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Sci. STKE, 9 September 2003
Vol. 2003, Issue 199, p. tw350
[DOI: 10.1126/stke.2003.199.tw350]


METHODOLOGY Receptor-Ligand Complexes Take a Spin

The conformational changes associated with receptor-ligand interactions and, in some cases, the internalization of those bound receptors remain challenging issues to study in live cells. Shafer et al. used electron paramagnetic resonance (EPR) spectroscopy of spin-labeled substance P, the ligand for the neurokinin-1 receptor (Nk1R), a G protein-coupled receptor, to study the local receptor environment surrounding the ligand. Differences in the spectrum were observed if binding was performed in the presence of guanosine diphosphate (GDP) or the nonhydrolyzable GTP analog Gpp(NH)p and between binding to intact cells and isolated membranes, consistent with different receptor conformations. The accessibility of oxygen, which detects nonpolar environments, or chromium oxalate, which detects polar environments, for the spin-labeled substance P bound to the receptor showed that, as previously reported using other methods, the ligand bound in a hydrophilic environment. Because the EPR signal is quenched by reducing environments, the loss of the EPR signal was used to report internalization of the receptor-ligand complex and showed similar kinetics to that observed for receptor internalization monitored by confocal microscopy of green fluorescent protein (GFP)-tagged Nk1R. The authors note that EPR requires a target cell line with high levels of expression to achieve significant signal strength, thus they used cells that had been sorted for expression of GFP-tagged Nk1R for these experiments. However, EPR is another tool in the arsenal for studying molecular interactions in living cells.

A. M. Shafer, V. J. Bennett, P. Kim, J. C. Voss, Probing the binding pocket and endocytosis of a G protein-coupled receptor in live cells reported by a spin-labeled substance P agonist. J. Biol. Chem. 278, 34203-34210 (2003). [Abstract] [Full Text]

Citation: Receptor-Ligand Complexes Take a Spin. Sci. STKE 2003, tw350 (2003).

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