Ligand autoradiographic receptor screening: receptor cDNA expression in replicas of transfected COS cells.
In this paper we validate a methodology, ligand autoradiographic receptor screening (LARS), for detecting expression of full length receptor cDNAs in COS cells. The method involves transfection of COS cells with receptor cDNAs by spheroplast fusion, production of filter replicas of the cell fragments, ligand binding to the receptors expressed in the membranes, and autoradiographic detection of bound ligand. A beta-adrenergic receptor cDNA cloned into a eukaryotic expression vector reliably induces high levels of beta-adrenergic receptor expression in 2-12% of COS cell colonies transfected with this plasmid after experimental conditions are optimized. Receptor expression is monitored by autoradiographic detection of 125iodocyanopindolol binding to COS cell fragments immobilized on polyester filter replicas. Binding displays appropriate pharmacological properties. The number of high-density binding spots per filter depends on the fraction of the spheroplasts in the fusion mixture that contain the beta-adrenergic receptor cDNA. A 100-plate LARS experiment can assess receptor expression in more than 10(4) transfected colonies. Thus detection of receptor-encoding sequences present in libraries in proportions as low as 1 in 10(4) should be possible. This technique may therefore be useful in detecting expression of other receptor cDNAs for which selective radioligands are available.
Rattray
M
M
Gene Neuroscience Unit, Addiction Research Center/NIDA, Baltimore, MD.
Lautar
S L
SL
Uhl
G R
GR
D000818:Animals; D001345:Autoradiography / Q000379:methods*; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D020120:Iodocyanopindolol; D010869:Pindolol / Q000031:analogs & derivatives* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011943:Receptors, Adrenergic, beta / Q000235:genetics* / Q000378:metabolism; D013104:Spheroplasts; D014162:Transfection
D011943:Receptors, Adrenergic, beta; D020120:Iodocyanopindolol; D004247:DNA; D010869:Pindolol