Tran Elizabeth, Fang Ye
Biochemical Technologies, Science and Technology Division, Corning Incorporated, Sullivan Park, Corning, New York, USA.
J Recept Signal Transduct Res. 2009;29(3-4):154-62. doi: 10.1080/10799890903052544.
Adenylyl cyclase is considered as an integrator for receptor signaling. However, its integrative role in receptor signaling is largely studied at the level of point of contacts in complex pathways. Here we used forskolin as a pharmacological probe and the resonant waveguide grating (RWG) biosensor to examine the signal integration of G protein-coupled receptors (GPCRs) at the cyclase-cyclic AMP-PKA module. The biosensor is a refractive index sensitive optical biosensor that is capable of detecting ligand-induced dynamic mass redistribution in cells without labels and cellular manipulations. Stimulation of seven cell lines with forskolin led to distinct optical responses, indicative of distinct expressions and/or organization of cyclase isoforms. The forskolin response in A431 was sensitive to the activities of protein kinase A, Rho kinase, and MAP kinases. Desensitization assays showed that the forskolin pretreatment heterologously desensitized G(s) signaling, partially attenuated G(q) signaling, but had complicate impacts on G(i) signaling. This study documents the integrative role of adenylyl cyclase in GPCR signaling and the power of forskolin as a pharmacological probe to differentiate receptor signaling using the label-free biosensor cellular assays.
腺苷酸环化酶被认为是受体信号的整合器。然而，其在受体信号中的整合作用主要是在复杂信号通路的接触点层面进行研究的。在此，我们使用福斯可林作为药理学探针，并利用共振波导光栅（RWG）生物传感器来检测G蛋白偶联受体（GPCRs）在环化酶 - 环磷酸腺苷 - 蛋白激酶A模块中的信号整合。该生物传感器是一种对折射率敏感的光学生物传感器，能够在不进行标记和细胞操作的情况下检测配体诱导的细胞内动态质量再分布。用福斯可林刺激七种细胞系会导致不同的光学响应，这表明环化酶同工型的表达和/或组织存在差异。A431细胞系中福斯可林的响应对蛋白激酶A、Rho激酶和丝裂原活化蛋白激酶的活性敏感。脱敏试验表明，福斯可林预处理可使G(s)信号发生异源脱敏，部分减弱G(q)信号，但对G(i)信号有复杂的影响。本研究证明了腺苷酸环化酶在GPCR信号中的整合作用，以及福斯可林作为一种药理学探针，利用无标记生物传感器细胞检测来区分受体信号的能力。
