Differentiation alters the expression of the two splice variants of the serotonin 5-HT3 receptor-A mRNA in NG108-15 cells.
The serotonin 5-HT3-A receptor (5-HT3R-A) mRNA has been shown recently to be expressed as two forms (5-HT3R-AL and 5-HT3R-AS) varying by the presence or the absence of a sequence of 18 bases in the region corresponding to the second cytoplasmic domain of the receptor, and generated by alternative splicing at the level of the 3' acceptor site of exon 9. As the long form of the receptor exhibits a potential phosphorylation site that is disrupted by the alternative splicing, the hypothesis of functional identity and stochastic expression of these two variants was questioned. In the present study, we used quantitative reverse transcriptase-polymerase chain reaction to examine the possible influence of culture conditions on the expression and the alternative splicing of 5-HT3R-A mRNA in NG108-15 clonal cells. Cell differentiation induced by dibutyryl cyclic AMP or theophyllin plus prostaglandin E1 in the presence of 10% serum reduced by threefold the expression of total 5-HT3R-A mRNA, and favored the short form of the message as the ratio S/L (5-HT3R-AS mRNA/5-HT3R-AL mRNA) shifted from 2.23 to 7.33 after 9 days of treatment. Culture with 0.3% serum (instead of 10%) lowered by 10-fold the level of expression of total 5-HT3R-A mRNA, but only slightly reduced the S/L ratio. However, this ratio fell to 0.06 in the presence of 0.3% serum plus 10 ng/ml basic fibroblast growth factor. These results demonstrate that external factors can influence the differential expression of the two variants of the 5-HT3R-A in NG108-15 cells. Appropriate culture conditions for the almost exclusive expression of 5-HT3R-AS mRNA or 5-HT3R-AL mRNA in NG108-15 cells should allow the identification of possible differences in the respective functional properties of each of these two forms of the native 5-HT3 receptor.
Emerit
M B
MB
INSERM U288, Neurobiologie Cellulaire et Fonctionnelle, Faculté de Médecine Pitié-Salpêtrière, Paris, France.
Martres
M P
MP
Miquel
M C
MC
el Mestikawy
S
S
Hamon
M
M
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002448:Cell Adhesion; D002454:Cell Differentiation; D002460:Cell Line; D016895:Culture Media, Serum-Free; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology; D006822:Hybrid Cells / Q000166:cytology / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D011985:Receptors, Serotonin / Q000235:genetics*
D016895:Culture Media, Serum-Free; D006133:Growth Substances; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011985:Receptors, Serotonin; D004121:Dimethyl Sulfoxide