Abstract

Functional characterization of the recombinant human 5-hydroxytryptamine_7(a) (h5-HT_7(a)) receptor isoform was performed using stably transfected LM(tk⁻) cells. Expression levels of the h5-HT_7(a) receptor determined from saturation studies using either a labeled agonist ([³H]5-HT) or antagonist ([³H]LSD) were very similar (B_max = 160–190 fmol/mg protein), suggesting that all receptors may exist in the high affinity (G protein-coupled) state. In intact cells, 5-HT produced a concentration-dependent elevation of intracellular cAMP levels ([cAMP]_i) with an EC₅₀ value of 80 nM and a maximal response of 5-fold increase above basal levels. The rank order of agonist potencies in the second messenger assay paralleled their rank order of binding affinities: 5-carboxamidotryptamine > 5-hydroxytryptamine ≥ 5-methoxytryptamine > 8-hydroxy N,N-dipropyl aminotetralin > sumatriptan. Agonist potencies (EC₅₀ values) to stimulate [cAMP]_i were more than 25-fold lower relative to their respective binding affinities (K_i values) obtained in [³H]5-HT competition assays. In contrast, antagonist potencies (K_b values) to block 5-HT-stimulated [cAMP]_i were in close agreement with their correspondingK_i values. These data may indicate low efficiency of receptor-effector coupling to adenylate cyclase stimulation. Pretreatment of stably transfected cells with cholera toxin abolished the 5-HT-mediated elevation of [cAMP]_i, indicating that the 5-HT_7(a) subtype directly interacts with G_αs protein(s) to activate adenylate cyclase(s). Clonal cell lines stably expressing h5-HT₇ receptor isoforms will serve as valuable cellular models to study their function and regulation, as well as assist in the development of selective 5-HT₇ receptor agents to uncover the biological roles and potential therapeutic applications of this novel receptor subtype.