Detection of ligand-induced CNTF receptor dimers in living cells by fluorescence cross correlation spectroscopy

Abstract

Ciliary neurotrophic factor (CNTF) signals via a receptor complex consisting of the specific CNTF receptor (CNTFR) and two promiscuous signal transducers, gp130 and leukemia inhibitory factor receptor (LIFR). Whereas earlier studies suggested that the signaling complex is a hexamer, more recent analyses strongly support a tetrameric structure. However, all studies so far analyzed the stoichiometry of the CNTF receptor complex in vitro and not in the context of living cells. We generated and expressed in mammalian cells acyl carrier protein-tagged versions of both CNTF and CNTFR. After labeling CNTF and CNTFR with different dyes we analyzed their diffusion behavior at the cell surface. Fluorescence (cross) correlation spectroscopy (FCS/FCCS) measurements reveal that CNTFR diffuses with a diffusion constant of about 2 x 10(-9) cm(2) s(-1) independent of whether CNTF is bound or not. FCS and FCCS measurements detect the formation of receptor complexes containing at least two CNTFs and CNTFRs. In addition, we measured Förster-type fluorescence resonance energy transfer between two differently labeled CNTFs within a receptor complex indicating a distance of 5-7 nm between the two. These findings are not consistent with a tetrameric structure of the CNTFR complex suggesting that either hexamers and or even higher-order structures (e.g. an octamer containing two tetramers) are formed.

Fluorescence Correlation Spectroscopy Analysis of Serotonin, Adrenergic, Muscarinic, and Dopamine Receptor Dimerization: The Oligomer Number Puzzle

“…Combining confocal microscopy with FCS led to the development of sensitive methods for monitoring protein dynamics in living cells (Qian and Elson, 1991;Rigler et al, 1993;Pramanik et al, 2001;Digman et al, 2005). FCS has been used to monitor diffusion and ligand binding for ion channels, tyrosine kinase receptors, and GPCRs (reviewed in Briddon and Hill, 2007), to examine neuropeptide Y and barrestin interactions (Kilpatrick et al, 2012), and to monitor the oligomer status of various receptors including somatostatin (Patel et al, 2002), epidermal growth factor (Liu et al, 2007), ciliary neurotrophic factor (Neugart et al, 2009), estrogen (Savatier et al, 2010), and serotonin 5-HT 1A (Ganguly and Chattopadhyay, 2010) and 5-HT 2C receptors (Herrick-Davis et al, 2012).…”

Oligomer Size of the Serotonin 5-Hydroxytryptamine 2C (5-HT2C) Receptor Revealed by Fluorescence Correlation Spectroscopy with Photon Counting Histogram Analysis

“…35). Fluorescence cross-correlation spectroscopy, which compares the autocorrelation signals from two different fluorescent probes, has been applied to study homo-and heterodimers of somatostatin receptors (36), epidermal growth factor receptors (37), ciliary neurotrophic factor receptors (38), and estrogen receptors (39).…”

Assembly and activation of neurotrophic factor receptor complexes

“…1(D)]. Recently, on the basis of evidence from fluorescence cross-correlation spectroscopy studies, it has been hypothesized that CNTF-induced CNTF-Ra dimerization in living cells could reflect higher order association of tetrameric CNTF receptor complexes (Neugart et al, 2009). Together, these observations suggest that different pre-formed complexes of CNTF-Ra with its partner receptors may exist at the cell membrane.…”