title: Functional expression of the human serotonin 5-HT1A receptor in Escherichia coli. Ligand binding properties and interaction with recombinant G protein alpha-subunits.
abstract: Signaling through serotonin 5-HT1A receptors involves multiple pathways. We have investigated the functional coupling of the human 5-HT1A receptor to different G proteins using an in vitro reconstitution system based on the expression of recombinant receptor (r5-HT1A) and G alpha-subunits (rG alpha) in Escherichia coli. The r5-HT1A receptor was expressed by insertion in a vector allowing its active expression in E. coli inner membranes. Binding of the selective agonist [3H] +/- 8-hydroxy-(2-N-dipropylamine)tetralin ([3H]8-OH-DPAT) to intact bacteria or E. coli membranes was saturable with a KD of approximately 8 nM and an average of 120 sites/bacterium. Binding properties of several serotoninergic ligands to r5-HT1A receptors were comparable with those measured in mammalian cells. Incubation of rG alpha.beta gamma with E. coli membranes resulted in high affinity agonist [3H]8-OH-DPAT binding (KD = 0.7 nM) and titration with a panel of rG alpha subtypes showed the order of potency: rGi alpha-3 greater than rGi alpha-2 greater than rGi alpha-1 much greater than rGo alpha, while rGs alpha appeared incapable of interacting with 5-HT1A receptors. Moreover, agonist-mediated enhancement of [35S]guanosine 5'-O-(3-thiotriphosphate) binding to rGi alpha confirmed the achievement of the functional interaction between receptor and G proteins. Our findings are in agreement with the in vivo ability of 5-HT1A receptors to activate Gi alpha related to adenylyl cyclase inhibition or K+ channel activation, but do not support previously reported adenylyl cyclase stimulation through interaction with Gs alpha.
authors.affiliation: Laboratoire d'Immunopharmacologie Moleculaire, Centre National de la Recherche Scientifique UPR 415, Paris, France.
authors.forename: B
authors.initials: B
authors.lastname: Bertin
authors.forename: M
authors.initials: M
authors.lastname: Freissmuth
authors.forename: R M
authors.initials: RM
authors.lastname: Breyer
authors.forename: W
authors.initials: W
authors.lastname: Schütz
authors.forename: A D
authors.initials: AD
authors.lastname: Strosberg
authors.forename: S
authors.initials: S
authors.lastname: Marullo
mesh_terms: D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010957:Plasmids; D011985:Receptors, Serotonin / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013764:Tetrahydronaphthalenes / Q000378:metabolism
chemical_list: D008024:Ligands; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D013764:Tetrahydronaphthalenes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; D019204:GTP-Binding Proteins