Identification of G protein-coupled receptors by RNase H-mediated hybrid depletion using Xenopus laevis oocytes as expression system.
A method has been developed for rapidly identifying putative G protein-coupled receptors isolated initially as small cDNA fragments, following reverse transcription and polymerase chain reaction (PCR) amplification of mRNA. The method is based upon the use of synthetic oligonucleotides deduced from the sequence of the amplified receptor fragments, to direct a RNase H-mediated specific degradation of hybrids formed between the oligonucleotides and the corresponding receptor-encoding mRNA. Loss of an agonist-dependent receptor response in the Xenopus laevis oocyte expression system identifies the amplified receptor fragment. Taking in vitro synthesised serotonin HT2-receptor (SR)-encoding mRNA as a model, it was shown that following incubation with RNase H and SR antisense oligonucleotides, injection of this message no longer caused the acquisition of agonist-dependent membrane currents in voltage-clamped oocytes. In contrast, when corresponding sense oligonucleotides were used, the serotonin-evoked membrane responses in oocytes were acquired as normal. The method should allow the identification of receptors which can functionally be expressed and measured in Xenopus oocytes.
Institut für Zellbiochemie und klinische Neurobiologie, UKE, Universität Hamburg, FRG.
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W
Meyerhof
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Richter
D000818:Animals; D001483:Base Sequence; D004553:Electric Conductivity; D004722:Endoribonucleases / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D007473:Ion Channels / Q000502:physiology; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D009865:Oocytes; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D011985:Receptors, Serotonin / Q000235:genetics* / Q000502:physiology; D016914:Ribonuclease H; D014982:Xenopus laevis
D007473:Ion Channels; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011985:Receptors, Serotonin; D004722:Endoribonucleases; D016914:Ribonuclease H; D019204:GTP-Binding Proteins