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

Pindon, Armelle; Hecke, Geert Van; Josson, Katty; Gompel, P. Van; Lesage, Anne; Leysen, Josée E.; Jurzak, Mirek

doi:10.1007/s10540-005-2582-5

Cited by 22 publications

(22 citation statements)

References 23 publications

(33 reference statements)

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“…We have introduced two point mutations in the extracellular extremity of rat NK2 (substitutions Thr 24 3 Ala and Phe 26 3 Ala). Y-NK2wt and Y-NK2mut chimeric receptors were generated by joining the carboxyl terminus of the yellow fluorescent protein EYFP to the amino-terminal part starting at residue Leu 16 of the wild-type and the mutant NK2 (Fig. 1A), as had previously been done for the wild-type NK2 receptor fused to the green fluorescent protein, EGFP (19,25).…”

Section: Construction and Expression Of The Fluorescent Y-nk2wt And Ymentioning

confidence: 99%

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Mutations in the Extracellular Amino-terminal Domain of the NK2 Neurokinin Receptor Abolish cAMP Signaling but Preserve Intracellular Calcium Responses

Lecat¹,

Bucher²,

Mély³

et al. 2002

Journal of Biological Chemistry

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G-protein-coupled receptors (GPCRs)1 represent the largest family of cell surface receptors and have multiple effects on the activated cell. Despite the variety of molecular types of ligands (hormones, proteins, small peptides, ions, lipids, and sensory stimuli such as odorants, pheromones, and photons), GPCRs share homology in their core domain composed of seven transmembrane ␣-helices. The conservation of the same overall hydrophobic structure exemplifies the essential role of this structure for the activation process.GPCR activation promotes GDP to GTP exchange on the ␣ subunit of the heterotrimeric G protein associated with its cytoplasmic loops, thereby initializing the intracellular cascade of signaling events. Among the theoretical models describing the activation mechanism, the two-state allosteric model of activation has been proposed, following the discovery of inverse agonists and of constitutive activity for GPCRs (1-4). In this model, a GPCR preexists in equilibrium between an active and an inactive state in the absence of ligand. As such, agonists, higher affinity for the active state having displace the equilibrium toward activation, whereas inverse agonists have higher affinities for the inactive state. Constitutive activity is explained by an intrinsic tendency for a receptor to spontaneously isomerize toward the active conformation. Although the multiple conformational states theory is an attractive model for describing GPCR activation, there is a need for more data correlating the parameters of possible intermediate conversion states with physiological responses.Indeed, several GPCRs have been reported to mediate multiple signaling pathways through activation of different heterotrimeric G proteins: the receptors for dopamine D1 (G s and G o (5), G s and G q (6)) and D5 (G s and G z (7)), the receptors for the parathyroid hormone (G s , G q , and G i (8)), for the corticotropinreleasing hormone (G s , G i , G q , G o , and G z (9)), for the melaninconcentrating hormone (G i and G o and maybe G q (10)), for the vasoactive intestinal peptide (G s and G i (11)), for prostacyclin (G s , G i , and G q (12)), the adenosine A1 receptor (G o and G i (13)), the ␤ 2 -adrenergic receptor (G s and G i (14)), the muscarinic m3 receptor (G q and G 12 (15)), the 5-hydroxytryptamine receptor type 4; Bpa, G o , and G s (16)), the endothelin subtype B receptor (G i , G o , and G q (17, 18)). These different couplings suggest that GPCRs may exist in more than a single active state.We have previously addressed the question of multiple receptor states by using fluorescence resonance energy transfer (FRET) to monitor ligand binding in parallel with cellular responses. A chimeric green fluorescent neurokinin NK2 receptor, EGFP-NK2R was expressed in HEK293 cells. Two of its neuropeptide agonists, the decapeptide neurokinin A (NKA) and its truncated form, NKA-(4 -10), were covalently linked to the fluorophore Texas Red (TR) (19). Kinetic measurements of the associations of TR1-NKA and TR7-NKA-(4 -10) to the EGFP-NK2 re...

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Section: Construction and Expression Of The Fluorescent Y-nk2wt And Ymentioning

confidence: 99%

“…(G i , G o , and G s (16)), the endothelin subtype B receptor (G i , G o , and G q (17,18)). These different couplings suggest that GPCRs may exist in more than a single active state.…”

mentioning

confidence: 99%

Mutations in the Extracellular Amino-terminal Domain of the NK2 Neurokinin Receptor Abolish cAMP Signaling but Preserve Intracellular Calcium Responses

Lecat¹,

Bucher²,

Mély³

et al. 2002

Journal of Biological Chemistry

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“…98 These two splice variants exhibit differential internalizations: the agonist stimulation results in a time-dependent internalization of the h5-HT 4b R, but not of the h5-HT 4a R. The h5-HT 4b R internalization is PTX insensitive, revealing that coupling to G is not necessary. 99 For the murine 5-HT 4 R (m5-HT 4 R) also, differences in desensitization kinetics were observed between splice variants. Serotonin induced a rapid desensitization of the adenylyl cyclase response mediated by the m5-HT 4d receptor that desensitized with a faster rate than the one induced by the m5-HT 4e receptor in CHO cells.…”

Section: -Ht 4 R Internalization and Desensitizationmentioning

confidence: 99%

Insights into Serotonin Receptor Trafficking

Darmon

Awabdh

Emerit

et al. 2015

Progress in Molecular Biology and Translational Science

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“…The second binding component had a lower apparent affinity with a K d of 8.5 ± 3.8 nM and B max of 385 ± 21 fmol/mg protein in CHO and a K d of 6.6 ± 3 nM and B max of 1052 ± 434 fmol/mg protein in COS-7 cells. Highand low-affinity 5-HT binding sites have also been identified in rat brain cells expressing the native 5-HT 4 receptor (Adham et al 1996) and in HEK-293 cells transiently expressing the h5-HT 4(a),(b) receptors (Pindon et al 2002).…”

Section: Fractions Of High-affinity H5-ht 4(e) Receptors Differ In Chmentioning

confidence: 99%

“…The B max for [ 3 H]GR113808 was 347 fmol/mg in CHO cells (Mialet et al 2000) and 375 fmol/mg in COS-7 cells (Bozon et al 2002). The agonist fraction (Pindon et al 2002) is determined by the fraction R * /R total corresponding to the B max of high-affinity [ 3 H]5-HT divided by the B max of [ 3 H]GR113808. The equilibrium constant J can be estimated by (R total − R * )/R * corresponding to the B max of [ 3 H]GR113808 minus the B max of high-affinity [ 3 H]5-HT divided by the B max of high-affinity [ 3 H]5-HT.…”

Section: Fractions Of High-affinity H5-ht 4(e) Receptors Differ In Chmentioning

confidence: 99%

Polyclonal Antibody Effects on the Human Cardiac 5-HT4(e) Receptors Depend Upon the Expression System

Scala¹,

Rose²,

Hérault³

et al. 2004

Receptors and Channels

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The initial objective of this work was to examine the effects of an antibody (Anti-G21V) directed against the second extracellular loop of human heart 5-HT4 receptors expressed in Chinese hamster ovary (CHO) cells. The antibody anti-G21V had no effect upon either basal cAMP-or 5-HT-evoked increases in cAMP in CHO cells, whereas it had shown an agonist-like effect in COS-7 cells. Analysis of agonist fractions of h5-HT4(e) receptors in CHO and COS-7 cells revealed that equilibrium constant could underlie the different responses of the receptor toward the anti-G21V antibody. Therefore, different expression systems could give rise to functional differences in 5-HT4 receptor behavior.

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

Cited by 22 publications

References 23 publications

Mutations in the Extracellular Amino-terminal Domain of the NK2 Neurokinin Receptor Abolish cAMP Signaling but Preserve Intracellular Calcium Responses

Mutations in the Extracellular Amino-terminal Domain of the NK2 Neurokinin Receptor Abolish cAMP Signaling but Preserve Intracellular Calcium Responses

Insights into Serotonin Receptor Trafficking

Polyclonal Antibody Effects on the Human Cardiac 5-HT4(e) Receptors Depend Upon the Expression System

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