Skip to main content
SpringerLink
Log in

Internalization of Human 5-HT4a and 5-HT4b Receptors is Splice Variant Dependent

  • Published:
Bioscience Reports

Abstract

The family of 5-HT4 receptors comprises 16 putative splice variants. We have previously shown that there are differences in signal transduction of the h5-HT4a and h5-HT4b receptors. In the present study, the internalization of these two splice variants following receptor stimulation was investigated with confocal microscopy on living cells. Chimeric receptors, h5-HT4a-GFP and h5-HT4b-GFP were generated by fusing the coding sequence of the 5-HT4 receptor with the coding sequence of the GFP. The agonist stimulation of fluorescent receptors resulted in a time-dependent internalization of the h5-HT4b-GFP receptor, but not of the h5-HT4a-GFP receptor. The h5-HT4b receptor displays a dual coupling to Gαi,o and Gαs proteins, in contrast to the h5-HT4a receptor, which couples to Gαs proteins only. We investigated whether the difference in internalization of the two splice variant receptors was related to their differential coupling. Therefore, we performed agonist-stimulation of the receptor following inhibition of the Gαi,o protein coupling using PTX. The h5-HT4b receptor internalization is PTX insensitive. We co-transfected the fluorescent chimeric receptors with other wild-type variants, which did not produce an alteration of the receptor trafficking. These findings provide the first evidence of differential internalization between the two splice variants, 5-HT4a and 5-HT4b receptors.

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

DMEM:

Dulbecco’s modified Eagle medium

FCS:

fetal calf serum

GFP:

green fluorescent protein

GPCR:

G-protein coupled receptor

GRK:

G-protein coupled receptor kinase

h5-HT4R:

human 5-hydroxytryptamine4receptor

5-HT:

hydroxytryptamine (serotonin)

K D :

dissociation constant

PTX:

Bordatella pertussis toxin.

References

  • L. S. Barak L. Mènard S. S. G. Ferguson A. -M. Colapietro M. G Caron (1995) ArticleTitleThe conserved seven-transmembrane sequence NP(X)2,,3Y of the G-protein-coupled receptor superfamily regulates properties of the β2-adrenergic receptor Biochemistry 34 15407–15414

    Google Scholar 

  • J. Barlic L. S. Khandaker M. H. Mahon J. Andrews M. E. Devries G. B. Mitchell R. Rahimpour S. S. G. Ferguson D. J Kevin (1999) ArticleTitleβ-Arrestins regulate interleukin-8 (IL8)-induced CXCR1 internalization J. Biol. Chem 274 1687–1694

    Google Scholar 

  • E. Bender A. Pindon I. Van Oers Y. B. Zhang W. Gommeren P. Verhasselt M. Jurzak J. Leysen W Luyten (2000) ArticleTitleStructure of the human serotonin 5-HT4 receptor gene and cloning of a novel 5-HT4 splice variant J. Neurochem 74 IssueID2 478–489

    Google Scholar 

  • M. Bouvier W. P. Hausdorff A. De Blasi B. F. O’dowd B. K. Kobilka M. G. Caron R. J Lefkowitz (1988) ArticleTitleRemoval of phosphorylation sites from the β2-adrenergic receptor delays the onset of agonist-promoted desensitization Nature (Lond) 333 370–373

    Google Scholar 

  • A. Dumuis R. Bouhelal M. Sebben R. Cory J Bockaert (1988) ArticleTitleA non-classical 5-hydroxytryptaminereceptor positively coupled with adenylate cyclase in the central nervous system Mol. Pharmacol 34 880–887

    Google Scholar 

  • S. S. G. Ferguson L. S. Barak J. Zhang M. G Caron (1996) ArticleTitleG-protein-coupled receptor regulation: Role of G-protein-coupled receptor kinases and arrestins Can. J. Physiol. Pharmacol 74 1095–1110

    Google Scholar 

  • S. S. G. Ferguson M. G Caron (1998) ArticleTitleG protein-coupled receptor adaptation mechanisms Semin. Cell Dev. Biol 9 119–127

    Google Scholar 

  • S. S. G. Ferguson (2001) ArticleTitleEvolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signalling Pharmacol. Rev 53 1–24

    Google Scholar 

  • D. J. Fontana S. E. Daniels E. H. Wong R. D. Clark R. M Eglen (1997) ArticleTitleThe effects of novel, selective 5-Hydroxytryptamine (5-HT)4 receptor ligands in rat spatial navigation Neuropharmacol 36 689–696

    Google Scholar 

  • APDW. Ford D. E. Clarke (1993) ArticleTitleThe 5-HT4 receptor Med. Res. Rev 13 633–662

    Google Scholar 

  • N. Galeotti C. Gherlardini A Bartolini (1998) ArticleTitleRole of 5-HT4 receptors in the mouse passive avoidance test J. Pharmacol. Exp. Ther 286 1115–1121

    Google Scholar 

  • R. Jockers A. Da Silva A. D. Strosberg M. Bouvier S Marullo (1996) ArticleTitleNew molecular and structural determinants involved in β2-adrenergic receptor desensitisation and sequestration: delineation using chimeric β3/β2-adrenergic receptors J. Biol. Chem 271 9355–9362

    Google Scholar 

  • T. Kirchhausen (1999) ArticleTitleAdaptors for clathrin-mediated traffic Annu. Rev. Cell Dev. Biol 15 705–732

    Google Scholar 

  • J. G. Krupnick J. L Benovic (1998) ArticleTitleThe role of receptor kinases and arrestins in G protein-coupled receptor regulation Annu. Rev. Pharmacol. Toxicol 38 289–319

    Google Scholar 

  • R. J. Lefkowitz (1993) ArticleTitleG protein-coupled receptor kinases Cell 74 409–412

    Google Scholar 

  • A. S. Lesage R. Wouters P. Van Gompel L. Heylen P. Vanhoenacker W. H. Luyten J.’E Leysen (1998) ArticleTitleAgonistic properties of alniditan,, sumatriptan and dihydroergotamine on human 5-HT1B and 5-HT1D receptors expressed in various mammalian cell lines Br. J. Pharmacol 123 1655–1665

    Google Scholar 

  • E. Leung M. T. Pulidos-Rios D. W. Bonhaus A. Perkins K. D. Zeitung S. A. O. Hsu R. D. Clark E. H. Wong R. M Eglen (1996) ArticleTitleComparison of 5-HT4 Receptors in guinea-pig colon and rat oesophagus: effects of novel agonists and antagonists Naunyn-Schmiedeberg’s Arch. Pharmacol 354 145–156

    Google Scholar 

  • S. Moffett L. Adam H. Bonin T. P. Loisel M. Bouvier B Mouillac (1996) ArticleTitlePalmitoylated cysteine 341 modulates phosphorylation of the beta 2-adrenergic receptor by the cAMP-dependent protein kinase J. Biol. Chem 271 21490–21497

    Google Scholar 

  • C. Moummi D. C. Yang G. W Gullikson (1992) ArticleTitle5-HT4 receptor activation induces relaxation and associated cAMP generation in rat esophagus Eur. J. Pharmacol 216 47–52

    Google Scholar 

  • A. Pindon G. Van Hecke P. Van Gompel A. S. Lesage J. E. Leysen M Jurzak (2002) ArticleTitleDifferences in signal transduction of two 5-HT4 receptor splice variants: compound specificity and dual coupling with Galphas- and Galphai/o-proteins Mol. Pharmacol 61 IssueID1 85–96

    Google Scholar 

  • R. R. Price D. P. Morris G. Biswas M. P. Smith D. A Schwinn (2002) ArticleTitleAcute agonist mediated desensitisation of the human α1a-adrenergic receptor is primarily independent of carboxyl terminus regulation J. Biol. Chem 277 IssueID11 9570–9579

    Google Scholar 

  • J. S. Silvestre A. G. Fernandez J. M Palacios (1996) ArticleTitleEffects of 5-HT4 receptor antagonists on rat behavior in the elevated plus-maze test Eur. J. Pharmacol 309 219–222

    Google Scholar 

  • M. T. Vilarò T. Domenech J. M. Palacios G Mengod (2002) ArticleTitleCloning and characterization of novel human 5-HT4 receptor variant that lacks the alternatively spliced carboxy terminal exon. RT-PCR distribution in human brain and periphery of multiple HT4 receptor variants Neuropharmacol 42 60–73

    Google Scholar 

  • N. Yuan J. Friedman B. S. Whaley R. B Clark (1994) ArticleTitlecAMP-dependent protein kinase and protein kinase C consensus site mutations of the beta-adrenergic receptor. Effect on desensitization and stimulation of adenylylcyclase J. Biol. Chem 269 23032–23038

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. A division of Janssen Pharmaceutica, JOHNSON & JOHNSON PHARMACEUTICAL RESEARCH & DEVELOPMENT, N.V. Turnhoutseweg 30, B-2340, Beerse, Belgium

    Armelle Pindon

  2. Department of Metabolic Disorder, J & J PRD, Belgium

    Geert Van Hecke

  3. De Clercq-Brants en Partners, Edgard Gevaertdreef 10/a, 9830, Sint-Martens-Latem, Belgium

    Katty Josson

  4. Department of Enabling Technology, J & J PRD, Belgium

    Paul Van Gompel

  5. Department of Central Nervous System, J & J PRD, Belgium

    Anne Lesage

  6. J & J Pharmaceutical Licensing, Belgium

    Josee E. Leysen

  7. CADS, Preclinical R & D, Biomedical Research Merck KgaA Darmstadt, Frankfurt Straße 250, 64293, Darmstadt, Germany

    Mirek Jurzak

Authors
  1. Armelle Pindon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Geert Van Hecke
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Katty Josson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Paul Van Gompel
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Anne Lesage
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Josee E. Leysen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mirek Jurzak
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Armelle Pindon.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pindon, A., Hecke, G.V., Josson, K. et al. Internalization of Human 5-HT4a and 5-HT4b Receptors is Splice Variant Dependent. Biosci Rep 24, 215–223 (2004). https://doi.org/10.1007/s10540-005-2582-5

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10540-005-2582-5

Key words

Search

Navigation