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Free keywords:
β-adrenergic receptor; Baculovirus; Expression; Glycosylation; Affinity chromatography; β2AR; β2-adrenergic receptor; CGP 12177; Ciba Geigy Product 12177; Con A; concanavalin A; Gs; stimulatory GTP binding protein; GTP[γ]S; guanosine 5′-O-(3-thiotriphosphate); [125I]ICYP; [125]iodocyanopindolol; [125I]ICYP-azided 2; [125I]iodocyanopindolol-azided 2; MOI; multiplicity of infection; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; Sf9-cells; Spodoptera frugiperda cells; WGA; wheat germ agglutini
Abstract:
A human cDNA fragment bearing the complete coding region for the β2‐adrenergic receptor was introduced into the genome of Autographa california nuclear polyhedrosis virus under the control of the polyhedrin promoter. Binding studies using [125I]iodocynnopindolol showed that Sf9 insect cells infected with the recombinant virus expressed ≈ 1 × 104 β2‐adrenergic receptors on their cell surface. Photoaffinity labeling of whole cells and membraines revealed a molecular weight of ≈ 46000 for the expressed receptor. The receptor produced in insect cells is glycosylated but the extent and pattern differ from that of the receptor from human tissue. The heterologously expressed receptor was purified by alprenolol affinity chromatography, and was able to activate isolated Gs‐protein.
