Characterization of pharmacologically active anti-peptide antibodies directed against the first and second extracellular loops of the serotonin 5-HT1A receptor.
The immunological properties and the functional role of the first (loop I) and second (loop II) extracellular loops of the human serotonin 5-HT1A receptor were studied with three populations of anti-peptide antibodies: Ab-1 (loop I; sequence Y-Q-V-L-N-K-W-T-L-G-Q-V-T-C-D-L; residues 96-111), Ab-2 (loop II; sequence G-W-R-T-P-E-D-R-S-D-P-D-A-C-T-I-S-K-D-H-G; residues 173-193), and Ab-12 (produced against loop I but cross-reacting with loop II). Chemical modification of peptide amino acid residues revealed the importance of the polyanionic stretch near the N-terminal domain of loop II for Ab-2 antibody binding and the role of the cysteine residues in both loops for the binding of Ab-1 and Ab-12 antibodies. Antibodies Ab-2 and Ab-12 recognized only the nonglycosylated form of the receptor (42 kDa) on immunoblots with transfected HeLa cells expressing the human 5-HT1A receptor but recognized the glycosylated forms (55 and 65 kDa) of rat 5-HT1A receptor from hippocampus membranes. The Ab-1 antibodies recognized no protein band from any cell type studied. Preincubation of transfected HeLa cell membranes with Ab-2 antibodies revealed two affinity binding sites of the 5-HT1A receptor (KDH = 0.54 +/- 0.09 nM and KDL = 13.74 +/- 4.9 nM) for the agonist 8-hydroxy-2-(di-n-[3H]propylamino) tetralin ([3H]8-OH-DPAT) binding, but Ab-1 and Ab-12 revealed only one site (KD of approximately 2.5 nM). In contrast to the Ab-2 antibodies, Ab-1 and Ab-12 antibodies decreased the Bmax of the [3H]8-OH-DPAT binding to 42 and 31%, respectively. These findings suggest that there are at least two epitopes on the extracellular loops: one inducing a high-affinity state for agonist binding and the other interfering with the accessibility of the ligand binding pocket.
Verdot
L
L
Laboratoire d'Enzymologie et de Chimie des Protéines, INSERM U316, Tours, Paris, France.
Bertin
B
B
Guilloteau
D
D
Strosberg
A D
AD
Hoebeke
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000939:Epitopes; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology*; D051381:Rats; D017208:Rats, Wistar; D011985:Receptors, Serotonin / Q000235:genetics* / Q000276:immunology*
D000906:Antibodies; D000939:Epitopes; D008024:Ligands; D010446:Peptide Fragments; D011985:Receptors, Serotonin