051P Brighton
Winter Meeting December 2008 |
Differences in the membrane organisation of human adenosine A1 receptors between apical and basolateral surfaces of living CHO cells
Jonathan Hern, Nicholas Holliday, Stephen Briddon, Stephen Hill
The University of Nottingham, Nottingham, UK
The human adenosine A1 receptor is a well characterised family A GPCR, and is a useful model in assays typically involving purified receptor preparations or considerable numbers of cells. As evidence of the molecular organisation of cell-surface receptors accumulates, techniques including fluorescence correlation spectroscopy (FCS) have been applied to elucidate the molecular pharmacology of GPCRs within membrane domains of single live cells (Briddon & Hill, 2007). The aim of the current study was to quantitatively examine the distribution, mobility and molecular organisation of the A1 receptor within small regions of the surface of whole live cells by means of FCS.
A stable clonal CHO cell line expressing a human adenosine A1 receptor-GFP (A1-GFP) fusion protein ([3H]DPCPX Bmax 18 ± 3 pmol/mg protein (n=4) at purified cell membranes), was chosen from a series previously characterised (Hern & Birdsall, 2004). FCS measurements were carried out and autocorrelation analysis performed as previously described (Briddon et al., 2004). Photon counting histogram (PCH) analysis of fluorescent particle brightness was performed using Zeiss AIM software. CHO-K1 cells were transiently transfected by Lipofectamine according to manufacturer’s instructions for 24 h with an EGFP construct modified with a poly basic and prenylation sequence from kRas in pcDNA3.1/Zeo. Microscopy was performed 48 h following transfection.
Table 1; Mobility, density and fluorescent brightness of A1-GFP and prenylated GFP particles at apical and basal surfaces of live CHO cells. Data shown as mean ± s.e.m. of n cells. * indicates significant (t-test; P<0.05) difference from corresponding apical measurements.
| | Diffusion coefficient (μm2.s-1) | Particle density (particles.μm-2) | n | Particle brightness (kHz) | n |
| A1-GFP |
apical |
0.113 ± 0.006 |
259 ± 27 |
51 |
16.3 ± 0.8 |
17 |
| basal |
0.081 ± 0.006 * |
460 ± 52 * |
47 |
10.1 ± 1.0 * |
15 |
| Prenylated GFP |
apical |
0.279 ± 0.041 |
310 ± 31 |
18 |
19.7 ± 1.5 |
18 |
| basal |
0.247 ± 0.028 |
354 ± 39 |
18 |
15.9 ± 1.9 |
18 |
An increased density of A1-GFP fluorescent particles was observed at the basal cell membrane (Table 1) compared to apical, however those basal particles were of reduced brightness and are postulated to contain fewer receptors. The increase in basal particle density was associated with a reduction in A1-GFP mobility. Prenylated GFP showed no significant difference between apical and basal membrane values.
These results illuminate novel details of GPCR expression, mobility and molecular organisation. FCS measurements performed within small regions of the cell surface reveal the polarisation of receptor organisation at different surfaces of single live cells.
Briddon SJ et al. (2004)Proc. Natl. Acad. Sci. (USA) 101, 4673
Briddon SJ & Hill SJ (2007) Trends in Pharmacological Sciences 28, 637
Hern J & Birdsall NJM (2004) Proceedings of the British Pharmacological Society at http://www.pA2online.org/Vol2Issue2abst004P.html
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