Heterologous expression of rat epitope-tagged histamine H₂ receptors in insect Sf9 cells

Abstract

1. Rat histamine H₂ receptors were epitope-tagged with six histidine residues at the C-terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope-tagged H₂ receptor were prepared and were used to infect insect Sf9 cells.

2. The His-tagged H₂ receptors expressed in insect Sf9 cells showed typical H₂ receptor characteristics as determined with [¹²⁵I]-aminopotentidine (APT) binding studies.

3. In Sf9 cells expressing the His-tagged H₂ receptor histamine was able to stimulate cyclic AMP production 9 fold (EC₅₀=2.1±0.1 μM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with K_i values of 0.60±0.43 μM, 0.25±0.15 μM and 28±7 nM, respectively (mean±s.e.mean, n=3).

4. The expression of the His-tagged H₂ receptors in infected Sf9 cells reached functional levels of 6.6±0.6 pmol mg⁻¹ protein (mean±s.e.mean, n=3) after 3 days of infection. This represents about 2×10⁶ copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol-β-cyclodextrin complex resulted in an increase of [¹²⁵I]-APT binding up to 169±5% (mean±s.e.mean, n=3).

5. The addition of 0.03 mM cholesterol-β-cyclodextrin complex did not affect histamine-induced cyclic AMP production. The EC₅₀ value of histamine was 3.1±1.7 μM in the absence of cholesterol-β-cyclodextrin complex and 11.1±5.5 μM in the presence of cholesterol-β-cyclodextrin complex (mean±s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 μM histamine was identical, 85±18 pmol/10⁶ cells in the absence and 81±11 pmol/10⁶ cells in the presence of 0.03 mM cholesterol-β-cyclodextrin complex (mean±s.e.mean, n=3).

6. Immunofluorescence studies with an antibody against the His-tag revealed that the majority of the His-tagged H₂ receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be identified as well.

7. These experiments demonstrate the successful expression of His-tagged histamine H₂ receptors in insect Sf9 cells. The H₂ receptors couple functionally to the insect cell adenylate cyclase. However, our studies with cholesterol complementation and with immunofluorescent detection of the His-tag reveal that only a limited amount of H₂ receptor protein is functional. These functional receptors are targeted to the plasma membrane.

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