Use of scintillation proximity assay (SPA) for the characterization of the specific G-protein subtypes stimulated by different GPCRs in postmortem human brain tissue

P Miranda-Azpiazu, R Diez-Alarcia, LF Callado, JJ Meana. University of the Basque Country (UPV/EHU), Department of Pharmacology and Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM). Leioa, Bizkaia 48940, Spain

[35S]GTPγS binding stimulation assays have been one of the most used experimental approaches in the study of G-protein coupled receptors (GPCRs) functionality. However, this technique has some limitations as the fact that it is only able to detect the activation of inhibitory G-proteins. A new experimental approach coupling [35S]GTPγS binding stimulation scintillation proximity assays (SPA) with immunoprecipitation has been developed, allowing the study of the different G-protein subunit subtypes implicated in GPCRs signalling processes.

The aim of the present study was to adapt this technology to the study of the activation of Gαi1-, Gαi2-, Gαi3-, Gαo-, Gαq/11-, Gαs-, and Gαz-protein subunits in postmortem human prefrontal cortex samples. CB1 and mGlu2, and 5HT2A receptors were chosen as models of Gαi/o- and Gαq/11-protein mediated signalling, respectively.

Stimulation data for each drug (at 10 µM) are shown as percentage over the basal binding value (BB) (considered as 100%) obtained for each Gα-protein subunit (mean±SEM of 3 to 7 independent experiments carried out in triplicate). Data analysis was carried out using GraphPad Prism® software and a two-tailed one sample Student´s t-test.

The CB1 receptor agonist WIN55,212-2 showed a statistically significant stimulation of [35S]GTPγS binding mediated by Gαi1 (137±9%, p=0.006), Gαi2 (133±8%, p=0.001), Gαi3 (196±19%, p=0.001) and Gαo (142±10%, p=0.001), while no stimulation was found for Gαq/11, Gαs or Gαz subunits. This stimulation was always reverted to BB by co-incubation with the CB1 receptor antagonist O-2050. When SR141716 (rimonabant) was used, no stimulation was observed for any subunit, while a statistically significant reduction of BB was found for Gαi3 (70±8%, p=0.005), Gαo (83±3%, p<0.001) and Gαz (89±2%, p<0.001) subtypes. This effect was not blocked by O-2050. These results demonstrate that SR141716 has CB1 receptor-independent inverse agonist properties.

The mGlu2/3 receptor agonist LY379268 stimulated all the Gα-protein subtypes (Gαi1: 125±4%, p=0.001; Gαi2: 125±5%, p=0.004; Gαi3: 127±4%, p<0.001; Gαo: 126±4%, p=0.001; Gαq/11: 121±6%, p=0.021; Gαs: 122±6%, p=0.026; and Gαz: 130±4% p<0.001). This effect was always reverted to BB by the mGlu2/3 receptor antagonist LY341495. SPA experiments carried out in mGlu2R knockout mice confirmed that the stimulation seen with LY379268 for human brain tissue is almost completely mGlu2 receptor-mediated.

The 5HT2A/C receptor agonist DOI triggered slight stimulations for Gαq/11 (115±2%, p<0.001) and for the Gαi/o-protein family subunits (Gαi1: 107±2%, p=0.005; Gαi2: 107±1%, p=0.007; Gαi3: 108±2%, p=0.003; and Gαo: 109±3%, p=0.005) while no stimulation was found for Gαs or Gαz subtypes. DOI-induced stimulations were decreased by co-incubation with the 5HT2A receptor antagonist ketanserin.

These findings demonstrate the usefulness of SPA assays in the study of GPCR signalling in postmortem human brain tissue. For instance, the characterization of the specific Gα-protein subunit subtypes involved or the detection of inverse agonist properties of different drugs.