The effect of agonist activation and homodimerisation on the membrane diffusion of the human histamine H1 receptor

Rachel Rose, Stephen Briddon, Stephen Hill

University of Nottingham, Nottingham, UK

There is considerable evidence to support the existence of dimers of class A G-protein-coupled receptors, including the histamine H1 receptor (H1R) (Bakker et al, 2004), although the functional significance of these dimers remains unclear. We have used bimolecular fluorescence complementation (BiFC) in combination with fluorescence correlation spectroscopy (FCS) to selectively monitor histamine-mediated changes in the diffusion characteristics of the dimeric human H1R (using BiFC), as well as those of the total H1R population (using labelling with the full length yellow fluorescent protein (YFP)).

Using standard molecular biology techniques, cDNAs encoding the full length wild type YFP or the C-terminal (Yc) and N-terminal (Yn) YFP fragments were cloned into pcDNA3.1 to produce fusions to the C-terminus of the H1R. CHO-K1 cells were transfected with the relevant cDNAs for 24h using Lipofectamine (Invitrogen) according to manufacturer’s instructions. Following a 24h incubation (30ºC/5%CO2), cells were maintained in HEPES-buffered saline and FCS measurements were carried out on the upper cell membrane and analysed as previously described (Briddon et al., 2004).

In each case, autocorrelation curves from FCS measurements of H1R diffusion showed two diffusion components (τD1 and τD2,). As previously described, τD1 represents a photophysical property of YFP, and did not differ significantly between data sets. However, translational diffusion of the H1R in the cell membrane (τD2) was significantly faster for oligomeric H1R than the total receptor population (Table 1). Following stimulation with 0.1mM histamine, there was a significant increase in both the diffusion time and particle number of the total receptor population after 10 minutes. This had returned to control values after 20 and 40 minutes. For the dimeric receptor population, however, there was no significant change in either translational receptor diffusion or particle number following histamine exposure.

Since FCS only detects the diffusion of mobile particles, the increase in particle number for H1YFP following 10 minutes agonist stimulation may reflect mobilisation of previously immobile receptors. The increased diffusion time could indicate association with larger protein complexes involved in receptor signalling, desensitisation or internalisation. The absence of such changes for the dimeric receptors suggests fundamental functional differences between monomeric and oligomeric receptor populations.

Bakker, R.A. et al. (2004) J Pharmacol Exp Ther, 311, 131-138

Briddon, S.J. et al. (2004) Proc. Nat. Acad. Sci. (USA), 101, 4673-4678.

RR holds an A J Clark studentship from BPS.