Vol. 57, Issue 5, 1034-1044, May 2000

Human 5-Hydroxytryptamine_5A Receptors Activate Coexpressed G_i and G_o Proteins in Spodoptera frugiperda 9 Cells

Bart J. B. Francken, Katty Josson, Peter Lijnen, Mirek Jurzak, Walter H. M. L. Luyten, and Josée E. Leysen

Departments of Biochemical Pharmacology (B.J.B.F., K.J., P.L., M.J., J.E.L.) and Functional Genomics (W.H.M.L.L.), Janssen Research Foundation, Beerse, Belgium

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 [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 (G_i or G_o plus G₁₂), the coupled phenotype was achieved: agonists bound with high affinity in a guanosine-5'-(,-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 G_i subunits without G₁₂ produced increases in both agonist affinity and maximum G protein activation that were smaller than those in the presence of G₁₂, suggesting that G₁₂ coexpression improves receptor-G protein coupling. Similarly, coexpression of receptors with G₁₂ 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.