Label-Free Detection of G Protein鈥揝NARE Interactions and Screening for Small Molecule Modulators

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• 作者：Christopher A. Wells ; Katherine M. Betke ; Craig W. Lindsley ; Heidi E. Hamm
• 刊名：ACS Chemical Neuroscience
• 出版年：2012
• 出版时间：January 18, 2012
• 年：2012
• 卷：3
• 期：1
• 页码：69-78
• 全文大小：511K
• 年卷期：v.3,no.1(January 18, 2012)
• ISSN：1948-7193

文摘

G_i/o-coupled presynaptic GPCRs are major targets in neuropsychiatric diseases. For example, presynaptic auto- or heteroreceptors include the D₂ dopamine receptor, H₃ histamine receptor, 5HT₁ serotonin receptors, M₄ acetylcholine receptors, GABA_B receptors, Class II and III metabotropic glutamate receptors, opioid receptors, as well as many other receptors. These GPCRs exert their influence by decreasing exocytosis of synaptic vesicles. One mechanism by which they act is through direct interaction of the G尾纬 subunit with members of the SNARE complex downstream of voltage-dependent calcium channels, and specifically with the C-terminus of SNAP25 and the H3 domain of syntaxin1A. (Gerachshenko, T., Blackmer, T., Yoon, E. J., Bartleson, C., Hamm, H. E., and Alford, S. (2005) G尾纬 acts at the C terminus of SNAP-25 to mediate presynaptic inhibition, Nat. Neurosci.8, 597鈥?05; Yoon, E. J., Gerachshenko, T., Spiegelberg, B. D., Alford, S., and Hamm, H. E. (2007) G尾纬 interferes with Ca2+-dependent binding of synaptotagmin to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, Mol. Pharmacol.72, 1210鈥?219; Blackmer, T., Larsen, E. C., Bartleson, C., Kowalchyk, J. A., Yoon, E. J., Preininger, A. M., Alford, S., Hamm, H. E., and Martin, T. F. (2005) G protein 尾纬 directly regulates SNARE protein fusion machinery for secretory granule exocytosis, Nat. Neurosci.8, 421鈥?25).(1-3) Small molecule inhibitors of the G尾纬鈥揝NARE interaction would allow the study of the relative importance of this mechanism in more detail. We have utilized novel, label-free technology to detect this protein鈥損rotein interaction and screen for several small molecule compounds that perturb the interaction, demonstrating the viability of this approach. Interestingly, the screen also produced enhancers of the G尾纬鈥揝NARE interaction.

Keywords:

Heterotrimeric G protein; protein鈭抪rotein interactions; label-free detection; high-throughput screening; SNARE proteins