Abstract

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 ofSpodoptera 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 [³⁵S]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 Gβ₁γ₂), the coupled phenotype was achieved: agonists bound with high affinity in a guanosine-5′-(β,γ-imido)triphosphate-sensitive manner and stimulated [³⁵S]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 Gβ₁γ₂produced increases in both agonist affinity and maximum G protein activation that were smaller than those in the presence of Gβ₁γ₂, suggesting that Gβ₁γ₂ coexpression improves receptor-G protein coupling. Similarly, coexpression of receptors with Gβ₁γ₂ 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.