mediates the interaction with TrkA. The effects of Kalirin on TrkA provide a molecular basis for the requirement of Kalirin in process extension from PC12 cells and for previously observed effects on axonal extension and dendritic maintenance. The interaction of TrkA with the pleckstrin homology domain of Kalirin may be one example of a general mechanism whereby receptor/Rho GEF pairings play an important role in receptor tyrosine kinase activation and signal transduction.

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