Functional expression of human 5-HT1A receptors and differential coupling to second messengers in CHO cells.
The signal transduction linkages of the cloned human 5-HT1A receptor as expressed stably in CHO cells were studied. A transfected clonal cell line which expresses 900 +/- 36 fmol 5-HT1A receptor/mg protein (designated CHO-5-HT1A/WT-27) responded to 5-HT and/or 8-OH-DPAT by coupling to several second messenger pathways. The 5-HT1A receptor inhibited, but did not stimulate, membrane adenylyl cyclase activity and whole cell cAMP accumulation in a dose-dependent manner (for 5-HT, IC50 = 146 +/- 27 and 55 +/- 12 nM, respectively). Activation of the receptor was associated with other signal transduction linkages: (i) a 40-50% increase in hydrolysis of inositol phosphates (for 5-HT, EC50 = 1.33 +/- 0.15 microM for 5-HT), (ii) a transient elevation of cytosolic Ca2+ levels (apparent at 1-100 microM 5-HT) which was not affected by chelation of extracellular Ca2+ by EGTA, and (iii) an augmentation of [3H]-arachidonic acid release pharmacologically with the calcium ionophore A23187 or by activation of endogenous thrombin or P2 purinergic receptors (for 5-HT, EC50 = 1.22 +/- 0.17 microM). This pathway may be an amplification mechanism for signaling in anatomic regions with high concentrations of several neuro-transmitters, hormones or autacoids, such as at neuronal junctions or near areas of platelet aggregation. All linkages were sensitive to pertussis toxin pre-treatment (IC50 approximately 0.5-0.6 ng/ml x 4.5 h for all pathways), suggesting the involvement of Gi protein(s) in these signal transduction pathways. Coupling to varied signal transduction pathways in a single cell system may be a common feature of receptors which classically inhibit adenylyl cyclase such as the 5-HT1A receptor.
Raymond
J R
JR
Department of Medicine (Nephrology), Duke University Medical Center, Durham, NC 27710.
Albers
F J
FJ
Middleton
J P
JP
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D016466:CHO Cells / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D011985:Receptors, Serotonin / Q000502:physiology*; D015290:Second Messenger Systems / Q000502:physiology*; D012680:Sensitivity and Specificity; D013268:Stimulation, Chemical; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D007295:Inositol Phosphates; D011985:Receptors, Serotonin; D010566:Virulence Factors, Bordetella; D016718:Arachidonic Acid; D037342:Pertussis Toxin; D000262:Adenylyl Cyclases; D002118:Calcium