Heterologous expression of rat epitope‐tagged histamine H 2 receptors in insect Sf9 cells

1 Rat histamine H 2 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 H 2 receptor were prepared and were used to infect insect Sf9 cells. 2 The His‐tagged H 2 receptors expressed in insect Sf9 cells showed typical H 2 receptor characteristics as determined with [ 125 I]‐aminopotentidine (APT) binding studies. 3 In Sf9 cells expressing the His‐tagged H 2 receptor histamine was able to stimulate cyclic AMP production 9 fold (EC 50 =2.1±0.1 μ M ) 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 μ M , 0.25±0.15 μ M and 28±7 n M , respectively (mean±s.e.mean, n =3). 4 The expression of the His‐tagged H 2 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 2×10 6 copies of receptor/cell. Preincubation of the cells with 0.03 m M cholesterol‐β‐cyclodextrin complex resulted in an increase of [ 125 I]‐APT binding up to 169±5% (mean±s.e.mean, n =3). 5 The addition of 0.03 m M cholesterol‐β‐cyclodextrin complex did not affect histamine‐induced cyclic AMP production. The EC 50 value of histamine was 3.1±1.7 μ M in the absence of cholesterol‐β‐cyclodextrin complex and 11.1±5.5 μ M 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 μ M histamine was identical, 85±18 pmol/10 6 cells in the absence and 81±11 pmol/10 6 cells in the presence of 0.03 m M 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 H 2 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 H 2 receptors in insect Sf9 cells. The H 2 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 H 2 receptor protein is functional. These functional receptors are targeted to the plasma membrane.British Journal of Pharmacology (1997) 122 , 867–874; doi: 10.1038/sj.bjp.0701466