Human 5-Hydroxytryptamine5A Receptors Activate Coexpressed Gi and Go Proteins inSpodoptera frugiperda 9 Cells

The ability of the human 5-hydroxytryptamine serotonin type 5A (h5-ht 5A ) receptor to couple to G proteins from distinct families was investigated through the simultaneous infection of Spodoptera frugiperda 9 insect cells with recombinant baculoviruses encoding the various proteins. Expression of G proteins was demonstrated in immunoblots. Receptor-G protein coupling was monitored by high-affinity agonist binding and agonist-induced stimulation of [ 35 S]guanosine-5′- O -(3-thio) triphosphate binding to membranes. Receptors expressed alone displayed low-affinity agonist binding, and endogenous G proteins were only poorly stimulated on the addition of 5-hydroxytryptamine. When receptors were coexpressed with mammalian G i /G o proteins (Gα i or Gα o plus Gβ 1 γ 2 ), the coupled phenotype was achieved: agonists bound with high affinity in a guanosine-5′-(β,γ-imido)triphosphate-sensitive manner and stimulated [ 35 S]guanosine-5′- O -(3-thio)triphosphate binding to high levels. These effects were not observed on coexpression with G z /G s /G q/11/16 or G 12/13 . Various ligands were evaluated for their agonistic, antagonistic, or inverse agonistic behavior in both receptor binding and activation assays. Although G o displayed different receptor coupling characteristics than G i proteins, no clear coupling preference was evident. Coexpression of receptors and Gα i subunits without Gβ 1 γ 2 produced increases in both agonist affinity and maximum G protein activation that were smaller than those in the presence of Gβ 1 γ 2 , suggesting that Gβ 1 γ 2 coexpression improves receptor-G protein coupling. Similarly, coexpression of receptors with Gβ 1 γ 2 alone resulted in an improved interaction with endogenous G proteins. Our results demonstrate that h5-ht 5A receptors expressed in Spodoptera frugiperda 9 cells selectively and functionally couple to coexpressed mammalian G i and G o proteins.