Characterisation and agonist stimulated internalisation of a GFP-tagged human histamine H3-receptor in CHO-K1 cells K. L. Powell and S. J. Hill. Institute of Cell Signalling, Medical School, Queen's Medical Centre, Nottingham, UK.

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The human histamine H₃-receptor (hH₃R) is a G_i/o-coupled presynaptic receptor that can regulate the synthesis and release of histamine and other neurotransmitters in the CNS (Arrang et al., 1983; Lovenberg et al., 1999).

In order to study the trafficking pathways of the H₃ receptor in live cells, we have created a fusion protein between the hH₃R and green fluorescent protein (GFP; attached to the C terminus of the receptor). The hH₃R cDNA was amplified by PCR using oligonucleotides designed to replace the stop codon with a serine residue using the proof reading enzyme, Pfu (Promega). The start codon of the GFP cDNA in pEGFP (Clontech) was then mutated to a valine by site-directed mutagenesis. The modified hH₃R cDNA was then cloned into the EcoR I and Kpn I sites of this pEGFP vector. CHO-K1 cells were transfected with the GFP-tagged receptor and dilution cloning was performed to generate a stable GFP-tagged hH₃R cell line. ³H-cAMP accumulation in cells expressing the wild-type or GFP-tagged hH₃R was measured as described previously (Baker et al., 2002). Confocal microscopy was performed with cells on glass coverslips using a Zeiss LSM 510 confocal microscope with excitation at 488nm and emission detected at 505nm.

Imetit, R--methylhistamine, N-methylhistamine and histamine inhibited forskolin-stimulated (3µM) cAMP accumulation in the presence of 1mM IBMX in both cell lines. Wild-type hH₃R LogEC₅₀ values were: Imetit -9.31±0.19 (n=6), R--methylhistamine -8.53±0.07 (n=6), N-methylhistamine -8.44±0.14 (n=5), Histamine -7.44±0.15 (n=7). GFP-tagged logEC₅₀ values were: Imetit -8.22±0.55 (n=6), R--methylhistamine -7.68±0.09 (n=8), N-methylhistamine -7.51±0.1 (n=6), Histamine -6.54±0.06 (n=7). In both cell lines the ability of R--methylhistamine to inhibit forskolin-stimulated cAMP accumulation was antagonised by clobenpropit (wild-type hH₃R log K_D = -8.96+0.04, Schild slope = 1.04 + 0.04, n=3; GFP-tagged hH₃R log K_D = -9.36+0.13, Schild slope = 1.06+0.04, n=3). 24 hour pretreatment with 100ng/ml pertussis toxin completely abolished the responses to histamine or imetit (wild-type hH₃R n=4; GFP-tagged hH₃R n=10).

Confocal imaging of GFP-tagged hH₃R in living cells showed internalisation of the receptor in response to 1µM histamine (n=9) or 100nM (n=5) imetit. This occured very slowly reaching maximum after 90 minutes of stimulation. Internalisation was blocked by pre-treatment of the cells with the H₃-antagonist clobenpropit (100nM) for 30 min prior to stimulation with 1mM histamine (n=4).

In conclusion we have constructed a functionally active GFP-tagged hH₃R which is expressed on the surface of transfected CHO cells and functions in a similar manner to the wild type receptor. This cell line should provide a useful tool for further characterisation of agonist-induced trafficking of the hH₃R.

Arrang, J.M. et al, (1983) Nature, 302,832-837.
Baker, J.G. et al, (2002) Br. J. Pharmacol. 137, 400-408.
Lovenberg, T.W. et al, (1999) Mol. Pharmacol., 55,1101-07.

We thank the Wellcome Trust for financial support (57199).