Competitive interaction of 5-HT1A 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-[³⁵S]thio)-triphosphate ([³⁵S]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.     

Dipeptide Tyr-Leu (YL) exhibits anxiolytic-like activity after oral administration via activating serotonin 5-HT1A, dopamine D1 and GABAA receptors in mice

We found that Tyr-Leu (YL) dose-dependently exhibits potent anxiolytic-like activity (0.1-1 mg/kg, i.p.) comparable to diazepam in the elevated plus-maze test in mice. YL was orally active (0.3-3 mg/kg). A retro-sequence peptide or a mixture of Tyr and Leu was inactive. The anxiolytic-like activity of YL was inhibited by antagonists for serotonin 5-HT_1A, dopamine D₁ and GABA_A receptors; however, YL had no affinity for them. We also determined the order of their activation is 5-HT_1A, D₁ and GABA_A receptors using selective agonists and antagonists. Taken together, YL may exhibit anxiolytic-like activity via activation of 5-HT_1A, D₁ and GABA_A receptors.     

The function and structural influence of selective relaxed constraint at functional intracellular loop3 of 5-HT1A serotonin-1 receptor family

Serotonin (5-HT) and its receptors have been involved in critical signal transduction mechanism and deregulation implicated in mood-related disorders. 5-HT activities are mediated through a family of transmembrane spanning serotonin receptors. Both within the family and species, 5-HT receptor protein sequence diversity and 7-transmembrane structural homogeneity have long been intriguing. In this study, we have analyzed the codon site constraint in 5-HT1 subclass receptors from 13 orthologous mammalian mRNA coding sequence. Further, the study was extended to computationally investigate the impact of non-synonymous sites with respect to function and structural significance through sequence homology algorithm and molecular dynamics simulation (MDS). Codon sites with significant posterior probability were observed in 5-HT_1A, 5‐HT_1B and 5-HT_1D receptor indicating variations in site constraint within the 5‐HT1 sub-class genes. In 5-HT_1A receptor, seven sites were detected at the functional intracellular loop₃ (ICL₃) with higher substitution rate through Codeml program. Sequence homology algorithm identifies that these sites were functionally tolerant within the mammals representing a selectively relaxed constraint at this domain. On the other hand, the root mean square deviation (rmsd) values from MDS suggest differences in structural conformation of ICL₃ models among the species. Specifically, the human ICL₃ model fluctuation was comparatively more stable than other species. Hence, we argue that these sites may have varying influence in G-proteins coupling and activation of effectors systems through downstream interacting accessory proteins of cell among the species. However, further experimental studies are required to elucidate the precise role and the seeming difference of these sites in 5-HT receptors between species.     

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function

A_2A adenosine receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca²⁺ signaling evoked by the P2Y₁R agonist, 2-methylthioladenosine 5’ diphosphate (2MeSADP) was significantly inhibited by the A_2AR antagonist (ZM241385 and SCH442416) and the P2Y₁₂R antagonist (ARC69931MX) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y₁R signaling by A_2AR and P2Y₁₂R antagonists was indeed mediated through A_2AR and P2Y₁₂R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.