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Effector coupling mechanisms of the cloned 5-HT1A receptor
A Fargin1, J R Raymond, J W Regan
1Department of Medicine, Howard Hughes Medical Institute, Durham, North Carolina 27710.
Abstract:
The signal transduction pathways of the cloned human 5-HT1A receptor have been examined in two mammalian cell lines transiently (COS-7) or permanently (HeLa) expressing this receptor gene. In both systems, 5-hydroxytryptamine (5-HT, serotonin) mediated a marked inhibition of beta 2-adrenergic agonist-stimulated (80% inhibition in COS-7 cells) or forskolin-stimulated cAMP formation (up to 90% inhibition in HeLa cells). This serotonin effect (EC50 = 20 nM) could be competitively antagonized by metitepine and spiperone (Ki = 81 and 31 nM, respectively) and could also be blocked by pretreatment of cells with pertussis toxin. In both cell types, 5-HT failed to stimulate adenylyl cyclase through the expressed receptors. In HeLa cells, 5-HT also stimulated phospholipase C (approximately 40-75% stimulation of formation of inositol phosphates). Again, this effect was inhibited by metitepine. However, the EC50 of 5-HT was considerably higher (approximately 3.2 microM) than that found for inhibition of adenylyl cyclase. Both pathways were demonstrated to be similarly affected by pertussis toxin. These findings indicate that like the M2 and M3 muscarinic cholinergic receptors, the 5-HT1A receptor can couple to multiple transduction pathways with varying efficiencies via pertussis toxin-sensitive G-proteins. The lack of stimulation of cAMP formation by this 5-HT1A receptor may suggest the existence of another pharmacologically closely related receptor.
Insights
The human 5-HT1A receptor, activated by serotonin, inhibits cAMP formation and stimulates phospholipase C in mammalian cells. These actions are mediated by pertussis toxin-sensitive G-proteins, suggesting diverse signaling pathways.
Area of Science:
- Molecular Pharmacology
- Cell Signaling
- Neuroscience
Background:
- The 5-HT1A receptor is a key serotonin receptor involved in various physiological processes.
- Understanding its signal transduction is crucial for developing targeted therapeutics.
Purpose of the Study:
- To investigate the signal transduction pathways utilized by the cloned human 5-HT1A receptor.
- To determine the coupling of 5-HT1A receptors to G-proteins and downstream effectors.
Main Methods:
- Transient and stable expression of the human 5-HT1A receptor in COS-7 and HeLa cells.
- Measurement of cAMP levels following stimulation with beta 2-adrenergic agonists or forskolin.
- Assessment of adenylyl cyclase and phospholipase C activity.
- Pharmacological characterization using antagonists (metitepine, spiperone) and pertussis toxin treatment.
Main Results:
- Serotonin (5-HT) significantly inhibited cAMP formation in both cell lines (up to 90% in HeLa cells).
- This inhibition was competitively antagonized by metitepine and spiperone and blocked by pertussis toxin.
- 5-HT failed to stimulate adenylyl cyclase directly but activated phospholipase C in HeLa cells (EC50 ~3.2 microM).
- Both pathways were sensitive to pertussis toxin, indicating coupling to G-proteins.
Conclusions:
- The 5-HT1A receptor couples to multiple signal transduction pathways, including inhibition of adenylyl cyclase and stimulation of phospholipase C.
- These pathways are mediated by pertussis toxin-sensitive G-proteins, similar to M2 and M3 muscarinic receptors.
- The distinct efficacies and EC50 values for these pathways suggest potential for distinct receptor subtypes or signaling complexes.