CHARACTERIZATION OF PHARMACOLOGICALLY ACTIVE ANTIPEPTIDE ANTIBODIES DIRECTED AGAINST THE FIRST AND 2ND EXTRACELLULAR LOOPS OF THE SEROTONIN5-HT1A RECEPTOR

The immunological properties and the functional role of the first (loo p I) and second (loop II) extracellular loops of the human serotonin 5 -HT1A receptor were studied with three populations of anti-peptide ant ibodies: Ab-1 (loop I; sequence Y-Q-V-L-N-K-W-T-L-G-Q-V-T-C-D-L; resid ues 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 cr oss-reacting with loop II). Chemical modification of peptide amino aci d residues revealed the importance of the polyanionic stretch near the N-terminal domain of loop II for Ab-2 antibody binding and the role o f the cysteine residues in both loops for the binding of Ab-1 and Ab-1 2 antibodies. Antibodies Ab-2 and Ab-12 recognized only the nonglycosy lated form of the receptor (42 kDa) on immunoblots with transfected He La cells expressing the human 5-HT1A receptor but recognized the glyco sylated forms (55 and 65 kDa) of rat 5-HT1A receptor from hippocampus membranes. The Ab-1 antibodies recognized no protein band from any cel l type studied. Preincubation of transfected HeLa cell membranes with Ab-2 antibodies revealed two affinity binding sites of the 5-HT1A rece ptor (K-DH = 0.54 +/- 0.09 nM and K-DL = 13.74 +/- 4.9 nM) for the ago nist 8-hydroxy-2-(di-n-[H-3]propylamino) tetralin ([H-3]8-OH-DPAT) bin ding, but Ab-1 and Ab-12 revealed only one site (K-D of approximate to 2.5 nM). In contrast to the Ab-2 antibodies, Ab-1 and Ab-12 antibodie s decreased the B-max of the [H-3]8-OH-DPAT binding to 42 and 31%, res pectively. These findings suggest that there are at least two epitopes on the extracellular loops: one inducing a high-affinity state for ag onist binding and the other interfering with the accessibility of the ligand binding pocket.