title: Photoaffinity labelling and solubilization on the central 5-HT1A receptor binding site.
abstract: Two complementary approaches, covalent labelling and solubilization, have been used to study the biochemical properties of the central 5-HT1A receptor binding site. We have first designed a photoaffinity ligand containing the structure of 8-OH-DPAT, a potent and specific agonist of 5-HT1A sites. Thus, 8-methoxy-2[N-n-propyl,N-3-(2-nitro-4-azido-phenyl)- aminopropyl]aminotetralin or 8-methoxy-3'-NAP-amino-PAT, was found to displace, in the dark, [3H]8-OH-DPAT from 5-HT1A sites in rat hippocampal membranes with an IC50 of 6.6 nM. Under two cumulative UV irradiations (366 nm, for 20 min at 4 degrees C), 8-methoxy-3-'-NAP-amino-PAT (30 nM) blocked irreversibly 55-60% of 5-HT1A binding sites. This blockade was specific of 5-HT1A sites since the other serotoninergic sites, 5-HT1B, 5-HT2 and also the presynaptic 5-HT3 sites were not affected by the treatment. In addition, the binding of [3H]Spiperone and [3H]7-OH-DPAT to striatal dopamine sites remained unchanged under similar photolysis conditions. The tritiated derivative of the photoaffinity ligand (92 Ci/mmol) was then synthesized for the identification of the covalently bound protein(s). SDS-PAGE of solubilized membranes irradiated in the presence of 20 nM 3H-8-methoxy-3'-NAP-amino-PAT allowed the detection of a 63 kD protein whose labelling appeared specific. Thus, 3H-incorporation into the 63 kD band could be prevented by microM concentrations of 5-HT, 8-OH-DPAT and other selective 5-HT1A ligands such as isapirone. In contrast, the 5-HT2 antagonist ketanserin, norepinephrine and dopamine-related ligands (including 7-OH-DPAT) were ineffective. Direct solubilization of 5-HT1A receptor binding sites was also attempted from rat hippocampal membranes. The best results were obtained using CHAPS (10 mM) plus NaCl (0.2 M), which led to 50% recovery of 5-HT1A sites in the 100,000 g supernatant. The pharmacological properties and sensitivity to N-ethyl-maleimide and GppNHp of soluble sites appeared near identical to those of membrane-bound 5-HT1A sites.
mesh_terms: D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D002412:Cations / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006624:Hippocampus / Q000032:analysis; D008297:Male; D010782:Photolysis; D051381:Rats; D011919:Rats, Inbred Strains; D011954:Receptors, Dopamine / Q000378:metabolism; D011985:Receptors, Serotonin / Q000145:classification / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism*; D012995:Solubility; D013134:Spiperone / Q000378:metabolism; D013764:Tetrahydronaphthalenes / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D001386:Azides; D002412:Cations; D003902:Detergents; D011954:Receptors, Dopamine; D011985:Receptors, Serotonin; D013764:Tetrahydronaphthalenes; C053146:8-methoxy-2-(N-n-propyl-N-3-(2-nitro-4-azidophenyl)aminopropyl)aminotetralin; D012701:Serotonin; D006165:Guanylyl Imidodiphosphate; D013134:Spiperone; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin