Abstract
The Sec1-Munc18 (SM) proteins are required for cellular exocytosis, but their mechanistic function remains poorly understood. We examined SM-syntaxin complexes in human platelets, which are terminally differentiated, anuclear cells that secrete the contents of their intracellular granules through syntaxin 2- and syntaxin 4-dependent mechanisms. Munc18a, Munc18b, and Munc18c were detected in human platelets by immunoblotting and/or PCR. The SM proteins and syntaxin 2 were found in the membrane and cytosolic fractions of cells, whereas syntaxin 4 was detected only in the membrane. Platelet membranes contain Munc18c-syntaxin 4 complexes, but minimal if any Munc18c-syntaxin 2 complexes were found. No significant amounts of Munc18a or Munc18b complexes were seen with either syntaxin. Munc18c-syntaxin 4 complexes were dissociated when cells were activated to secrete. Two potential inhibitors of Munc18c-syntaxin 4 complexes were generated to examine whether complex dissociation may lead to exocytosis. Peptides that mimic the projected intermolecular contact sites of Munc18c with syntaxin enhanced Ca2+-triggered dense granule exocytosis in permeabilized cells. Similarly, an anti-Munc18c monoclonal antibody that inhibited the Munc18c-syntaxin complex potently amplified Ca2+-induced platelet granule secretion. In summary, Munc18 proteins bind to specific syntaxin isoforms in platelets despite the presence of other potential binding partners. Acute inhibition of the SM-syntaxin complex promotes Ca2+-induced exocytosis, suggesting that complex formation per se has a regulatory effect on triggered secretion.
Highlights
The Sec1-Munc18 (SM)1 proteins are a conserved family of ϳ65-kDa molecules that play a central but still enigmatic role in exocytosis in yeast, neurons, and specialized secretory cells
Our results indicate that SM proteins selectively interact with specific syntaxins in platelets and through these interactions play a critical role in platelet exocytosis
Platelets are well suited to examine the role of SM-syntaxin complex formation on exocytosis, relatively separate from the potential roles SM proteins may play in other vesicle trafficking processes
Summary
The Sec1-Munc18 (SM)1 proteins are a conserved family of ϳ65-kDa molecules that play a central but still enigmatic role in exocytosis in yeast, neurons, and specialized secretory cells (reviewed in Ref. 1). This study used human platelets as a model system to examine the expression of SM proteins, the specificity of their interactions with the syntaxins, and the effect of syntaxin-SM complex formation on exocytosis. Anti-Munc18c monoclonal antibodies (mAbs) were generated that inhibit the SM-syntaxin complex in order to determine the acute effects of this complex on platelet granule secretion.
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