2 MATERIALS AND METHODS
3.3 Changes in the GABA B receptor signaling in the developing mouse…
GABAB receptor functionality and selectivity to diverse G proteins subtypes was studied by baclofen stimulated [35S]-GTPγS (Harrison and Traynor 2003) and GTP-Eu binding assay.
The [35S]-GTPγS-binding assay measures the level of the G protein activation, following agonist occupation of a GPCR, by determining the binding of [35S]-GTPγS to the Gα subunits. In general, the assay is experimentally more feasible for receptors coupled to the abundant Gαi/o proteins. Nevertheless, [35S]-GTPγS binding assays are used also with GPCRs that couple to the Gαs and Gαq families of G proteins. For the measurement of the labeled Gα the labeled-Gα subunits are immunoprecipitation with Gα-antibodies which are cross-linked with protein A-Sepharose.
[35S]-GTPγS binding to GABAB receptor was performed in young and adult mouse brainstem homogenates. To evaluate whether receptor activation could increase GTPγS binding, cells were incubated in the presence of a GABAB receptor agonist and receptor antagonist. The following immunoprecipitation was then performed with antibodies against Gαi/o, Gαi3, Gαs, Gαq/11, Gα12 and Gα13 as described under chapter 2.2.6.1. Fused proteins were then isolated on protein A-Sepharose, and bound [35 S]-GTPγS was eluted and quantified by scintillation counting.
The experiments were done in duplicate. The figure 3.7 shows the results as the percentage of mean control values. The Gαi/o subunit showed increase of the bounded [35S]-GTPγS after treatment with baclofen and a decrease after the addition of an antagonist, compared to the control (inactivated GABAB receptor). It is known that GABAB receptors interact with Gαi/o proteins, but in addition was observed an
receptors binding affinity to Gαi3, Gα12 and Gα13 shows no interactions between the receptor and those Gα proteins.
The figure 3.7.B shows the GABAB receptors interaction to Gαs. In this case an unexpected dramatical increase of the immunoprecipitated Gαs in young animals was observed in contrast to the near negligble shift at adult mouse brainstem after activation with baclofen. As the results showed an unexpected difference of GABAB
receptor interaction to the Gαs protein subtypes between the two ages (P0 and adult), additional experiments were necessary.
Further experiments for the GABAB receptor functionality and selectivity to diverse G proteins subtypes were done with a non-radioactive, non-hydrolyzable, europium
Figure 3.7. The diagram shows GABAB receptor agonist-stimulated [35S]-GTPγS binding to specific Gα protein subtypes in brainstem.
Membranes prepared from brainstem of P0 and adult mice were incubated with [35 S]-GTPγS in presence of buffer, 100 μM baclofen and 10 μM CGP 55845A.
Immunoprecipitation was per-formed with appropriate antibodies against different Gα subunits and bounded [35 S]-GTPγS was quantified by
A) Shows, as expected, an increase of the immuno-precipitated Gαi/o at both ages after stimulation with baclofen.
B) Shows an unexpected dramatical increase of the immunoprecipitated Gαs in young animals, which hardly appears at adult mouse brain-stem after activation with baclofen.
labeled GTP analogue, the GTP-Eu. The GTP-Eu binding assay yielded similar values to the traditional [35S]-GTPγS assay (Frang, Mukkala et al. 2003; Labrecque, Anastassov et al. 2005) but with less intensity. We developed Eu-GTP-binding assay with immunoprecipitation in order to avoid problems involved in working with radioactivity and to help us to increase the number of experiments.
The assay was optimized as described under ‘Material and methods’. The young and adult mouse brainstem homogenates were incubated in the presence of a GABAB
receptor agonist and antagonist together with the europium-labeled GTP.
The samples were further incubated with antibodies against Gαi/o, Gαi3, Gαs, Gαq/11, Gα12 and Gα13. The fused proteins were isolated on protein A-Sepharose, and bound Eu-GTP was washed, eluted and quantifie by using the autofluorescent properties of europium (340 nm excitation and 615 nm emission).
Figure 3.8. The diagram shows GABAB receptor agonist-stimulated Eu-GTP binding to specific Gαi/o protein subtypes in brainstem. Membranes prepared from brainstem of P0 and adult mice were incubated with Eu-GTP in presence of buffer, 100 μM baclofen and 10 μM CGP 55845A. Immunoprecipitation was performed with an antibody against Gαi/o subunits and bounded Eu-GTP was quantifie using the autofluorescent properties of Eu (340 nm excitation/615 nm emission) by using the GENios pro plate reader. The experiments were done in fourplicate and the figure shows the means ± standard deviation.
Figure 3.9. The diagram shows GABAB receptor agonist-stimulated Eu-GTP binding to specific Gαs protein subtypes in brainstem. Membranes prepared from brainstem of P0 and adult mice were incubated with Eu-GTP in presence of buffer, 100 μM baclofen and 10 μM CGP 55845A. Immunoprecipitation was performed with an antibody against Gαs subunits and bounded Eu-GTP was quantifie using the autofluorescent properties of Eu (340 nm excitation/615 nm emission) by using the GENios pro plate reader. The experiments were done in fourplicate and the figure shows the means ± standard deviation.
The experiments were done in fourplicate. The figures 3.8, 3.9 and 3.10 show the means ± standard deviation.
The G protein subunits alpha i3, alpha 12 and alpha 13 did not bind appreciable levels of Eu-GTP, at both ages (P0 and adult), consistent with a minimal level of GDP/Eu-GTP exchange. In contrast, the subunits alpha i/o (see figure 3.8) and alpha s (see figure 3.9) bound measurable levels at the age of P0.
In mature brainstems only the Gαi/o shows measurable levels of Eu-GTP, the Gαs
binding in adult brainstems was not different compare to control. Binding to Gαq/11
was weak and not significant.
In summary, the results of the Eu-GTP-binding and [35S]-GTPγS binding assays with immunoprecipitation confirm the coupling of the GABAB receptor to Gαi/o at both ages in brainstem with more robust increases at younger ages. The Gα , Gα and
Gα13 did not show any interaction to GABAB receptor. The Gαq/11 was in both cases not significant (Eu-GTP and [35S]-GTPγS), although there was a weak signal after activation with baclofen. The surprisingly result was the Gαs interaction with the GABAB receptors only in new born mice, something which disappears in adult animals.
3.4 Maturation of the mouse brain is associated with a differential expression