Purification and characterization of the adenosine A2-like binding site from human placental membrane

Hutchison, Kevin A.; Fox, Irving H.

doi:10.1016/s0021-9258(19)47195-0

Cited by 43 publications

(20 citation statements)

References 57 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Fitting the experimental data to a biphasic competition curve (R 2 ϭ 0.9297) showed that there was a population of binding sites with affinity values (IC 50 : 3.24 M) that were very close to those calculated from the monophasic analysis and a second population of binding sites with affinity characteristics in the nanomolar range (IC 50 : 10.23 nM). This IC 50 value corresponds well with those described for NECA binding to glucose-regulated protein of 94 kDa (GRP94) in rabbit gastric parietal cell membranes at 4°C (5) and to purified human (36) or bovine (57) GRP94. This chaperone is normally confined to the endoplasmic reticulum but escapes the KDELmediated retention system and anchors to the plasma membrane in some cell types and circumstances, but the significance of surface GRP94 expression and of its ability to bind adenosine analogs with high affinity (57) remains unclear.…”

Section: Resultssupporting

confidence: 84%

Stimulation of gastric acid secretion by rabbit parietal cell A_2B adenosine receptor activation

Arin

Vallejo

Rueda

et al. 2015

American Journal of Physiology-Cell Physiology

View full text Add to dashboard Cite

Adenosine modulates different functional activities in many cells of the gastrointestinal tract; some of them are believed to be mediated by interaction with its four G protein-coupled receptors. The renewed interest in the adenosine A2B receptor (A2BR) subtype can be traced by studies in which the introduction of new genetic and chemical tools has widened the pharmacological and structural knowledge of this receptor as well as its potential therapeutic use in cancer and inflammation- or hypoxia-related pathologies. In the acid-secreting parietal cells of the gastric mucosa, the use of various radioligands for adenosine receptors suggested the presence of the A2 adenosine receptor subtype(s) on the cell surface. Recently, we confirmed A2BR expression in native, nontransformed parietal cells at rest by using flow cytometry and confocal microscopy. In this study, we show that A2BR is functional in primary rabbit gastric parietal cells, as indicated by the fact that agonist binding to A2BR increased adenylate cyclase activity and acid production. In addition, both acid production and radioligand binding of adenosine analogs to isolated cell membranes were potently blocked by selective A2BR antagonists, whereas ligands for A1, A2A, and A3 adenosine receptors failed to abolish activation. We conclude that rabbit gastric parietal cells possess functional A2BR proteins that are coupled to Gs and stimulate HCl production upon activation. Whether adenosine- and A2BR-mediated functional responses play a role in human gastric pathophysiology is yet to be elucidated.

show abstract

Section: Resultssupporting

confidence: 84%

Stimulation of gastric acid secretion by rabbit parietal cell A_2B adenosine receptor activation

Arin

Vallejo

Rueda

et al. 2015

American Journal of Physiology-Cell Physiology

View full text Add to dashboard Cite

show abstract

“…11 Previous studies utilizing a high throughput screen identified 5′-(N-ethylcarboxamido)adenosine (NECA) as one of the first molecules to exhibit isoform selectivity, as it bound Grp94 but displayed no affinity toward the cytosolic isoform, Hsp90α. 12,13 Differential binding to Grp94 is best explained upon analysis of the N-terminal ATP-binding domain. While the ATP-binding pocket is highly conserved across all four isoforms, Grp94 contains a five amino acid insertion (QEDGQ) into the primary sequence that leads to the formation of a hydrophobic binding pocket that is not present in the other Hsp90 isoforms.…”

mentioning

confidence: 99%

“…Like NECA (11), the small alkyl carboxamides tended to be selective for Grp94 as compared to Hsp90α. The methyl (12) and cyclopropyl (14) analogues were 46-and >42-fold selective, respectively. However, the substitution of fluorines for hydrogens resulted in altered selectivity.…”

mentioning

confidence: 99%

Biological Evaluation of 5′-(N-Ethylcarboxamido)adenosine Analogues as Grp94-Selective Inhibitors

Tosh

Brackett

Jung

et al. 2021

ACS Med. Chem. Lett.

View full text Add to dashboard Cite

The heat shock protein 90 kDa (Hsp90) family of chaperones is highly sought-after for the treatment of cancer and neurodegenerative diseases. Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum localized isoform that is responsible for the maturation of proteins involved in cell adhesion and the immune response, including Toll-like receptors, immunoglobulins, and integrins. Consequently, Grp94 has been implicated in many different diseases including cancer metastasis, glaucoma, and viral infection. 5′-(N-Ethylcarboxamido)adenosine (NECA) was identified from a high-throughput screen as one of the first molecules to exhibit isoform selectivity toward Grp94, with the ethyl group projecting into a unique pocket within the ATP binding site of Grp94. This pocket has since been exploited by several groups to develop Grp94 selective inhibitors. Despite success in the development of other classes of inhibitors, relatively little work has been done to further develop inhibitors with the NECA scaffold. Unfortunately, NECA is also a potent adenosine receptor agonist, which is likely to confound any biological activity. Therefore, structure–activity relationship studies were performed on the NECA scaffold leading to the discovery of several molecules that displayed similar selectivity and affinity as the parent compound.

show abstract

“…[3H]NECA, 5'-(7V-ethylcarbamoyl)[2,8-3H]adenosine; CHAPS,dimethylammonio]-lpropanesulfonate; EDTA, ethylenediamine-iV,A',A",A"-tetraacetic acid; EGTA, ethylene glycol bis(i3-aminoethyl ether)-A',A',A",A"-tetraacetic acid; ERp99, 99-kDa murine endoplasmic reticulum protein; GP96, 96-kDa murine tumor rejection antigen; GRP94, 94-kDa hamster glucoseregulated protein; HEPES, Ar-(2-hydroxyethyl)piperazine-Ar -2-ethanesulfonic acid; HSP108, 108-kDa chicken heat shock protein; NECA, 5'-(A'-ethylcarbamoyl)adenosine; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; Tris-HCl, tris(hydroxymethyl)aminomethane hydrochloride. To gain insight into the function of this low-affinity adenosine binding protein, we purified it to homogeneity from human placental membranes (Hutchison & Fox, 1989). This ubiquitous protein is a homodimer of M, 98 000 subunits and is asymmetric with a frictional ratio of 1.5 (Hutchison & Fox, 1989).…”

mentioning

confidence: 99%

Soluble and membrane-associated human low-affinity adenosine binding protein (adenotin): properties and homology with mammalian and avian stress proteins

Hutchison¹,

Nevins²,

Perini³

et al. 1990

Biochemistry

View full text Add to dashboard Cite

A low-affinity adenosine binding protein has recently been distinguished from the adenosine A2 receptor and purified from human placental membranes. Soluble human placental extracts contain an adenosine binding activity that has properties similar to those of the membrane low-affinity adenosine binding protein. The binding protein was purified from soluble human placental extracts 134-fold to 89% purity with a Bmax of 2.5 nmol/mg. It comprises 0.7-0.9% of the soluble protein. The major purified soluble protein has a subunit molecular mass of 98 kDa and a Stokes radius identical with that of the membrane-bound adenosine binding protein. Competition analysis of the soluble protein revealed similar affinities and an identical potency order for displacement of 5'-(N-ethylcarbamoyl)[2,8-3H]adenosine ([3H]NECA) as follows: NECA greater than 2-chloroadenosine greater than adenosine greater than (R)-N6-(2-phenylisopropyl)adenosine. The soluble binding protein was more acidic than the membrane binding protein as revealed by a comparison of the elution properties during ion exchange chromatography. A second form of soluble adenosine binding activity comprised 17% of the major form and had a charge similar to that of the membrane binding protein, a smaller Stokes radius, and a subunit molecular mass of 74 kDa. Carbohydrate composition analysis revealed that the major soluble form has 4.3% carbohydrate by weight as compared to the membrane-associated form, which has 5.5% carbohydrate by weight.(ABSTRACT TRUNCATED AT 250 WORDS)

show abstract

Purification and characterization of the adenosine A2-like binding site from human placental membrane

Cited by 43 publications

References 57 publications

Stimulation of gastric acid secretion by rabbit parietal cell A_2B adenosine receptor activation

Stimulation of gastric acid secretion by rabbit parietal cell A_2B adenosine receptor activation

Biological Evaluation of 5′-(N-Ethylcarboxamido)adenosine Analogues as Grp94-Selective Inhibitors

Soluble and membrane-associated human low-affinity adenosine binding protein (adenotin): properties and homology with mammalian and avian stress proteins

Contact Info

Product

Resources

About

Purification and characterization of the adenosine A2-like binding site from human placental membrane

Cited by 43 publications

References 57 publications

Stimulation of gastric acid secretion by rabbit parietal cell A2B adenosine receptor activation

Stimulation of gastric acid secretion by rabbit parietal cell A2B adenosine receptor activation

Biological Evaluation of 5′-(N-Ethylcarboxamido)adenosine Analogues as Grp94-Selective Inhibitors

Soluble and membrane-associated human low-affinity adenosine binding protein (adenotin): properties and homology with mammalian and avian stress proteins

Contact Info

Product

Resources

About

Stimulation of gastric acid secretion by rabbit parietal cell A_2B adenosine receptor activation

Stimulation of gastric acid secretion by rabbit parietal cell A_2B adenosine receptor activation