Ligand-Induced Phosphorylation, Clustering, and Desensitization of A<sub>1</sub>Adenosine Receptors

Ciruela, Francisco; Saura, Carles; Canela, Enric I.; Mallol, Josefa; Lluı́s, Carmen

doi:10.1124/mol.52.5.788

Cited by 76 publications

(56 citation statements)

References 33 publications

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“…Previous studies showed that A 1 R agonist (R)-PIA induced a time-dependent reduction in cell surface adenosine A1 receptor radioligand binding sites, which reached a maximum at 48-72 h [31,32] . To examine whether heterologous desensitization of the DOR by CHA was attributed to receptor down-regulation, saturation binding was used to assess receptor affinity (K d ) and receptor density (B max ) of DOR in plasma membranes prepared from cells pretreated with or without 1 μmol/L CHA for 72 h. Saturation curves of DOR ( Figure 5A) and the Scatchard analysis of the saturation binding ( Figure 5B) were present in Figure 5, which showed no significant change of receptor numbers and affinity after chronic CHA exposure.…”

Section: 92±443mentioning

confidence: 99%

Adenosine A1 receptor agonist N6-cyclohexyl-adenosine induced phosphorylation of delta opioid receptor and desensitization of its signaling

et al. 2010

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Aim:To define the effect of adenosine A 1 receptor (A 1 R) on delta opioid receptor (DOR)-mediated signal transduction. Methods: CHO cells stably expressing HA-tagged A 1 R and DOR-CFP fusion protein were used. The localization of receptors was observed using confocal microscope. DOR-mediated inhibition of adenylyl cyclase was measured using cyclic AMP assay. Western blots were employed to detect the phosphorylation of Akt and the DOR. The effect of A 1 R agonist N 6 -cyclohexyladenosine (CHA) on DOR down-regulation was assessed using radioligand binding assay.]enkephalin (DPDPE)-induced inhibition of intracellular cAMP accumulation with a t 1/2 =2.56 (2.09-3.31) h. Pretreatment with 1 μmol/L CHA for 24 h caused a right shift of the dose-response curve of DPDPE-mediated inhibition of cAMP accumulation, with a significant increase in EC 50 but no change in E max . Pretreatment with 1 μmol/L CHA for 1 h also induced a significant attenuation of DPDPE-stimulated phosphorylation of Akt. Moreover, CHA time-dependently phosphorylated DOR (Ser363), and this effect was inhibited by A 1 R antagonist 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) but not by DOR antagonist naloxone. However, CHA failed to produce the down-regulation of DOR, as neither receptor affinity (K d ) nor receptor density (B max ) of DOR showed significant change after chronic CHA exposure. Conclusion: Activation of A 1 R by its agonist caused heterologous desensitization of DOR-mediated inhibition of intracellular cAMP accumulation and phosphorylation of Akt. Activation of A 1 R by its agonist also induced heterologous phosphorylation but not downregulation of DOR.

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Section: 92±443mentioning

confidence: 99%

Adenosine A1 receptor agonist N6-cyclohexyl-adenosine induced phosphorylation of delta opioid receptor and desensitization of its signaling

et al. 2010

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“…Untreated and R-PIA-treated cells were fixed, incubated with fluorescein-conjugated rabbit anti-A 1 R antibody (PC21-FITC), and analyzed by confocal microscopy. The PC21 antibody is directed against the second extracellular loop of A 1 R and its specificity for cell surface-expressed A 1 R has been previously demonstrated (32). Receptor distribution in untreated cells revealed homogenous bright staining on the surface of DDT 1 MF-2 cells (Fig.…”

Section: Effect Of Ada On Cell Surface Distribution Of a 1 Rs In Thementioning

confidence: 99%

“…2, bottom). ADA Affects Ligand-induced Phosphorylation of A 1 Rs-In a previous study, we have shown that ligand-induced phosphorylation of A 1 R correlates with the clustering of receptors on the DDT 1 MF-2 cell surface (32). To determine whether the potentiation of the clustering by ADA could involve changes in receptor phosphorylation, we immunoprecipitated the receptor from cells metabolically labeled with [ 32 P]orthophosphate.…”

Section: Effect Of Ada On Cell Surface Distribution Of a 1 Rs In Thementioning

confidence: 99%

Adenosine Deaminase and A1 Adenosine Receptors Internalize Together following Agonist-induced Receptor Desensitization

Saura

Mallol

Canela

et al. 1998

Journal of Biological Chemistry

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Adenosine is an autacoid that exerts its physiological actions through specific cell surface receptors. Four adenosine receptors (A 1 , A 2A , A 2B , and A 3 ), belonging to the family of G protein-coupled receptors, have been cloned and pharmacologically characterized (1). Acting through different adenosine receptors, adenosine is a neuromodulator in both the central and peripheral nervous systems (2, 3). Via A 1 adenosine receptors (A 1 Rs), 1 adenosine reduces heart rate (4), glomerular filtration rate, and renin release in the kidney (5); it induces bronchoconstriction (6, 7) and inhibits lipolysis. A 1 Rs can be coupled to different pertussis toxin-sensitive G proteins (8 -10), which mediate inhibition of adenylate cyclase (11) and regulate Ca 2ϩ and K ϩ channels and inositol phosphate metabolism (1). A 1 R displays two different affinities for agonist, which have classically been attributed to a different coupling to heterotrimeric G proteins (12). According to this two-independent site model, coupled receptor-G protein complexes display high affinity for agonists (K d ϭ 0.1-0.2 nM), and uncoupled receptors display low affinity (1-2 nM) (12, 13). The recently reported cluster-arranged cooperative model predicts that the high and low affinity sites are a consequence of the negative cooperativity of agonist binding and do not seem to be related to the content of free and G protein-coupled receptors (14). According to the cluster-arranged cooperative model, the agonist-induced conversion of the high to the low affinity state may partially explain the ligand-induced desensitization of A 1 R (14, 15).Receptors belonging to the G protein-coupled receptor family are desensitized and down-regulated in response to agonist stimulation. ␤ 2 -Adrenergic receptors (␤ 2 -ARs) constitute the best characterized system within the G protein-coupled receptor family. Agonist-induced ␤ 2 -AR desensitization is caused by a conformational change of the agonist-occupied receptor that facilitates receptor phosphorylation by second messenger-activated kinases or G protein-coupled receptor kinases (16,17). Following ␤ 2 -AR phosphorylation, ␤-arrestin binds to the phosphorylated receptor and uncouples the receptor from the heterotrimeric G proteins (18,19). ␤-Arrestin not only desensitizes the receptor but also functions as a clathrin adaptor, mediating receptor sequestration, i.e. receptor internalization toward intracellular compartments (20,21). Although sequestration is not required for phosphorylation and desensitization, it appears to be necessary for dephosphorylation and resensitization of ␤ 2 -ARs (22, 23).Like other G protein-coupled receptor members, A 1 R expression is regulated in response to agonist or antagonist stimulation. Desensitization of A 1 R has been described in intact animals and in cell cultures. Prolonged administration of A 1 R agonists to animals leads to functional desensitization of A 1 R in guinea pig heart (24), rat adipocytes (25), rat atrial muscle (26), and rat brain (27,28). The reduced functiona...

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“…Partially purified receptors were reported to undergo rapid (5 min) [35] or slow (18 h) agonist-stimulated phosphorylation [36]. However, in this type of experiment it may be difficult to distinguish minor contaminating $#P-labelled phosphoproteins from phosphorylated receptors in instances in which receptors are impure.…”

Section: Discussionmentioning

confidence: 99%

“…Phosphorylated receptors cannot be resolved from contaminating non-receptor phosphoproteins unless they are deglycosylated. Using deglycosylation to confirm the identity of the putative receptor phosphoprotein has not been used in previous studies, such as in the study by Ciruela et al [35], who reported that native A " ARs partially purified by immunoprecipitation from DDT " MF # cells display a 7-fold increase in [$#P]phosphotyrosine into a 36 kDa putative receptor within minutes. These results are surprising in view of studies which show that desensitization of A " ARs on DDT " MF # cells takes several hours [36].…”

Section: Discussionmentioning

confidence: 99%

Purification of A1 adenosine receptor–G-protein complexes: effects of receptor down-regulation and phosphorylation on coupling

Gao

Robeva

Linden

1999

Biochemical Journal

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We examined the effects of exposing A1 adenosine receptors (A1ARs) to an agonist on the stability and phosphorylation state of receptor–guanine nucleotide-binding regulatory protein (R–G-protein) complexes. Non-denatured recombinant human A1ARs extended on the N-terminus with hexahistidine (His6) and the FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) epitope (H/F) were purified to near homogeneity from stably transfected Chinese-hamster ovary (CHO)-K1 cells. Purified receptors have pharmacological properties similar to receptors in membranes. G-proteins were co-purified with 15±2% of H/F-A1AR unless receptor–G-protein (R–G) complexes were uncoupled by pre-treating cell membranes with GTP. By silver staining, purified A1AR–G-protein complexes contain receptors, G-protein α and β subunits and an unidentified 97 kDa protein. Pretreating intact cells with N6-cyclopentyladenosine (CPA) for 24 h decreased both the total number of receptors measured in membranes and the number of purified A1ARs by about 50%. In contrast, pretreating cells with CPA decreased the number of R–G complexes measured in membranes (54±6%) significantly less than it decreased the number of purified R–G complexes (78±3%) as detected by 125I-N6-(4-aminobenzyl)adenosine binding or by Western blotting Giα2. The effect of CPA to decrease the fraction of receptors purified as R–G complexes was not associated with any change in low-level A1AR phosphorylation (found on serine), or low-level phosphorylation of G-protein α or β subunits or the 97 kDa protein. These experiments reveal a novel aspect of agonist-induced down-regulation, namely a diminished stability of receptor–G-protein complexes that is manifested as uncoupling during receptor purification.

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Ligand-Induced Phosphorylation, Clustering, and Desensitization of A₁Adenosine Receptors

Cited by 76 publications

References 33 publications

Adenosine A1 receptor agonist N6-cyclohexyl-adenosine induced phosphorylation of delta opioid receptor and desensitization of its signaling

Adenosine A1 receptor agonist N6-cyclohexyl-adenosine induced phosphorylation of delta opioid receptor and desensitization of its signaling

Adenosine Deaminase and A1 Adenosine Receptors Internalize Together following Agonist-induced Receptor Desensitization

Purification of A1 adenosine receptor–G-protein complexes: effects of receptor down-regulation and phosphorylation on coupling

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Ligand-Induced Phosphorylation, Clustering, and Desensitization of A1Adenosine Receptors

Cited by 76 publications

References 33 publications

Adenosine A1 receptor agonist N6-cyclohexyl-adenosine induced phosphorylation of delta opioid receptor and desensitization of its signaling

Adenosine A1 receptor agonist N6-cyclohexyl-adenosine induced phosphorylation of delta opioid receptor and desensitization of its signaling

Adenosine Deaminase and A1 Adenosine Receptors Internalize Together following Agonist-induced Receptor Desensitization

Purification of A1 adenosine receptor–G-protein complexes: effects of receptor down-regulation and phosphorylation on coupling

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Ligand-Induced Phosphorylation, Clustering, and Desensitization of A₁Adenosine Receptors