Cell-specific coupling of the cloned human 5-HT1F receptor to multiple signal transduction pathways

We recently described the cloning of a fifth member of the 5-hydroxytryptamine (5-HT)₁ (serotonin₁) receptor class that inhibits adenylyl cyclase, namely the human 5-HT_1F receptor (Adham et al. 1993 a). In the present study we have examined in greater detail the functional coupling of the 5-HT_1F receptor in two different cell lines, NIH-3T3 and LM(tk^–) fibroblasts (receptor densities of 1.7 and 4.4 pmol/mg protein, respectively). The maximal inhibitory response elicited by 5-HT was significantly greater in NIH-3T3 as compared to LM(tk^–) cells, whereas the EC₅₀ values were comparable.To investigate the relationship between receptor occupancy and inhibition of cAMP accumulation mediated by 5-HT_1F receptors in NIH-3T3 cells (and hence the degree of receptor reserve), we used the irreversible receptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The half-maximal response required only about 10% receptor occupancy, consistent with a receptor reserve of 90% (88±2.1%, n = 4) for 5-HT-induced inhibition of FSCA. Despite the presence of such a high degree of receptor reserve, a range of intrinsic activities was displayed by structurally diverse classes of compounds. For example, sumatriptan and lysergol were as efficacious as 5-HT itself and thus acted as full agonists, whereas metergoline and 1-NP behaved as partial agonists and as shown previously (Adham et al. 1993a), methiothepin was a silent antagonist (K_b = 438 nM).We have also investigated activation of additional signal transduction pathways by the 5-HT_1F receptor and found that the responses differ in the two cell lines with respect to stimulation of phospholipase C. For example, in NIH-3T3 cells no elevation of inositol phosphates (IP) of [Ca²⁺]_i was observed even at very high agonist concentrations (100 M). In contrast, in LM(tk^–) cells concentrations of 5-HT as low as 10 nM induced stimulation of IP and a rapid increase of [Ca²⁺]i. The 5-HT_1F receptor failed to alter arachidonic acid release in either cell line.The maximal increase in IP accumulation in LM(tk^–) cells was modest, averaging about 100% above basal. The increases of IP and [Ca²⁺]_i required 5-HT concentrations less than one order of magnitude greater than those inhibiting FSCA (EC₅₀ = 17, 55 and 8 nM, respectively), and both responses were blocked by 100 M methiothepin. All three responses (cAMP, IP, and [Ca²⁺]_i) were sensitive to pertussis toxin pre-treatment, suggesting the involvement of G_i/G_o protein(s) in these signal transduction pathways. [Ca²⁺]_i was also elevated by sumatriptan, which may provide a mechanism by which this drug causes constriction of the vasculature. In conclusion, these data indicate that the human 5-HT_1F receptor can couple to multiple effectors, and that this coupling is cell-type dependent.Abbreviations FSCA forskolin-stimulated cAMP accumulation - [Ca²⁺] intracellular free calcium concentration - AA arachidonic acid - EEDQ N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline - CHO chinese hamster ovary cell - LM(tk^–) mouse fibroblast cell - B_max maximal binding site density - K_i apparent dissociation constant obtained from competition binding studies - G protein guanine nucleotide-binding protein - HBS HEPES-buffered saline - IP inositol phosphates - IP₃ inositol 1,4,5 trisphosphate - PLC phospholipase C - K_b antagonist dissociation constant - K_d equilibrium dissociation constant - N-1F-6 stable NIH-3T3 cells expressing the cloned 5-HT_1F receptor - L-1F-3 stable LM(tk^–) cells expressing the cloned 5-HT_1F receptor - PTX pertussis toxin - BSA bovine serum albumin - METH methiothepin - SUMA sumatriptan - 5-MeO-DMT 5-methoxy-N,N-dimethyltryptamine - 1-NP 1-(1-napthyl)piperazine - 5-CT 5-carboxyamidotryptamineCorrespondence to: N. Adham at the above address