Competitive interaction of 5-HT(1A) receptors with G-protein subtypes in CHO cells demonstrated by RNA interference.
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.
Rauly-Lestienne
Isabelle
I
Department of Cellular and Molecular Biology, Centre de Recherche Pierre Fabre, 17 avenue Jean Moulin, Castres Cedex, France. isabelle.rauly.lestienne@pierre-fabre.com
Lestienne
Fabrice
F
Ailhaud
Marie-Christine
MC
Binesse
Johan
J
Newman-Tancredi
Adrian
A
Cussac
Didier
D
D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D051960:GTP-Binding Protein alpha Subunit, Gi2 / Q000235:genetics / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D044282:Receptor, Serotonin, 5-HT1A / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D044282:Receptor, Serotonin, 5-HT1A; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C498819:GNAI3 protein, human; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go