Woehler Andrew, Wlodarczyk Jakub, Ponimaskin Evgeni G
Department of Neuro and Sensory Physiology, University of Göttingen, Gottingen, Germany.
Glycoconj J. 2009 Aug;26(6):749-56. doi: 10.1007/s10719-008-9187-8. Epub 2008 Oct 14.
In the present study we analyze the oligomerization of the 5-HT1A receptor within living cells at the sub-cellular level. Using a 2-excitation Förster Resonance Energy Transfer (FRET) method combined with spectral microscopy we are able to estimate the efficiency of energy transfer based on donor quenching as well as acceptor sensitization between CFP-and YFP-tagged 5-HT1A receptors at the plasma membrane. Through the analysis of the level of apparent FRET efficiency over the various relative amounts of donor and acceptor, as well as over a range of total surface expressions of the receptor, we verify the specific interaction of these receptors. Furthermore we study the role of acylation in this interaction through measurements of a palmitoylation-deficient 5-HT(1A) receptor mutant. Palmitoylation increases the tendency of a receptor to localize in lipid rich microdomains of the plasma membrane. This increases the effective surface density of the receptor and provides for a higher level of stochastic interaction.
在本研究中，我们在亚细胞水平分析了活细胞内5-HT1A受体的寡聚化。使用结合光谱显微镜的双激发Förster共振能量转移（FRET）方法，我们能够基于供体淬灭以及质膜上CFP和YFP标记的5-HT1A受体之间的受体敏化来估计能量转移效率。通过分析在供体和受体的各种相对量以及受体的一系列总表面表达上的表观FRET效率水平，我们验证了这些受体的特异性相互作用。此外，我们通过测量棕榈酰化缺陷型5-HT(1A)受体突变体来研究酰化在这种相互作用中的作用。棕榈酰化增加了受体定位于质膜富含脂质微区的倾向。这增加了受体的有效表面密度，并提供了更高水平的随机相互作用。
