Allosteric modulation and constitutive activity of fusion proteins between the adenosine A1 receptor and different 351Cys-mutated Gi α-subunits

Klaasse, Elisabeth; Ligt, Rianne A.F. de; Roerink, Sophie F.; Lorenzen, Anna; Milligan, Graeme; Leurs, Rob; IJzerman, Adriaan P.

doi:10.1016/j.ejphar.2004.07.108

Cited by 5 publications

(5 citation statements)

References 20 publications

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“…The extent of stimulation is less than the maximal agonist-stimulated increase detected in membranes containing an equivalent amount (58.6 fmol) of C351I fusion protein (basal: 4.8 ± 1 pmol (mg of protein) -1 min -1 ; stimulated: 24 ± 3 pmol (mg of protein) -1 min -1 ). This result is consistent with less efficient stimulation of wt as compared to C351I fusion proteins for equivalent amounts of receptor as reported previously using radiolabeled GTP as the substrate 3 Agonist-stimulated specific GTPase activity can be modulated with RGS8.…”

Section: Resultssupporting

confidence: 92%

“…An added advantage is that the extent of nucleotide binding in the presence of agonist is significantly elevated in fusion systems as compared to unfused co-expressed receptors and G α subunits. The use of G α o1 with the 351 Cys→Ile mutation has been shown to further enhance GTP binding and enhance sensitivity of GPCR activation assays …”

Section: Resultsmentioning

confidence: 99%

“…One area of improvement is assay reproducibility. The reproducibility of assays used in high-throughput screening may be assessed using the Z ‘-factor, which is calculated as Z ‘ = 1.0 − (3.0 × ( s neg + s pos )/ R ) where s neg is the standard deviation of the response of a negative control (no drug), s pos is the standard deviation of the response to a positive control (active drug), and R is the difference in signal between the mean of positive and negative controls. − The maximum Z ‘ is 1.0, and Z ‘ over 0.5 is considered necessary for high-throughput screens. The assay described had a Z ‘ = 0.4 suggesting that improvements were needed to make this assay viable for high-throughput screens.…”

Section: Resultsmentioning

confidence: 99%

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Capillary Electrophoresis Assay for G Protein-Coupled Receptor-Mediated GTPase Activity

et al. 2006

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We describe a capillary electrophoresis (CE) assay to detect G protein-coupled receptor (GPCR)-stimulated G protein GTPase activity in cell membranes expressing alpha2A adrenoreceptor-Galphao1 wild-type (wt) or C351I mutant fusion proteins using a fluorescent, hydrolyzable GTP analogue. As no change in total fluorescence is observed by conversion of substrate to product, CE is used to separate the fluorescent substrate (*GTP) from the fluorescent product (*GDP). Using the assay, the alpha2a adrenoceptor agonist UK14,304 was shown to simulate specific production of *GDP in membranes from HEK293T cells expressing receptor-G protein fusion to 525% of basal levels with an EC50 of 0.48 +/- 0.20 microM. The EC50 increased to 9.4 +/- 5 muM with addition of the antagonist yohimbine. Nucleotide hydrolysis was increased further over agonist-stimulated levels with addition of the in vivo modulator protein RGS (regulator of G protein signaling). It is envisioned that this technique could be used for screening for novel GPCR ligands or other G protein signaling modifiers.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Capillary Electrophoresis Assay for G Protein-Coupled Receptor-Mediated GTPase Activity

et al. 2006

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“…Coexpression of the wt rAdeR, or its mutants, respectively, with mammalian G i proteins allowed the performance of [ 35 S]GTPγS binding studies, which can indicate the degree of receptor activation [42][43][44]. As already mentioned, G proteins can allosterically modulate the binding of an agonist to its receptor.…”

Section: Discussionmentioning

confidence: 99%

The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site

Knospe

Müller

Rosa

et al. 2013

Purinergic Signalling

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The rat adenine receptor (rAdeR) was the first member of a family of G protein-coupled receptors (GPCRs) activated by adenine and designated as P0-purine receptors. The present study aimed at gaining insights into structural aspects of ligand binding and function of the rAdeR. We exchanged amino acid residues predicted to be involved in ligand binding (Phe110 3.24 47 , were found to be crucial for activation since their alanine mutants did not respond to adenine. Moreover we showed that-in contrast to most other rhodopsin-like GPCRs-the rAdeR does not contain essential disulfide bonds since preincubation with dithiothreitol neither altered adenine binding in Sf9 cell membranes, nor adenineinduced inhibition of adenylate cyclase in 1321N1 astrocytoma cells transfected with the rAdeR. To detect rAdeRs by Western blot analysis, we developed a specific antibody. Finally, we were able to show that the extended N-terminal sequence of the rAdeR constitutes a putative signal peptide of unknown function that is cleaved off in the mature receptor. Our results provide important insights into this new, poorly investigated family of purinergic receptors.

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“…This increase in binding was abrogated in the presence of DPCPX and not influenced by the addition of adenosine deaminase, excluding a role for endogenous adenosine in these observations. In yet another approach, Klaasse et al (2004) studied the behavior of PD81,723 on receptor-Gα i protein fusion products. The effects of 10 μM PD81,723 on ligand binding were rather similar for both the unfused A 1 AR (expressed in G protein-poor COS cells) and the different fusion proteins (mutations in the α-subunit and expressed in the same cell line).…”

Section: ‘Translational’ Assessment Of a Prototypical Pam Of A1 Armentioning

confidence: 99%

Allosteric Modulation of Purine and Pyrimidine Receptors

Gao¹,

Göblyös²,

IJzerman³

2011

Advances in Pharmacology

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Among the purine and pyrimidine receptors, the discovery of small molecular allosteric modulators has been most highly advanced for the A1 and A3 ARs. These AR modulators have allosteric effects that are structurally separated from the orthosteric effects in SAR studies. The benzoylthiophene derivatives tend to act as allosteric agonists, as well as selective positive allosteric modulators (PAMs) of the A1 AR. A 2-amino-3-aroylthiophene derivative T-62 has been under development as a PAM of the A1 AR for the treatment of chronic pain. Several structurally distinct classes of allosteric modulators of the human A3 AR have been reported: 3-(2-pyridinyl)isoquinolines, 2,4-disubstituted quinolines, 1H-imidazo-[4,5-c]quinolin-4-amines, endocannabinoid 2-arachidonylglycerol and the food dye Brilliant Black BN. Site-directed mutagenesis of A1 and A3 ARs has identified residues associated with the allosteric effect, distinct from those that affect orthosteric binding. A few small molecular allosteric modulators have been reported for several of the P2X ligand-gated ion channels and the G protein-coupled P2Y receptor nucleotides. Metal ion modulation of the P2X receptors has been extensively explored. The allosteric approach to modulation of purine and pyrimidine receptors looks promising for development of drugs that are event-specific and site-specific in action.

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Allosteric modulation and constitutive activity of fusion proteins between the adenosine A1 receptor and different 351Cys-mutated Gi α-subunits

Cited by 5 publications

References 20 publications

Capillary Electrophoresis Assay for G Protein-Coupled Receptor-Mediated GTPase Activity

Capillary Electrophoresis Assay for G Protein-Coupled Receptor-Mediated GTPase Activity

The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site

Allosteric Modulation of Purine and Pyrimidine Receptors

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