Immunolabelling of the rat intestinal tract with antibodies specific to the long form of the 5-hydroxytryptamine3 receptor.
The mouse 5-hydroxytryptamine3 (5-HT3) type of serotonin receptors is expressed as two forms, 5-HT3R-A(L) and 5-HT3R-A(S), generated by alternative splicing of its primary transcript, that differ by a stretch of six amino acids in the second intracellular loop domain. Because this six-amino acid region contains a putative phosphorylation site that may be important for the function and/or regulation of 5HT3R-A(L) receptor, specifically, we developed polyclonal antibodies as appropriate tools for studies relevant to this question. Antibodies against a 20-amino acid peptide corresponding to the sequence of 5-HT3R-A(L) at the level of this six-amino acid region were obtained as soon as one month after injection of this synthetic peptide to rabbits. Immunocytochemistry with these antibodies led to a strong positive labelling of plasma membrane, reticulum and Golgi apparatus of COS-7 cells expressing cloned murine 5-HT3R-A(L), whereas COS-7 cells expressing similar levels of 5-HT3R-A(S) exhibited only a very weak labelling. Immunoblots of fusion proteins combining glutathion-S-transferase and the second cytoplasmic loop of 5-HT3R-A(L) or 5-HT3R-A(S) revealed a c. 20-fold selectivity of the antibodies for the first, long form, as evaluated by densitometric analysis of enhanced chemiluminescence detection. Similarly, immunoblots of COS-7 cells transfected with cloned 5-HT3 receptors showed that the anti-peptide antibodies detected a band at 53,000 mol. wt only in cells transfected with 5-HT3R-A(L). Under optimal conditions, antibodies immunoprecipitated 52% of 5-HT3R-A(L), but only 11% of 5-HT3R-A(S), solubilized from COS-7 cells transfected with the respective encoding plasmids. In the rat, no immunoautoradiographic labelling by the anti-peptide antibodies could be detected in brain structures which had previously been described to express preferentially a short form of the 5-HT3 receptor. In contrast, a strong immunolabelling was found in the intestinal mucosa, especially in the rat fetus (at the 17th embryonic day), suggesting the possible participation of the 5-HT3R-A(L) isoform in the development of this tissue. These results show that specific antibodies are useful tools for the visualization of the least abundant 5-HT3 receptor isoform in rat tissue.
Doucet
E
E
INSERM U288, Neuropsychopharmacologie Moléculaire, Cellulaire et Fonctionnelle, Faculté de Médecine Pitié Salpêtrière, Paris, France.
Hamon
M
M
Emerit
M B
MB
D017398:Alternative Splicing / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001345:Autoradiography; D001549:Benzamides / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D019556:COS Cells; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005333:Fetus / Q000166:cytology; D005982:Glutathione Transferase; D015151:Immunoblotting; D007422:Intestines / Q000294:innervation*; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D017933:Peripheral Nervous System / Q000737:chemistry* / Q000502:physiology; D011233:Precipitin Tests; D011247:Pregnancy; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011985:Receptors, Serotonin / Q000032:analysis / Q000235:genetics* / Q000276:immunology*; D044406:Receptors, Serotonin, 5-HT3; D011994:Recombinant Proteins; D012702:Serotonin Antagonists / Q000494:pharmacology; D014162:Transfection; D014316:Tritium
D001549:Benzamides; D019086:Bridged Bicyclo Compounds, Heterocyclic; D010446:Peptide Fragments; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D011994:Recombinant Proteins; D012702:Serotonin Antagonists; D014316:Tritium; C055971:zacopride; D005982:Glutathione Transferase