Munc18-syntaxin complexes and exocytosis in human platelets.
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.
Houng
Aiilyan
A
Cardiovascular Biology Laboratory, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Polgar
Janos
J
Reed
Guy L
GL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001792:Blood Platelets / Q000378:metabolism*; D017931:DNA Primers; D005089:Exocytosis; D005260:Female; D006801:Humans; D008565:Membrane Proteins / Q000097:blood*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins / Q000378:metabolism*; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D033921:Vesicular Transport Proteins
D017931:DNA Primers; D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins; C497937:STXBP1 protein, human; C497940:STXBP2 protein, human; C497941:Stxbp2 protein, mouse; C497943:Stxbp3 protein, mouse; D033921:Vesicular Transport Proteins