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

Rauly-Lestienne I, Lestienne F, Ailhaud MC, Binesse J, Newman-Tancredi A, Cussac D.
Cell Signal. 2010 Aug 17. [Epub ahead of print]

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 Galpha subunits in Chinese hamster ovary (CHO) cells expressing human (h)5-hydroxytryptamine1A receptors (CHO-h5-HT1A). siRNAs against Galpha-i2 and Galpha-i3 transfected alone or in combination caused a large decrease in the corresponding mRNA level (64-80%) and also at the protein level for Galpha-i3 (60-70%), whereas a non-specific siRNA showed no effect. In membranes of CHO-h5-HT1A, 5-HT stimulated guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding was differentially affected by transfection of siRNAs against Galpha-i protein, siRNAs against Galpha-i2 inducing a more important decrease in the efficacy of 5-HT than transfection of siRNAs against Galpha-i3. The high potency component was abolished after transfection of siRNAs against Galpha-i3 and the lower potency component was suppressed after transfection of siRNAs against Galpha-i2. To directly investigate Galpha-i3 activation we used an antibody-capture/scintillation proximity assay. (+)8-OH-DPAT yielded bell-shaped curves for Galpha-i3 activation, a response that was abolished after transfection of siRNAs against Galpha-i3 protein. Interestingly, (+)8-OH-DPAT yielded a sigmoidal response when only Galpha-i3 protein was expressed. These data suggest that when efficacious agonists attain a high level of occupation of h5-HT1A receptors, a change occurs that induces coupling to Galpha-i2 protein and suppresses signalling through Galpha-i3 subunits.