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Gs couples thyrotropin-releasing hormone receptors expressed in Xenopus oocytes to phospholipase C
P de la Peña1, D del Camino, L A Pardo
1Departamento de Biología Functional, Facultad de Medicina, Universidad de Qviedo, Spain.
Abstract:
Coupling of thyrotropin-releasing hormone (TRH) receptors to individual G-proteins has been studied in Xenopus oocytes injected with receptor cRNA and antisense oligonucleotides to mRNA encoding different G-protein alpha- and beta-subunits. Injection of antisenses which target mRNA sequences shared by several G-protein alpha or beta gamma polypeptides effectively blocked Ca(2+)-dependent Cl- currents induced by TRH through activation of phospholipase C. Three different alpha s-specific antisense oligonucleotides complementary to sequences located in different positions along the coding region of the alpha s protein mRNA were highly effective in inhibiting TRH-induced responses. Anti-alpha o, -alpha q, -alpha i, or -alpha z oligonucleotides were not able to modify the TRH-evoked response. In contrast, anti-alpha o, but not anti-alpha s, oligonucleotides blocked the response to serotonin in oocytes injected with serotonin 5-HT1c receptor cRNA. Cholera toxin catalyzed the [32P]ADP-ribosylation of 40-42- and 50-52-kDa proteins in GH3 cell plasma membranes. [32P]ADP-ribosylation of oocyte membranes with the toxin labeled several proteins. These include a single 50-55-kDa substrate, which is clearly diminished in membranes from anti-alpha s-injected oocytes. Amplification of oocyte RNA in a polymerase chain reaction system and sequencing of the amplified products demonstrated that anti-alpha s oligonucleotides selectively recognize the message for the Xenopus alpha s polypeptide. It is concluded that Gs, but not Go, Gq, Gi, or Gz, couples TRH receptors expressed in oocytes to activation of phospholipase C and subsequent inositol 1,4,5-trisphosphate-dependent stimulation of Ca(2+)-dependent Cl- currents.
Insights
Thyrotropin-releasing hormone (TRH) receptors couple to Gs proteins to activate phospholipase C in Xenopus oocytes. This coupling is essential for TRH-induced calcium-dependent chloride currents, mediated by Gs, not other G-proteins.
Area of Science:
- Molecular Pharmacology
- Cellular Signaling
- G-Protein Coupled Receptors
Background:
- Thyrotropin-releasing hormone (TRH) receptors are known to activate intracellular signaling pathways.
- G-proteins are critical mediators of signal transduction from cell surface receptors to intracellular effectors.
- Understanding the specific G-protein subtypes coupled to TRH receptors is crucial for elucidating TRH signaling mechanisms.
Purpose of the Study:
- To identify the specific G-protein alpha- and beta-subunits that couple to thyrotropin-releasing hormone (TRH) receptors in Xenopus oocytes.
- To investigate the role of different G-protein subtypes in mediating TRH-induced activation of phospholipase C and subsequent calcium-dependent chloride currents.
Main Methods:
- Xenopus oocytes were injected with receptor cRNA and antisense oligonucleotides targeting specific G-protein alpha- and beta-subunit mRNAs.
- TRH-induced calcium-dependent chloride currents were measured electrophysiologically.
- G-protein alpha-subunit expression was confirmed using RT-PCR and sequencing.
- [32P]ADP-ribosylation assays with cholera toxin were performed on oocyte membranes.
Main Results:
- TRH-induced currents were effectively blocked by antisense oligonucleotides targeting Gs alpha-subunit mRNA, but not by those targeting Go, Gq, Gi, or Gz alpha-subunits.
- Antisense oligonucleotides against Gs alpha selectively inhibited TRH responses, while anti-Go oligonucleotides blocked serotonin responses mediated by the 5-HT1c receptor.
- ADP-ribosylation assays showed a diminished 50-55 kDa protein substrate in membranes from oocytes injected with anti-Gs alpha oligonucleotides.
Conclusions:
- The Gs G-protein subtype, but not Go, Gq, Gi, or Gz, couples TRH receptors to the activation of phospholipase C in Xenopus oocytes.
- This Gs-mediated signaling pathway leads to the stimulation of inositol 1,4,5-trisphosphate production and subsequent calcium-dependent chloride currents.
- The findings clarify the specific G-protein involvement in TRH receptor signaling, providing a molecular basis for TRH-mediated cellular responses.