Functional expression of the human serotonin 5-HT1A receptor in Escherichia coli. Ligand binding properties and interaction with recombinant G protein alpha-subunits.

摘要: Signaling through serotonin 5-HT1A receptors involves multiple pathways. We have investigated the functional coupling of human receptor to different G proteins using an in vitro reconstitution system based on expression recombinant (r5-HT1A) and alpha-subunits (rG alpha) Escherichia coli. The r5-HT1A was expressed by insertion a vector allowing its active E. coli inner membranes. Binding selective agonist [3H] +/- 8-hydroxy-(2-N-dipropylamine)tetralin ([3H]8-OH-DPAT) intact bacteria or membranes saturable with KD approximately 8 nM average 120 sites/bacterium. properties several serotoninergic ligands were comparable those measured mammalian cells. Incubation rG alpha.beta gamma resulted high affinity [3H]8-OH-DPAT binding (KD = 0.7 nM) titration panel alpha subtypes showed order potency: rGi alpha-3 greater than alpha-2 alpha-1 much rGo alpha, while rGs appeared incapable interacting receptors. Moreover, agonist-mediated enhancement [35S]guanosine 5'-O-(3-thiotriphosphate) confirmed achievement interaction between proteins. Our findings are agreement vivo ability activate Gi related adenylyl cyclase inhibition K+ channel activation, but do not support previously reported stimulation Gs alpha.