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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