GTPgammaS Binding Stimulation Research Articles

5-Hydroxytryptamine1A and 5-hydroxytryptamine1B receptors stimulate [35S]guanosine-5'-O-(3-thio)triphosphate binding to rodent brain sections as visualized by in vitro autoradiography.

[35S]Guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding to G proteins was measured by in vitro autoradiography in guinea pig and rat brain sections after activation by 5-hydroxytryptamine (5-HT) receptor agonists. 5-Carboxamidotryptamine stimulated binding strongly in hippocampus and lateral septum and weakly in substantia nigra. This effect was blocked in the substantia nigra by the 5-HT1B/1D receptor antagonist GR-127,935 and in the former two regions by the 5-HT1A antagonist NAN-190. 5-HT1B/1D receptor agonists stimulated binding in substantia nigra and in areas containing 5-HT1A receptors. In guinea pig substantia nigra, 5-(nonyloxy)-tryptamine maximally stimulated [35S]GTPgammaS binding by 54%, with an EC50 value of 62 nM; at 100 microM, this agonist increased binding by approximately 200% in hippocampus (with a 2-fold weaker EC50 value). The distribution of [3H]8-OH-DPAT binding sites was identical to that of the [35S]GTPgammaS labeling stimulated by the 5-HT1A agonist (R)-8-hydroxy-2-dipropylaminotetralin [(R)-8-OH-DPAT)]. (R)-8-OH-DPAT, (S)-8-OH-DPAT, and buspirone stimulated [35S]GTPgammaS binding in hippocampus by 340%, 140%, and 78%, with EC50 values of 71, 51, and 132 nM. Enhanced [35S]GTPgammaS binding was not detected in the presence of 5-HT1F, 5-HT2, 5-HT4, and 5-HT7 receptor agonists. Because activation of mu-opioid, muscarinic M2, histamine H3, and cannabinoid receptors was also visualized successfully, these data suggest that only receptors coupled to pertussis toxin-sensitive G proteins can be seen by [35S]GTPgammaS binding autoradiography. This study also shows that different 5-HT receptors coupled to these proteins can show a wide range of [35S]GTPgammaS binding stimulation. Although the functional significance of these variations is unclear, this technique offers advantages over receptor autoradiography because it does not require high affinity radioligands and provides a measure of agonist efficacies in various brain regions.

Expression of human M2 muscarinic receptors in Sf9 cells: characterisation and reconstitution with G-proteins.

The gene for the human m2 muscarinic receptor was expressed in Sf9 cells using the baculovirus expression system. As assessed by [3H]NMS binding, Sf9 cells expressed receptor at levels of 3.3 pmoles/mg protein. The receptor was identified on western blots using an anti-muscarinic receptor antibody and was shown to have the pharmacological characteristics of an m2 muscarinic receptor. Membranes from Sf9 cells were examined to identify endogenous G-proteins by immuno-blotting and by ADP-ribosylation, indicating the presence of Gq, and a pertussis-toxin substrate which was not recognised by antibodies raised against the alpha-subunits of Gi1, Gi2, Gi3 or Go. Gsalpha was not detected, neither were there any cholera toxin substrates in Sf9 membranes. Sf9 membranes expressing m2 receptors did not show carbachol-stimulated GTPgammaS binding to endogenous G-proteins; however, when membranes were reconstituted with a mixture of purified Gi and Go, a maximum 8-fold stimulation of GTPgammaS binding was observed in response to carbachol that could be reduced by atropine. These data show that the human muscarinic m2 receptor expressed in Sf9 cells is functional.