ausblenden:
Schlagwörter:
Animals
Base Sequence
Calcium/metabolism
Cell Line
Chlorides/metabolism
Enzyme Activation
GTP-Binding Proteins/*metabolism
Molecular Sequence Data
Oligonucleotides, Antisense
Oocytes
RNA, Complementary
Receptors, Serotonin/genetics
Receptors, Thyrotropin-Releasing Hormone/genetics/*metabolism
Recombinant Proteins/metabolism
Type C Phospholipases/*metabolism
Xenopus
Zusammenfassung:
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.
