Stable, high-level expression of human serotonin receptors in L929 cells using an inducible expression system.
Heterologous expression of cloned receptor subtypes for screening programs has become a real necessity for a modern pharmaceutical company. As the expression levels obtained so far are often low or unstable, we addressed this problem by using an inducible promoter system, i.e. the interferon-inducible mouse Mxl promoter. Using the gene coding for chloramphenicol acetyltransferase (CAT) as a reporter gene, we tested the inducibility of this promoter in the murine cell line L929. We found that background expression was low and that a distinct interferon-induced expression could be obtained. CAT expression reached its maximum at approximately 15 ng CAT/mg protein after induction for 24 hr with 1000 U/ml murine interferon-beta; the induction ratio was 150-fold. Next, L929 cells were transfected with four different human serotonin (5HT) receptor cDNAs (5HT1A, 5HT2A, 5HT1D beta and 5HT1E) under the control of the same Mxl promoter fragment. Also in this case well-regulated serotonin receptor-expressing clones were isolated. Bmax values varied from 3100 fmol/mg protein for the 5HT2A receptor, 3300 fmol/mg protein for the 5HT1D beta receptor, 9800 fmol/mg protein for the 5HT1E receptor, and even up to 10,400 fmol/mg protein for the 5HT1A receptor. Furthermore, the expression levels were shown to remain stable during serial propagation for at least one year, demonstrating the usefulness of this expression system. In fact, the 5HT1D beta receptor-expressing cells were used in the characterization of a new antimigraine agent, viz. alniditan.
Vanhoenacker
P
P
Laboratory of Molecular Biology, University of Gent-VIB, Belgium. PETERVH@LMB1.RUG.AC.BE
Gommeren
W
W
Luyten
W H
WH
Leysen
J E
JE
Haegeman
G
G
D000818:Animals; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D019204:GTP-Binding Proteins; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D064547:Myxovirus Resistance Proteins; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics*; D044302:Receptor, Serotonin, 5-HT1B; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin / Q000235:genetics*; D044263:Receptors, Serotonin, 5-HT1
D064547:Myxovirus Resistance Proteins; D011506:Proteins; D044302:Receptor, Serotonin, 5-HT1B; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; C076396:serotonin 1E receptor; D015500:Chloramphenicol O-Acetyltransferase; D019204:GTP-Binding Proteins