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Jaakola VP Vainio M Sen S Rehn M Heimo H Scheinin M Goldman A 《Journal of receptor and signal transduction research》2005,25(2):99-124
All three alpha2-adrenoceptor subtypes have a long third intracellular loop (3i), which is conserved by overall size and charge-hydrophobic properties but not by amino acid sequence similarity. These properties must be relevant for function and structure, because they have been preserved during hundreds of millions of years of evolution. The contribution of different loop portions to agonist/antagonist binding properties and G protein coupling of the human alpha2B-adrenoceptor (alpha2B-AR) was investigated with a series of 3i truncated constructs (delta3i). We used a variety of agonists/antagonists in competition binding assays. We stimulated alpha2B-AR delta3i with various agonists and measured [35S]GTPgammaS binding in isolated cell membranes with or without antagonist inhibition. We also evaluated the ability of oligopeptides, analogous to the amino and carboxyl terminal parts of 3i, to promote G protein activation, monitored with the [35S]GTPgammaS assay. Our results reveal that the carboxyl end residues of 3i, R360(6.24) to V372(6.36), are important for Gi/Go protein activation. Deletions in regions from G206(5.72) to R245(5.110) altered the binding of some alpha2B-AR agonists, indicating that agonist binding is dependent on the conformation of the 3i domain, possibly through the involvement of G protein interactions. The truncated receptor constructs may be more stable on purification and thus be useful for structural characterization of alpha2B-AR. 相似文献
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Kapat A Jaakola VP Heimo H Liitti S Heikinheimo P Glumoff T Goldman A 《Bioseparation》2000,9(3):167-172
The objective is to generate milligram quantities of recombinant human 2C2 adrenergic receptor for X-ray crystallographic studies. It has been cloned in Saccharomyces cerevisiae, and the production level is at best about 13 pmol/mg of membrane protein, as estimated by radio-ligand binding assay. The receptor is solubilized with sucrose monolaurate followed by immunoaffinity purification and reconstitution into phospholipid vesicles. The efficiency of solubilization and immuno-purification are 60% and 91%, respectively. 相似文献
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We analyzed a lacustrine sediment core covering the Holocene from Lake Einstaken, Nordaustlandet, for its fossil Cladocera
(Crustacea) with an aim to reconstruct past aquatic communities in this environmentally extreme and unexplored region. In
the analysis, we encountered remains (carapaces, ephippia, headshields, and postabdomens) of an unknown chydorid (Chydoridae,
Aloninae) species during two separate periods in the early Holocene. The remains had some comparable morphological characters
with the European Alona guttata s.str. Sars, 1862 and with the glacial relict Alona werestschagini Sinev, 1999, but they differed clearly from the previous species; the headshield had broadly rounded rostrum and narrow fornices, the
ephippium was heavily pigmented and reticulated, and the postabdomen had convex dorsal and ventral margins. The postabdomen
had evidently similar morphology with Alona bergi R?en, 1992, which has been described, although inadequately, from arctic Canada and northern Greenland. We conclude, based on the morphology
of the postabdomen, that the unknown remains belong to species closely resembling A. bergi, named here Alona cf. bergi, and assume that the species, whether the true A. bergi or some other cryptic species of the A.
guttata group, is a postglacial relict of the high arctic adapted to cold climate. Herewith, we emphasize the need for extensive
biogeographical investigations into both fossil and intact specimens of chydorids in the arctic. 相似文献