Double tagging recombinant A1- and A2A-adenosine receptors with hexahistidine and the FLAG epitope

Robeva, Anna; Woodard, Robin L.; Luthin, David R.; Taylor, Heidi E.; Linden, Joel

doi:10.1016/0006-2952(95)02235-x

Cited by 69 publications

(78 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Rat A 2B receptor cDNA was prepared by reverse transcription polymerase chain reaction from rat bladder and sequenced on both strands in the University of Virginia biomolecular core laboratory. A2B and A3 cDNAs were subcloned into pDoubleTrouble (25). The plasmids were amplified in competent JM109 cells, and plasmid DNA was isolated using Wizard Megaprep columns (Promega; Madison, WI).…”

Section: Methodsmentioning

confidence: 99%

Selective A_2Aadenosine receptor activation reduces skin pressure ulcer formation and inflammation

Peirce

Skalak

Rieger³

et al. 2001

American Journal of Physiology-Heart and Circulatory Physiology

Self Cite

View full text Add to dashboard Cite

Activation of A2A adenosine receptors (A2A-AR) by ATL-146e (formerly DWH-146e) prevents inflammatory cell activation and adhesion. Recurrent ischemia-reperfusion (I/R) of the skin results in pressure ulcer formation, a major clinical problem. ATL-146e was evaluated in a novel reproducible rat model of pressure ulcer. A 9-cm2 region of dorsal rat skin was cyclically compressed at 50 mmHg using a surgically implanted metal plate and an overlying magnet to generate reproducible tissue necrosis. Osmotic minipumps were implanted into 24 rats divided into four equal groups to infuse vehicle (control), ATL-146e (0.004 μg · kg−1 · min−1), ATL-146e plus an equimolar concentration of A2A antagonist, ZM-241385, or ZM-241385 alone. Each group received 10 I/R cycles. In non-I/R-treated skin, ATL-146e has no effect on blood flow. I/R-treated skin of the ATL-146e group compared with the vehicle group had 65% less necrotic area, 31% less inhibition of average skin blood flow, and fewer extravasated leukocytes (23 ± 3 vs. 49 ± 6 per 500 μm2). These data suggest that ATL-146e, acting via an A2A-AR, reduces leukocyte infiltration and is a potent prophylactic for I/R injury in skin.

show abstract

Section: Methodsmentioning

confidence: 99%

Selective A_2Aadenosine receptor activation reduces skin pressure ulcer formation and inflammation

Peirce

Skalak

Rieger³

et al. 2001

American Journal of Physiology-Heart and Circulatory Physiology

Self Cite

View full text Add to dashboard Cite

show abstract

“…The purification of H\F-A " ARs by sequential anti-FLAG antibody and Ni-NTA affinity chromatography results in a good yield of very highly purified receptors, as described previously [9]. In the present study, we sought to identify conditions optimal for the purification of receptors that are coupled to G-proteins (R-G) and receptors uncoupled from G-proteins (R).…”

Section: Affinity Purification Of G-protein-coupled and Uncoupled H/fmentioning

confidence: 97%

“…N'-Cyclopentyladenosine (CPA), 5h-N-ethylcarboxamidoadenosine (NECA), 9-chloro-2-(2-furyl)-5,6-dihydro [1,2,4] ' \FLAG-tagged human A " ARs (H\F-A " ARs) [9] have been described previously.…”

Section: Experimental Materialsmentioning

confidence: 99%

“…We have previously demonstrated that extension of the Ntermini of ARs with His ' and the FLAG epitope has no effect on the binding affinity of ligands, the coupling of receptors to G- proteins or the receptor-mediated changes in cyclic AMP accumulation [9]. Here we show that, with the exception of very hydrophobic ligands (CPX and CGS15943), which show decreased affinity for detergent-solubilized receptors, purified H\F-A " ARs have pharmacological properties similar to those in membranes ( Table 2).…”

Section: Figure 1 Silver-stained Sds/page and Western Analysis Of Purmentioning

confidence: 99%

“…Higher-efficiency receptor purification has been achieved recently using receptors genetically engineered to facilitate purification. Recombinant human adenosine receptors were double-tagged with hexahistidine (His ' ) and the FLAG epitope (Asp-Tyr-Lys-Asp-AspAsp-Asp-Lys) (H\F) and purified to near homogeneity by sequential anti-FLAG-antibody and Ni-nitrilotriacetate (NTA)-affinity-chromatography steps [9]. Of the four G-protein-coupled adenosine receptors that have been cloned (A " , A #A , A #B and A $ [10]), the A " and the A $ receptors are coupled to the G i/o subfamily of G-proteins.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Gao

Robeva

Linden

1999

Biochemical Journal

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Immunohistochemical localization of adenosine A2A receptors in the rat central nervous system

et al. 1998

Self Cite

View full text Add to dashboard Cite

The A2A adenosine receptor (A2A-AR) transcript and radioligand binding sites have a distinct distribution in rat brain, restricted primarily to the striatum, nucleus accumbens and olfactory tubercles. We describe here the use of purified recombinant human A2A-ARs to generate a monoclonal antibody that has been used to better resolve the distribution of A2A-ARs in rat brain. The antibody can detect 1 ng of purified recombinant receptor by Western blotting and is potent (EC50 = 0.62 microg/ml) and highly selective for the A2A-AR subtype. By Western blotting, the apparent molecular mass of recombinant and rat striatal receptors shifts upon deglycosylation from 43-48 to 42 kilodaltons. Analyses of chimeric A1/A2A-ARs and synthesis of a blocking peptide pinpointed the epitope (SQPLPGER) of the antibody to the center of the third intracellular loop of the receptor. Incubation of rat striatal membranes with antibody reduces receptor coupling to G-proteins. In rat brain, dense A2A-AR-like immunoreactivity that is eliminated by the blocking peptide was found in the neuropil of the striatum, nucleus accumbens (rostral pole, core and shell), cell bridges of the striatum, olfactory tubercles, and areas of extended amygdala with somewhat lighter labeling in the globus pallidus and nucleus of the solitary tract. Light perikaryal labeling was found in other areas of the brain, including the cortex, hippocampus, thalamus, cerebellum, and portions of the hindbrain. The observed distribution of A2A-AR immunoreactivity throughout the neuraxis is consistent with the receptors' role in modulating dopaminergic neurotransmission and central control of cardiovascular function.

show abstract

Double tagging recombinant A1- and A2A-adenosine receptors with hexahistidine and the FLAG epitope

Cited by 69 publications

References 31 publications

Selective A_2Aadenosine receptor activation reduces skin pressure ulcer formation and inflammation

Selective A_2Aadenosine receptor activation reduces skin pressure ulcer formation and inflammation

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

Immunohistochemical localization of adenosine A2A receptors in the rat central nervous system

Contact Info

Product

Resources

About

Double tagging recombinant A1- and A2A-adenosine receptors with hexahistidine and the FLAG epitope

Cited by 69 publications

References 31 publications

Selective A2Aadenosine receptor activation reduces skin pressure ulcer formation and inflammation

Selective A2Aadenosine receptor activation reduces skin pressure ulcer formation and inflammation

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

Immunohistochemical localization of adenosine A2A receptors in the rat central nervous system

Contact Info

Product

Resources

About

Selective A_2Aadenosine receptor activation reduces skin pressure ulcer formation and inflammation

Selective A_2Aadenosine receptor activation reduces skin pressure ulcer formation and inflammation