Trends in Pharmacological Sciences
OpinionIs Gα16 the optimal tool for fishing ligands of orphan G-protein-coupled receptors?
Section snippets
G proteins
Heterotrimeric (αβγ) guanine nucleotide-binding proteins (G proteins) relay signals from activated seven-transmembrane GPCRs to effector polypeptides such as ion channels or enzymes 9. The α-subunits currently define the different G-protein subtypes and are divided into four major groups (Gαs, Gαi, Gαq and Gα12/13) on the basis of sequence homology 10, 11. The Gαq family comprises five different members (Gαq, Gα11, Gα14, Gα15 and Gα16) that regulate the activity of PLC-β isoforms 12. Gα15 and Gα
Gα16 versus GαΔ6qi4myr
Fig. 2a–c shows ligand activation of a set of Gi-linked GPCRs (CCR5, and dopamine D2 and sst1 receptors) co-transfected with either vector DNA, Gα16 or GαΔ6qi4myr in a microtiter plate-based FLIPR Ca2+ mobilization assay.
It is evident that unlike Gαqi5, GαΔ6qi4myr couples the sst1 receptor to the PLC-β pathway 15. In addition, the chimeric GαΔ6qi4myr is significantly more potent than Gα16 in channeling Gi-linked GPCRs to the release of intracellular Ca2+. These findings agree with recent
Gα16 versus GαΔ6qs5myr
Fig. 2d–f shows ligand activation of selected Gs-coupled receptors (dopamine D1 receptors, and β2- and β1-adrenoceptors) co-transfected with either vector DNA, Gα16 or GαΔ6qs5myr in a FLIPR Ca2+ assay. As expected, and unlike Gαqs5, GαΔ6qs5myr links the β2-adrenoceptor to intracellular Ca2+ mobilization 15. However, Gα16 is clearly superior to GαΔ6qs5myr in the enhancement of the Ca2+ mobilization signal induced by Gs-linked receptors.
These results imply that the obvious solution for a
Gα16 plus GαΔ6qi4myr: does competition for βγ-subunits play a role?
This strategy for the identification of orphan GPCR ligands should raise concern among experts, owing to the competition among Gα-subunits for the βγ-subunit complexes to form αβγ heterotrimers. Formation of αβγ heterotrimers is necessary for a G protein to perceive and transduce signals from activated GPCRs. Given the scenario – that only one Gα-subunit efficiently transduces signals from an activated GPCR – the presence of a second Gα-subunit would only impair signaling caused by partial
Dominant-negative effects of Gα16 on GPCR signaling
The thyrotropin releasing hormone (TRH) receptor might provide an example of dominant-negative effects of Gα16 on GPCR signaling: oocytes, co-injected with the TRH receptor and vector or Gαq cRNA, reveal potent Cl− conduction following stimulation with an agonist 22. Co-injection of TRH receptor and Gα16 cRNA completely abolishes agonist-induced Cl− conduction, which cannot be restored by concomitant co-transfection of Gαq (Ref. 22). The M1 and NK2 receptors stimulate the release of [3
Melanin-concentrating hormone
Melanin-concentrating hormone (MCH) has been reported to be the natural ligand for the orphan receptor SLC-1 (Ref. 23). Using total brain extracts, high-performance liquid chromatography (HPLC) fractions were tested for their ability to mobilize intracellular Ca2+ in CHO cells transfected with the SLC-1 receptor alone or co-transfected with SLC-1 and the Gαqi3 chimera. Subsequently, MCH could be identified as the component responsible for the robust release of intracellular Ca2+ with an EC50 of
Concluding remarks
Gα16 is still the most notable example of a promiscuous G protein, linking a wider range of GPCRs to the PLC-β pathway than any chimeric G-protein α-subunit available to date. However, chimeric G proteins that link Gi-coupled receptors to the PLC-β pathway are clearly more effective than Gα16 (5, 18; Fig. 2a–c); by contrast, Gα16 is clearly superior to G-protein chimeras in coupling to the Gs class of receptors (Fig. 2d–f). Mody et al. 3 have shown that incorporation of Gαz sequences into Gα16
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