Skip to main content
SpringerLink
Log in

Functional expression of human 5-HT1A receptors and differential coupling to second messengers in CHO cells

  • Published:
Naunyn-Schmiedeberg's Archives of Pharmacology Aims and scope Submit manuscript

Summary

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 μM for 5-HT), (ii) a transient elevation of cytosolic Ca2+ levels (apparent at 1–100 μM 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 μM). 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 pertussistoxinpre-treatment (IC50≈0.5–0.6 ng/ml × 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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

cAMP:

adenosine 3′,5′-cyclic monophosphate

PKC:

protein kinase C (calcium and phospholipid-dependent kinase)

5-HT:

serotonin

8-OH-DPAT:

8-OH-2-(di-n-propylamino)1,2,3,4-tetrahydronaphthalene

PBS:

phosphate buffered saline

EDTA:

ethylenediaminetetraacetic acid

BSA:

bovine serum albumin

Hepes:

N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid

CHAPS:

3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfate

[125I]-N3-NAPS:

N-(p-azido-m-[125I]iodophenethyl)-spiperone

G protein:

(guanine nucleotide binding protein)

mCPP:

1-(3-chlorophenyl)piperazine

TFMPP:

m-trifluoromethylphenyl-piperazine

PAPP (LY-165,163):

p-aminophenylethyl-m-trifluoromethylphenyl piperazine

mCPP:

1-(3-chlorophenyl)piperazinedihydrochloride

WB 4101:

2-(2,6-dimethoxyphenoxyethyl)aminoethyl1,4-benzodioxane hydrochloride

NAN-190:

1-(2-methoxyphenyl)-4-(2-phthalimido)butyl]piperazine

H-7:

1-(5-isoquinolinesulfonyl)-2-methylpiperazine

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

References

  • Albert PR, Zhou Q-Y, Van Tol HHM, Bunzow JR, Civelli O (1990) Cloning, functional expression and messenger RNA tissue distribution of the rat 5 hydroxytryptamine-l-A receptor gene. J Biol Chem 265:5825–5832

    Google Scholar 

  • Andrade R, Nicoll RA (1987) Pharmacologically distinct actions of serotonin on single pyramidal neurons of the rat hippocampus recorded in vitro. J Physiol (Lond) 349:99–124

    Google Scholar 

  • Ashkenazi A, Winslow JW, Peralta EG, Peterson GL, Schimmerlik MI, Capon DJ, Ramachandran J (1987) An M2 muscarinic receptor subtype coupled to both adenylyl cyclase and phosphoinositide turnover. Science 238:672–675

    Google Scholar 

  • Ashkenazi A, Peralta EG, Winslow JW, Ramachandran J, Capon DJ (1990) Functionally distinct G proteins selectively couple different receptors to PI hydrolysis in the same cell. Cell 56:487–493

    Google Scholar 

  • Axelrod J (1990) Receptor-mediated activation of phospholipase A2 and arachidonic acid release in signal transduction. Biochem Soc Trans 18:503–507

    Google Scholar 

  • Blier P, de Montigny C (1990) Electrophysiological investigation of the adaptive response of the 5-HT system to the administration of 5-HT1A receptor agonist. J Cardiovasc Pharmacol 15 [Suppl]:S42-S48

    Google Scholar 

  • Bockaert J, Dumuis A, Bouhelal R, Sebben M, Cory RN (1987) Piperazine derivatives including the putative anxiolytic drugs, buspirone and ipsapirone, are agonists at 5-HT1a receptors negatively coupled to adenylyl cyclase in hippocampal neurons. Naunyn-Schmiedeberg's Arch Pharmacol 335:588–592

    Google Scholar 

  • Boddeke HWGM, Fargin A, Raymond JR, Schoeffter P, Hoyer D (1992) Agonist/antagonist interactions with cloned human 5-HT1A receptors: variations in intrinsic activity in transfected HeLa cells. Naunyn-Schmiedeberg's Arch Pharmacol 345: 257–263

    Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye binding. Anal Biochem 72:248–254

    Google Scholar 

  • Claustre Y, Bénavides J, Seatton B (1988) 5-HT1A receptor agonists inhibit carbachol-stimulation of phosphoinositide turnover in the rat hippocampus. Eur J Pharmacol 149:149–153

    Google Scholar 

  • Colino A, Haliwell JV (1987) Differential modulation of three separate K-conductances in hippocampal CAI neurons by serotonin. Nature 328:73–77

    Google Scholar 

  • Cotecchia S, Kobilka BK, Daniel KW, Nolan R, Lapetina EY, Caron MG, Lefkowitz RJ, Regan JW (1990) Multiple second messenger pathways of α-adrenergic receptor subtypes expressed in eukaryotic cells. J Biol Chem 265:63–69

    Google Scholar 

  • Cullen BR (1987) Use of eukaryotic expression techniques in the functional analysis of cloned genes. Methods Enzymol 152:684–704

    Google Scholar 

  • DeLean A, Munson PJ, Rodbard D (1978) Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose-response curves. Am J Physiol 235:E97-E102

    Google Scholar 

  • DeLean A, Hancock AA, Lefkowitz RJ (1982) Validation and statistical analysis of a computer modeling method for quantitative analysis of radioligand binding data for mixtures of pharmacological receptor subtypes. Mol Pharmacol 21:5–16

    Google Scholar 

  • DeVivo M, Maayani S (1986) Characterization of 5-hydroxytryptamine receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes. J Pharmacol Exp Ther 238:248–253

    Google Scholar 

  • Dumuis A, Sebben M, Bockaert J (1988) Pharmacology of 5-hydroxytryptamine-l-A receptors which inhibit cAMP production in hippocampal and cortical neurons in primary culture. Mol Pharmacol 33:178–186

    Google Scholar 

  • Fargin A, Raymond JR, Lohse MJ, Kobilka BK, Caron MG, Lefkowitz RJ (1988) The genomic clone G-21 which resembles a β-adrenergic receptor sequence encodes the 5-HT1A receptor. Nature 335:358–360

    Google Scholar 

  • Fargin A, Raymond JR, Regan JW, Cotecchia S, Lefkowitz RJ, Caron MG (1989) Effector coupling mechanisms of the cloned 5-HT1A receptor. J Biol Chem 264:14848–14852

    Google Scholar 

  • Fargin A, Yamamoto K, Cotecchia S, Goldsmith PK, Spiegel AM, Lapetina EG, Caron MG, Lefkowitz RJ (1991) Dual coupling of the cloned 5-HT1A receptor to both adenylyl cyclase and phospholipase C is modulated by the same Gi protein. Cell Signal 3:547–557

    Google Scholar 

  • Felder CC, Dieter P, Kinsella J, Takamura K, Kanterman R, Axelrod J (1990) A transfected M5 muscarinic acetylcholine receptor stimulates phospholipase A2 by inducing both calcium influx and activation of protein kinase C. J Pharmacol Exp Ther 255:1140–1147

    Google Scholar 

  • Felder CC, Williams HL, Axelrod J (1991) A novel signal transduction pathway associated with Gi coupled receptors that amplifies ATP-mediated arachidonic acid release. Proc Natl Acad Sci USA 88:6477–6480

    Google Scholar 

  • Fraser CM, Arakawa S, McCombie WR, Venter JC (1989) Cloning, sequence analysis, and permanent expression of a human α2-adrenergic receptor in Chinese hamster ovary cells. J Biol Chem 264:11754–11761

    Google Scholar 

  • Gerhardt MA, Neubig RR (1991) Multiple Gi protein subtypes regulate a single effector mechanism. Mol Pharmacol 40:707–711

    Google Scholar 

  • Godson C, Weiss BA, Insel PA (1990) Differential activation of protein kinase C alpha′ is associated with arachidonic acid release in Madin-Darby canine kidney cells. J Biol Chem 265:8369–8372

    Google Scholar 

  • Gozlan H, El Mestikawy S, Pichat S, Glowinski J, Hamon M (1983) Identification of presynaptic serotonin autoreceptors using a new ligand: 3H-PAT. Nature 305:140–142

    Google Scholar 

  • Gupta SK, Diez E, Heasly LE, Osawa S, Johnson GL (1990) A G protein mutant that inhibits thrombin and purinergic receptor activation of phospholipase A2. Science 249:662–666

    Google Scholar 

  • Jones SB, Halenda SP, Bylund DB (1991) α2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese ovary cells is mediated by different mechanisms. Mol Pharmacol 39:239–245

    Google Scholar 

  • Jones SVP, Helman CJ, Brann MR (1991) Functional responses of cloned muscarinic receptors expressed in CHO-K1 cells. Mol Pharmacol 40:242–247

    Google Scholar 

  • Hamblin MW, Metcalf MA (1991) Primary structure and functional characterization of a human 5-HT1D-type serotonin receptor. Mol Pharmacol 40:143–148

    Google Scholar 

  • Hoyer D, Engel G, Kalkman HO (1985) Molecular pharmacology of 5-HT1 and 5-HT2 recognition sites in rat and pig brain membranes: radioligand binding studies with [3H]5-HT, [3H]8-OH-DPAT, (−)[125I]iodocyanopindolol, [3H]mesulergine and [3H]ketanserin. Eur J Pharmacol 118:13–23

    Google Scholar 

  • Kanterman RY, Mahan LC, Briley EM, Monsma FJ, Sibley DR, Axelrod J, Felder CC (1991) Transfected D2 dopamine receptors mediate the potentiation of aracbidonic acid release in Chinese hamster ovary cells. Mol Pharmacol 39:364–369

    Google Scholar 

  • Kobilka BK, Frielle T, Collins S, Yang-Feng T, Kobilka TS, Francke U, Lefkowitz RJ, Caron MG (1987) An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins. Nature 329:75–79

    Google Scholar 

  • Law SF, Manning D, Reisine T (1991) Identification of the subunits of GTP-binding proteins coupled to somatostatin receptors. J Biol Chem 266:17885–17897

    Google Scholar 

  • Liu YF, Albert PR (1991) Cell-specific signaling of the 5-HTIA receptor. J Biol Chem 266:23689–23697

    Google Scholar 

  • Limbird LE (1988) Receptors linked to the inhibition of adenylate cyclase: additional signaling mechanisms. FASEB J 2:2686–2695

    Google Scholar 

  • Markstein R, Hoyer D, Engel G (1986) 5-HTla-receptors mediate stimulation of adenylate cyclase in rat hippocampus. Naunyn-Schmiedeberg's Arch Pharmacol 333:335–341

    Google Scholar 

  • Matesic DF, Manning DR, Luthin GR (1991) Tissue-dependent association of muscarinic acetylcholine receptors with guanine nucleotide-binding regulatory proteins. Mol Pharmacol 40: 347–353

    Google Scholar 

  • Middleton JP, Raymond JR, Whorton AR, Dennis VW (1990) Short-term regulation of Na+/K+-ATPase by recombinant human 5-HT1A receptor expressed in HeLa cells. J Clin Invest 86:1799–1805

    Google Scholar 

  • Milligan G, Carr C, Gould GW, Mullaney I, Lavan BE (1991) Agonist-dependent, cholera toxin-catalyzed ADP-ribosylation of pertussis toxin-sensitive G-proteins following transfection of the human α2-C10 adrenergic receptor into rat 1 fibroblasts. J Biol Chem 266:6447–6455

    Google Scholar 

  • Peralta EG, Ashkenazi A, Winslow JW, Ramachandran JS, Capon DJ (1988) Differential regulation of phosphatidylinositol hydrolysis and adenylyl cyclase by muscarinic receptor subtypes. Nature (Lond) 334:434–437

    Google Scholar 

  • Piomelli D, Greengard P (1990) Lipoxygenase metabolites of arachidonic acid in neural transmembrane signalling. Trends Pharmacol Sci 11:367–373

    Google Scholar 

  • Piomelli D, Pilon C, Giros B, Sokoloff P, Martres M-P, Schwartz JC (1991) Dopamine activation of the arachidonic acid cascade as a basis for D1/D2 receptor synergism. Nature 353:164–167

    Google Scholar 

  • Raymond JR (1991) Protein kinase C induces phosphorylation and desensitization of the human 5-HT1A receptor. J Biol Chem 266:14747–14753

    Google Scholar 

  • Raymond JR, Fargin A, Middleton JP, Graff JM, Haupt DM, Caron MG, Lefkowitz RJ, Dennis VW (1989a) The human 5-HT1A receptor expressed in HeLa cells stimulates sodium-dependent phosphate uptake via protein kinase C. J Biol Chem 264:21943–21950

    Google Scholar 

  • Raymond JR, Fargin A, Lohse MJ, Regan JW, Senogles S, Lefkowitz RJ, Caron MG (1989b) Identification of the ligand binding subunit of the human 5-hydroxy-tryptamine1A receptor with N-(p-azido-m-[125I]iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe. Mol Pharmacol 35:15–21

    Google Scholar 

  • Raymond JR, Hnatowich M, Caron MG, Lefkowitz RJ (1990) Structure-function relationships of G-protein-coupled receptors. In: Moss J, Vaughan J (eds) ADP-Ribosylating toxins and G proteins. Am Soc Microbiol, Washington DC, pp 163–176

    Google Scholar 

  • Raymond JR, Albers FJ, Middleton JP, Caron MG, Lefkowitz RJ, Obeid LM, Dennis VW (1991) 5-HT1A and histamine H1 receptors in HeLa cells stimulate phosphoinositide hydrolysis and phosphate uptake via distinct G protein pools. J Biol Chem 266:372–379

    Google Scholar 

  • Rydelek-Fitzgerald L, Teitler M, Fletcher PW, Ismaiel AM, Glennon RA (1990) NAN-190, agonist and antagonist interactions with brain 5-HT1A receptors. Brain Res 532:191–196

    Google Scholar 

  • Salomon Y, Londos C, Rodbell M (1974) A highly sensitive adenylate cyclase assay. Anal Biochem 58:541–548

    Google Scholar 

  • Shenker A, Maayani S, Weinstein H, Green JP (1985) Two 5-HT receptors linked to adenylate cyclase in guinea pig hippocampus are discriminated by 5-carboxamidotryptamine and spiperone. Eur J Pharmacol 109:427–429

    Google Scholar 

  • Voight MM, McCune SK, Kanterman RY, Felder CC (1991) The rat α2-C4 adrenergic receptor gene encodes a novel pharmacological subtype. FEBS Lett 278:45–50

    Google Scholar 

  • Weiss BA, Insel PA (1991) Intracellular Ca2+ and protein kinase C interact to regulate α1-adrenergic- and bradykinin receptor-stimulated phospholipase A2 activation in Madin-Darby canine kidney cells. J Biol Chem 266:2126–2133

    Google Scholar 

  • Weiss S, Sebben M, Kempe D, Bockaert J (1986) Serotonin 5-HT1 receptors mediate inhibition of cyclic-AMP production in neurons. Eur J Pharmacol 120:227–230

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Medicine (Nephrology), Duke University Medical Center, 27710, Durham, NC, USA

    John R. Raymond, Frank J. Albers & John P. Middleton

  2. Medical Service (Nephrology), Department of Veterans Affairs Medical Center, 27705, Durham, NC, USA

    John R. Raymond & Frank J. Albers

Authors
  1. John R. Raymond
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Frank J. Albers
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. John P. Middleton
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Send offprint requests to J. R. Raymond

Rights and permissions

Reprints and permissions

About this article

Cite this article

Raymond, J.R., Albers, F.J. & Middleton, J.P. Functional expression of human 5-HT1A receptors and differential coupling to second messengers in CHO cells. Naunyn-Schmiedeberg's Arch Pharmacol 346, 127–137 (1992). https://doi.org/10.1007/BF00165293

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00165293

Key words

Search

Navigation