International Carl Zeiss Workshop on International Carl Zeiss ...

<strong>International</strong> <strong>Carl</strong> <strong>Zeiss</strong> <strong>Workshop</strong> on Fluorescence Correlation Spectroscopy and Related Methods
March, 30 to March 31, Jena / Germany
FCS-analysis of ligand-receptor interactions in the membrane of cultured cells
Aladdin Pramanik and Rudolf Rigler
Dept. of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden
Receptor binding studies on peptide/hormone ligands most often require the use of
radioactively labeled ligands and include several washing steps to remove unbound
ligands. In certain cases, the numbers of receptors are few per cell and no specific
binding is detected because of high background. In addition, the half-life of the receptorligand
complex is often shorter or similar to the time required for separations of free and
bound ligands, respectively. Specific interactions between certain ligands (e.g. peptides,
hormones, natural products) and their different receptor subtypes are, therefore, often
overlooked by the conventional equilibrium binding technique. In FCS the small laser
volume element (0.2 fl) allows the detection of single molecules as well as the
measurement of molecular properties at specific coordinates in the cell membrane (Rigler
et al. (1999) PNAS 96:13318-13323). FCS allows detection of the interaction of ligands
with binding sites of receptors on the molecular level in their native environment on cell
surfaces with single molecule detection sensitivity (Rigler et al. (1999) PNAS 96:13318-
13323); Pramanik et al. (1999) Biomed. Chromatogr. 13:119-120; Boonen et al. (2000)
Planta Med. 66:7-10). This technique permits the identification of receptors or target
molecules which were not possible before to detect by isotope labeling. With FCS one
can analyze a mixture of multiple ligand-receptor complexes possessing different
molecular weights (M 1
, M 2
, M 3
) and different diffusion times ( τ D1
, τ D2
, τ D3
), respectively.
The beauty of FCS technique is that there is no need for separating unbound ligand from
bound one to calculate the receptor bound and free ligand fractions y
1,
y 2
and y 3
corresponding to M 1
/τ D1
, M 2
/τ D2
and M 3
/τ D3
, respectively or to calculate other parameters
describing binding of the fluorescent labeled ligand. Thus, it is possible to perform largescale
drug and/or active compound screening in cell cultures using FCS technique.
To demonstrate receptor binding in the membranes of living cells we will report our
studies conducted on interactions between the ligands galanin (GAL), a neuropeptide
that displays a variety of important biological actions and is thought to be implicated in
several human disorders such as Alzheimer’s disease, Depression and Feeding Disorders
(Bartfai et al. (1993) Crit. Rev. Neurobiol. 7:229-274) and the epidermal growth factor
(EGF) that plays a crucial role in the molecular network communication of physiological
processes (Boonstra et al. (1995) Cell Biol. Int. 19:413-430; Zwick et al. (1999) Trends
Pharmacol. Sci. 20:408-412). Both GAL and EGF have been labeled by a fluorophore
tetramethyl rhodamine isothiocyanate (Rh) which provides the fluorescence signal in the
interaction of the ligands with their respective receptors. The results demonstrate the
presence of specific binding of GAL and EGF to their respective membrane-bound
receptors in cultured cells. The specificity of the binding is attested by the consistent
displacement of bound Rh-GAL and Rh-EGF following addition of 1,000 -fold molar
excess of unlabeled GAL and EGF, respectively.
Cont.
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