RECEPTOR CDNA EXPRESSION CLONING USING XENOPUS OOCYTE EXPRESSION

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Expression cloning is an attractive approach to identifying the genes and cDNAs encoding receptors for abused substances and for the neurotransmitters implicated in drug abuse (see above). The ability of the Xenopus oocyte to appropriately translate, post-translationally modify, and appropriately insert several receptors into its membrane has led us to establish this system as a screening tool for these cDNAs and receptors. Progress in this project has included: 1) Obtaining electrophysiologic signals for neurotensin, cholecystokinin, and kainic acid from oocytes injected with these synthetic mRNA transcripts, 2) Cloning of a single cDNA that induces CCK-A responses in oocytes that are pharmacologically-appropriate, but likely due to upregulation of the endogenous CCK receptor gene by the injected transcript. This cDNA "CCK-UP" is expressed in several brain regions, and may provide important insights into the upregulation mechanisms important for one of the chief dopamine cotransmitters in the brain, 3) Characterizing interesting properties of the GABA-Rho1 receptor expressed in this system, including bicucculing-insensitivity. This approach thus enhances abilities to clone interestingc DNAs through "sib selection" techniques, and improves abilities to characterize the properties of expressed cDNAs.