The G protein G_i1 exhibits basal coupling but not preassembly with G protein-coupled receptors

The G_i/o protein family transduces signals from a diverse group of G protein-coupled receptors (GPCRs). The observed specificity of G_i/o-GPCR coupling and the high rate of G_i/o signal transduction have been hypothesized to be enabled by the existence of stable associates between G_i/o proteins and their cognate GPCRs in the inactive state (G_i/o-GPCR preassembly). To test this hypothesis, we applied the recently developed technique of two-photon polarization microscopy (2PPM) to Gα_i1 subunits labeled with fluorescent proteins and four GPCRs: the α_2A-adrenergic receptor, GABA_B, cannabinoid receptor type 1 (CB₁R), and dopamine receptor type 2. Our experiments with non-dissociating mutants of fluorescently labeled Gα_i1 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_i1 subunits and α_2A-adrenergic receptor, GABA_B, or dopamine receptor type 2 receptors did not reveal any interaction between the G_i1 protein and the non-stimulated GPCRs. In contrast, non-stimulated CB₁R exhibited an interaction with the G_i1 protein. Further experiments revealed that this interaction is caused solely by CB₁R basal activity; no preassembly between CB₁R and the G_i1 protein could be observed. Our results demonstrate that four diverse GPCRs do not preassemble with non-active G_i1 However, we also show that basal GPCR activity allows interactions between non-stimulated GPCRs and G_i1 (basal coupling). These findings suggest that G_i1 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.