HETEROLOGOUS EXPRESSION OF RAT EPITOPE-TAGGED HISTAMINE H-2 RECEPTORSIN INSECT SF9 CELLS

1 Rat histamine H-2 receptors were epitope-tagged with six histidine r esidues at the C-terminus to allow immunological detection of the rece ptor. Recombinant baculoviruses containing the epitope-tagged H-2 rece ptor 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 [I-125]-aminopotentidine (APT) binding studies. 3 In Sf9 cells expressing the His-tagged H-2 re ceptor histamine was able to stimulate cyclic AMP production 9 fold (E C50=2.1+/-0.1 mu M) by use of the endogenous signalling pathway. The c lassical antagonists cimetidine, ranitidine and tiotidine inhibited hi stamine induced cyclic AMP production with K-i values of 0.60+/-0.43 m u M, 0.25+/-0.15 mu M and 28+/-7 nM, respectively (mean+/-s.e.mean, n= 3). 4 The expression of the His-tagged H-2 receptors in infected Sf9 c ells 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 2x10 (6) copies of receptor/cell. Preincubation of the cells with 0.03 mM c holesterol-beta-cyclodextrin complex resulted in an increase of [I-125 ]-APT binding up to 169+/-5% (mean+/-s.e.mean, n=3). 5 The addition of 0.03 mM cholesterol-beta-cyclodextrin complex did not affect histamin e-induced cyclic AMP production. The EC50 value of histamine was 3.1+/ -1.7 mu M in the absence of cholesterol-beta-cyclodextrin complex and 11.1+/-5.5 mu M in the presence of cholesterol-beta-cyclodextrin compl ex (mean+/-s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 mu M histamine was identical, 85+/-18 pmol/10(6) c ells in the absence and 81+/-11 pmol/10(6) cells in the presence of 0. 03 mM cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). 6 Immunofluorescence studies with an antibody against the His-tag reveal ed that the majority of the His-tagged H-2 receptors was localized ins ide the insect Sf9 cells, although plasma membrane labelling could be identified as well. 7 These experiments demonstrate the successful exp ression of His-tagged histamine H-2 receptors in insect Sf9 cells. The H-2 receptors couple functionally to the insect cell adenylate cyclas e. However, our studies with cholesterol complementation and with immu nofluorescent detection of the His-tag reveal that only a limited amou nt of H-2 receptor protein is functional. These functional receptors a re targeted to the plasma membrane.