Competitive interaction of 5-HT1A receptors with G-protein subtypes in CHO cells demonstrated by RNA interference

Abstract Following agonist action, G-protein-coupled receptors may exhibit differential coupling to G-proteins or second messenger pathways, supporting the notion of agonist-directed trafficking. To explore these mechanisms, we have designed and transfected synthetic siRNA duplexes to knockdown different G α subunits in Chinese hamster ovary (CHO) cells expressing human (h)5-hydroxytryptamine 1A receptors (CHO-h5-HT 1A ). siRNAs against G αi2 and G αi3 transfected alone or in combination caused a large decrease in the corresponding mRNA level (64–80%) and also at the protein level for G αi3 (60–70%), whereas a non-specific siRNA showed no effect. In membranes of CHO-h5-HT 1A , 5-HT stimulated guanosine-5′-O-(3-[ 35 S]thio)-triphosphate ([ 35 S]GTPγS) binding was differentially affected by transfection of siRNAs against G αi protein, siRNAs against G αi2 inducing a more important decrease in the efficacy of 5-HT than transfection of siRNAs against G αi3 . The high potency component was abolished after transfection of siRNAs against G αi3 and the lower potency component was suppressed after transfection of siRNAs against G αi2 . To directly investigate G αi3 activation we used an antibody-capture/scintillation proximity assay. (+)8-OH-DPAT yielded bell-shaped curves for G αi3 activation, a response that was abolished after transfection of siRNAs against G αi3 protein. Interestingly, (+)8-OH-DPAT yielded a sigmoidal response when only G αi3 protein was expressed. These data suggest that when efficacious agonists attain a high level of occupation of h5-HT 1A receptors, a change occurs that induces coupling to G αi2 protein and suppresses signalling through G αi3 subunits.