Degenerate suppression PCR identifies the beta2-adrenergic receptor as upregulated by neuronal differentiation.
Communication between cells is necessary for the functioning of a multicellular organism. Cells process a large amount of information through G-protein-coupled receptors, and activation of this receptor class has been implicated in neuronal differentiation. In this study, we used a method based on PCR with degenerated primers to identify G-protein-coupled receptors regulated by retinoic acid-induced differentiation of the human teratocarcinoma cell line NTera-2/D1. Subtracted cDNA libraries and control cDNA served as templates in half-sided PCR with a forward degenerate primer based on a conserved sequence from human serotonergic, adrenergic, and dopaminergic receptors and reverse primers on adaptors with long terminal repeats commonly employed in subtractive suppression hybridization. We developed conditions to amplify G-protein-coupled receptors from adaptor-ligated cDNA and found the beta2-adrenergic receptor to be upregulated fourfold. This seems to be physiologically relevant, as it could also be shown in rat primary cortical cultures maturing in vitro. The method presented here makes use of the otherwise unused control cDNA from subtractive suppression hybridization experiments and could be easily adapted to other gene families.
Lewerenz
Jan
J
Research Group Protective Signaling, Zentrum für Molekulare Neurobiologie and Department of Neurology, University Hospital Hamburg, Martinistr. 52, D-20246 Hamburg, Germany.
Leypoldt
Frank
F
Methner
Axel
A
D000818:Animals; D015152:Blotting, Northern; D002454:Cell Differentiation / Q000502:physiology*; D002540:Cerebral Cortex / Q000378:metabolism / Q000473:pathology; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009474:Neurons / Q000166:cytology* / Q000378:metabolism* / Q000473:pathology; D016133:Polymerase Chain Reaction; D051381:Rats; D017208:Rats, Wistar; D011943:Receptors, Adrenergic, beta / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics; D013382:Subtraction Technique; D018243:Teratocarcinoma / Q000378:metabolism / Q000473:pathology; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D017931:DNA Primers; D018076:DNA, Complementary; D011943:Receptors, Adrenergic, beta; D043562:Receptors, G-Protein-Coupled