Oocyte Expression Cloning

cDNAs encoding several low abundance cell membrane receptors have been isolated by functional expression either in Xenopus oocytes or in mammalian cells using specific assays to detect receptor-ligand interactions. These assays varied from radioligand binding to detection of intracellular calcium mobilization or secretion of a particular hormone (6,7). Cloning of the GHS-R was hampered by the relative paucity of biochemical information on the receptor protein, because of its low abundance (6 fmol/mg membrane protein), and the requirement to use primary pituitary tissue as a source for mRNA or protein since cell lines expressing the receptor were lacking. GHS-R cloning required the development of a sensitive and robust high-throughput screening assay. The ability to functionally express the GHS-R in Xenopus oocytes, injected with swine pituitary poly

(A)+ RNA, was shown by the detection of a rapidly activating Ca2+-dependent chloride current in response to MK-0677 administration. Because only a small fraction of the Xenopus frogs tested (4 out of 50) had oocytes that gave positive responses, the authors determined whether the expression of a requisite G protein a subunit was limiting in some batches of Xenopus oocytes resulting in inefficient receptor-effector coupling.

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