Bondar Alexey, Lazar Josef
From the Center for Nanobiology and Structural Biology, Institute of Microbiology, Academy of Sciences of the Czech Republic, 37333 Nove Hrady,
From the Center for Nanobiology and Structural Biology, Institute of Microbiology, Academy of Sciences of the Czech Republic, 37333 Nove Hrady.
J Biol Chem. 2017 Jun 9;292(23):9690-9698. doi: 10.1074/jbc.M116.768127. Epub 2017 Apr 24.
The G protein family transduces signals from a diverse group of G protein-coupled receptors (GPCRs). The observed specificity of G-GPCR coupling and the high rate of G signal transduction have been hypothesized to be enabled by the existence of stable associates between G proteins and their cognate GPCRs in the inactive state (G-GPCR preassembly). To test this hypothesis, we applied the recently developed technique of two-photon polarization microscopy (2PPM) to Gα subunits labeled with fluorescent proteins and four GPCRs: the α-adrenergic receptor, GABA, cannabinoid receptor type 1 (CBR), and dopamine receptor type 2. Our experiments with non-dissociating mutants of fluorescently labeled Gα subunits (exhibiting impaired dissociation from activated GPCRs) showed that 2PPM is capable of detecting GPCR-G protein interactions. 2PPM experiments with non-mutated fluorescently labeled Gα subunits and α-adrenergic receptor, GABA, or dopamine receptor type 2 receptors did not reveal any interaction between the G protein and the non-stimulated GPCRs. In contrast, non-stimulated CBR exhibited an interaction with the G protein. Further experiments revealed that this interaction is caused solely by CBR basal activity; no preassembly between CBR and the G protein could be observed. Our results demonstrate that four diverse GPCRs do not preassemble with non-active G However, we also show that basal GPCR activity allows interactions between non-stimulated GPCRs and G (basal coupling). These findings suggest that G interacts only with active GPCRs and that the well known high speed of GPCR signal transduction does not require preassembly between G proteins and GPCRs.
G蛋白家族可转导来自多种G蛋白偶联受体（GPCR）的信号。据推测，G蛋白与GPCR在非活性状态下存在稳定的缔合物（G-GPCR预组装），这使得G-GPCR偶联具有特异性，且G信号转导速率较高。为验证这一假设，我们将最近开发的双光子偏振显微镜技术（2PPM）应用于标记有荧光蛋白的Gα亚基和四种GPCR：α-肾上腺素能受体、GABA、1型大麻素受体（CBR）和2型多巴胺受体。我们对荧光标记的Gα亚基非解离突变体（与活化的GPCR解离受损）进行的实验表明，2PPM能够检测GPCR与G蛋白的相互作用。对未突变的荧光标记Gα亚基与α-肾上腺素能受体、GABA或2型多巴胺受体进行的2PPM实验未发现G蛋白与未受刺激的GPCR之间存在任何相互作用。相比之下，未受刺激的CBR与G蛋白存在相互作用。进一步的实验表明，这种相互作用完全是由CBR的基础活性引起的；未观察到CBR与G蛋白之间的预组装。我们的结果表明，四种不同的GPCR不会与非活性G蛋白预组装。然而，我们还表明，GPCR的基础活性允许未受刺激的GPCR与G蛋白之间发生相互作用（基础偶联）。这些发现表明，G蛋白仅与活性GPCR相互作用，且众所周知的GPCR信号转导的高速性并不需要G蛋白与GPCR之间的预组装。
