Characterization of a 5‐HT<sub>1B</sub> receptor on CHO cells: functional responses in the absence of radioligand binding

Giles, Heather; Lansdell, Stuart J.; Bolofo, Mary L.; Wilson, Heather L.; Martin, Graeme R.

doi:10.1111/j.1476-5381.1996.tb16705.x

Cited by 62 publications

(54 citation statements)

References 19 publications

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“…However, the efficiency with which the receptor is coupled to the signaling pathway must also be taken into account. A similar phenomenon has been observed for 5HT 1B receptors endogenously expressed in Chinese hamster ovary cells (Giles et al, 1996). In this case, the inability to detect receptor expression with radioligand binding indicated a very low level of receptor expression.…”

Section: Use Of Modified a 2a Ar Constructs With A Flash Tag Within Tsupporting

confidence: 81%

A Novel Nonribose Agonist, LUF5834, Engages Residues That Are Distinct from Those of Adenosine-Like Ligands to Activate the Adenosine A_2a Receptor

Lane¹,

Herenbrink²,

Westen³

et al. 2011

Mol Pharmacol

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The recent publication of both the antagonist-and agonist-bound structures of the adenosine A 2A receptor have revealed much about how a ligand may bind to a receptor and cause the conformational changes associated with agonist-mediated activation. In particular, the agonist-bound structure revealed key interactions between the ribose group of adenosine-derived agonists and amino acids in the receptor binding pocket that lead to receptor activation. However, agonists without a ribose group also exist, and we wondered whether such compounds occupy the same agonist binding site. Therefore we used a mutagenesis approach in this study to investigate the mode of binding of 2-amino-4-(4-hydroxyphenyl)-6-(1H-imidazol-2-ylmethylsulfanyl)pyridine-3,5-dicarbonitrile (LUF5834), a potent partial agonist without a ribose moiety, compared with the adenosine-derived reference agonist 2-[p-(2-carboxyethyl)phenyl-ethylamino]-5Ј-N-ethylcarboxamidoadenosine (CGS21680). Mutation of the orthosteric residue Phe168 to alanine abrogated the function of both agonists. However, mutation to alanine of residues Thr88 and Ser277 shown by the crystal structures to interact with the ribose group of adenosine-like ligands had no effect on the potency of LUF5834. Furthermore, alanine mutation of Asn253, which makes a hydrogen-bonding interaction with the exocyclic nitrogen of the adenine ring, had minimal effect on LUF5834 affinity but removed agonist activity of this ligand. Mutation of other residues, such as the highly conserved Trp246 or Glu13, had significant deleterious effects on the function of CGS21680 but little effect on LUF5834. In summary, our findings suggest that this class of agonist interacts with distinct residues to activate the receptor compared with classic adenosine derived agonists.

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Section: Use Of Modified a 2a Ar Constructs With A Flash Tag Within Tsupporting

confidence: 81%

A Novel Nonribose Agonist, LUF5834, Engages Residues That Are Distinct from Those of Adenosine-Like Ligands to Activate the Adenosine A_2a Receptor

Lane¹,

Herenbrink²,

Westen³

et al. 2011

Mol Pharmacol

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“…F 13640 and F14679, in contrast to 5-CT, are highly selective for the 5-HT 1A receptor. Table 2 compares binding affinities between 5-HT 1A and two receptor subtypes (5-HT 1B and 5-HT 2A ) that have been postulated to be endogenously expressed in CHO-K1 cells and C6-glial cells (Giles et al, 1996;Pauwels et al, 1996). F 13640 and F 14679 do not significantly bind to 5-HT 1B and 5-HT 2A receptors.…”

Section: Resultsmentioning

confidence: 99%

“…The Ca 2ϩ response induced by the native ligand 5-HT is likely to occur via activation of the recombinant 5-HT 1A receptor; no Ca 2ϩ signal could be detected in nontransfected CHO-K1 cells, although the Ca 2ϩ pathway was adequately responsive to bradykinin. CHO cells have previously been reported to express endogenous 5-HT 1B receptors, which are negatively coupled to adenylyl cyclase and positively coupled to increases in intracellular Ca 2ϩ formation (Dickenson and Hill, 1995;Giles et al, 1996). In the present study, the selective 5-HT 1B receptor antagonist SB 224289 (Gaster et al, 1998) affected the 5-HT-mediated Ca 2ϩ response little, if at all, whereas the selective 5-HT 1A receptor antagonist WAY 100635 (Fletcher et al, 1996) (Fargin et al, 1991), this may be different for CHO-K1 and C6-glial cells.…”

Section: Discussionmentioning

confidence: 99%

Ca²⁺Responses in Chinese Hamster Ovary-K1 Cells Demonstrate an Atypical Pattern of Ligand-Induced 5-HT_1AReceptor Activation

Pauwels¹,

Colpaert²

2003

J Pharmacol Exp Ther

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Little experimental evidence has been reported for diverse signaling via 5-hydroxytryptamine (5-HT) 1A receptors despite the fact that agonists seem to be more efficacious at dorsal raphe somatodendritic 5-HT 1A autoreceptors than at postsynaptic 5-HT 1A receptors. The present study investigated Ca 2ϩ responses in Chinese hamster ovary (CHO)-K1 cells expressing a human 5-HT 1A receptor by 5-HT, prototypical 5-HT

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“…Then, cells were preincubated for 15 min with serum-free medium supplemented with 5 mM theophylline, 10 lM pargyline and 1 lM GR127935 to block the activity of endogenous 5-HT 1B receptor [22]. 5-HT 4 R antagonists were also added during this preincubation period.…”

Section: Cyclic Amp Radioimmunoassaymentioning

confidence: 99%

Constitutive dimerization of human serotonin 5‐HT₄ receptors in living cells

et al. 2005

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Serotonin 5-HT 4 receptor isoforms are G proteincoupled receptors (GPCRs) with distinct pharmacological properties and may represent a valuable target for the treatment of many human disorders. Here, we have explored the process of dimerization of human 5-HT 4 receptor (h5-HT 4 R) by means of co-immunoprecipitation and bioluminescence resonance energy transfer (BRET). Constitutive h5-HT 4(d) R dimer was observed in living cells and membrane preparation of CHO and HEK293 cells. 5-HT 4 R ligands did not influence the constitutive energy transfer of the h5-HT 4(d) R splice variant in intact cells and isolated plasma membranes. In addition, we found that h5-HT 4(d) R and h5-HT 4(g) R which structurally differ in the length of their C-terminal tails were able to form constitutive heterodimers independently of their activation state. Finally, we found that coexpression of h5-HT 4 R and b 2 -adrenergic receptor (b 2 AR) led to their heterodimerization. Given the large number of h5-HT 4 R isoforms which are coexpressed in a same tissue, our results points out the complexity by which this 5-HTR sub-type mediates its biological effects.

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Characterization of a 5‐HT_1B receptor on CHO cells: functional responses in the absence of radioligand binding

Cited by 62 publications

References 19 publications

A Novel Nonribose Agonist, LUF5834, Engages Residues That Are Distinct from Those of Adenosine-Like Ligands to Activate the Adenosine A_2a Receptor

A Novel Nonribose Agonist, LUF5834, Engages Residues That Are Distinct from Those of Adenosine-Like Ligands to Activate the Adenosine A_2a Receptor

Ca²⁺Responses in Chinese Hamster Ovary-K1 Cells Demonstrate an Atypical Pattern of Ligand-Induced 5-HT_1AReceptor Activation

Constitutive dimerization of human serotonin 5‐HT₄ receptors in living cells

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Characterization of a 5‐HT1B receptor on CHO cells: functional responses in the absence of radioligand binding

Cited by 62 publications

References 19 publications

A Novel Nonribose Agonist, LUF5834, Engages Residues That Are Distinct from Those of Adenosine-Like Ligands to Activate the Adenosine A2a Receptor

A Novel Nonribose Agonist, LUF5834, Engages Residues That Are Distinct from Those of Adenosine-Like Ligands to Activate the Adenosine A2a Receptor

Ca2+Responses in Chinese Hamster Ovary-K1 Cells Demonstrate an Atypical Pattern of Ligand-Induced 5-HT1AReceptor Activation

Constitutive dimerization of human serotonin 5‐HT4 receptors in living cells

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Characterization of a 5‐HT_1B receptor on CHO cells: functional responses in the absence of radioligand binding

A Novel Nonribose Agonist, LUF5834, Engages Residues That Are Distinct from Those of Adenosine-Like Ligands to Activate the Adenosine A_2a Receptor

A Novel Nonribose Agonist, LUF5834, Engages Residues That Are Distinct from Those of Adenosine-Like Ligands to Activate the Adenosine A_2a Receptor

Ca²⁺Responses in Chinese Hamster Ovary-K1 Cells Demonstrate an Atypical Pattern of Ligand-Induced 5-HT_1AReceptor Activation

Constitutive dimerization of human serotonin 5‐HT₄ receptors in living cells