Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232-6600, USA.
Mol Pharmacol. 2012 Dec;82(6):1136-49. doi: 10.1124/mol.112.080507. Epub 2012 Sep 7.
Spatial and temporal regulation of neurotransmitter release is a complex process accomplished by the exocytotic machinery working in tandem with numerous regulatory proteins. G-protein βγ dimers regulate the core process of exocytosis by interacting with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins soluble N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25), syntaxin 1A, and synaptobrevin. Gβγ binding to ternary SNAREs overlaps with calcium-dependent binding of synaptotagmin, inhibiting synaptotagmin-1 binding and fusion of the synaptic vesicle. To further explore the binding sites of Gβγ on SNAP-25, peptides based on the sequence of SNAP-25 were screened for Gβγ binding. Peptides that bound Gβγ were subjected to alanine scanning mutagenesis to determine their relevance to the Gβγ-SNAP-25 interaction. Peptides from this screen were tested in protein-protein interaction assays for their ability to modulate the interaction of Gβγ with SNAP-25. A peptide from the C terminus, residues 193 to 206, significantly inhibited the interaction. In addition, Ala mutants of SNAP-25 residues from the C terminus of SNAP-25, as well as from the amino-terminal region decreased binding to Gβ₁γ₁. When SNAP-25 with eight residues mutated to alanine was assembled with syntaxin 1A, there was significantly reduced affinity of this mutated t-SNARE for Gβγ, but it still interacted with synaptotagmin-1 in a Ca²⁺ -dependent manner and reconstituted evoked exocytosis in botulinum neurotoxin E-treated neurons. However, the mutant SNAP-25 could no longer support 5-hydroxytryptamine-mediated inhibition of exocytosis.
神经递质释放的时空调节是一个复杂的过程，由协同工作的胞吐机制和众多调节蛋白共同完成。G 蛋白 βγ 二聚体通过与可溶性 N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）蛋白可溶性 N-乙基马来酰亚胺敏感因子附着蛋白-25（SNAP-25）、突触融合蛋白 1A 和突触融合蛋白结合，调节胞吐的核心过程。Gβγ 与三元 SNARE 的结合与钙依赖性突触融合蛋白结合重叠，抑制突触融合蛋白-1 的结合和突触小泡融合。为了进一步探索 Gβγ 在 SNAP-25 上的结合位点，基于 SNAP-25 序列筛选了与 Gβγ 结合的肽段。与 Gβγ 结合的肽段进行丙氨酸扫描突变，以确定其与 Gβγ-SNAP-25 相互作用的相关性。从该筛选中获得的肽段在蛋白质-蛋白质相互作用测定中测试其调节 Gβγ 与 SNAP-25 相互作用的能力。来自 C 端的肽段，残基 193 到 206，显著抑制了相互作用。此外，来自 SNAP-25 的 C 端以及氨基末端区域的 SNAP-25 残基的 Ala 突变体减少了与 Gβ₁γ₁ 的结合。当与 8 个残基突变为丙氨酸的 SNAP-25 组装在一起时，该突变 t-SNARE 与 Gβγ 的亲和力显著降低，但它仍然以 Ca²⁺ 依赖性方式与突触融合蛋白-1 相互作用，并在肉毒神经毒素 E 处理的神经元中重新构建了诱发的胞吐作用。然而，突变的 SNAP-25 不再能够支持 5-羟色胺介导的胞吐抑制。
