Dey Sandeepa, Zhan Senmiao, Matsunami Hiroaki
Department of Molecular Genetics and Molecular Biology, Duke University, USA.
J Vis Exp. 2011 Feb 23(48):2405. doi: 10.3791/2405.
The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors (GPCRs) that are postulated to bind volatile odorant molecules and converting the extracellular signal into an intracellular signal by coupling with G protein Gαolf. Binding of the odorants to the receptors is thought to follow a combinatorial rule, that is, one odorant may bind several receptors and one receptor may bind several odorants to varying degrees. Biochemical, signaling and ligand binding studies have been conveniently carried out for most GPCRs using heterologous cells. However use of heterologous cells for study of odorant receptors, was precluded for a long time since on transfection they failed to export to the surface. Saito et al have demonstrated single membrane pass Receptor Transporting Protein (RTP) family chaperones show enhanced expression in the olfactory sensory neurons and act as chaperones to traffic odorant receptors to the surface in heterologous cells, when co transfected together. To carry out biochemical assays for receptors using heterologous cells, one must first determine if the receptor shows robust surface expression in the cell line. This can be assayed by overexpressing the receptors with the chaperone RTP1S followed by live cell staining to fluorescently label the extracellular domain or a tag in the extracellular domain exclusively. Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.
气味的生动世界是由嗅觉感知的。小鼠的嗅觉由大约1200种G蛋白偶联受体（GPCR）介导，这些受体被假定与挥发性气味分子结合，并通过与G蛋白Gαolf偶联将细胞外信号转化为细胞内信号。气味分子与受体的结合被认为遵循组合规则，即一种气味分子可能与几种受体结合，而一种受体可能在不同程度上与几种气味分子结合。对于大多数GPCR，已经使用异源细胞方便地进行了生化、信号传导和配体结合研究。然而，长期以来，异源细胞一直无法用于研究气味受体，因为转染后它们无法转运到细胞表面。Saito等人证明，单跨膜受体转运蛋白（RTP）家族伴侣在嗅觉感觉神经元中表达增强，当与它们共转染时，可作为伴侣将气味受体转运到异源细胞的表面。为了使用异源细胞对受体进行生化分析，首先必须确定受体在细胞系中是否有强大的表面表达。这可以通过用伴侣RTP1S过表达受体，然后进行活细胞染色以荧光标记细胞外结构域或仅标记细胞外结构域中的标签来进行检测。在这里，我们展示了一种进行活细胞染色的方案，可方便地用于检测HEK293T细胞表面的气味受体。此外，它还可用于检测其他化学感应受体或GPCR的表面表达。
