Neuroblastoma SH-SY5Y (SH) cells endogenously express A 2A adenosine receptors and can be differentiated into a sympathetic neuronal phenotype, capable of depolarisation-dependent noradrenaline release. Using differentiated SH culture, we here explored the link between A 2A -receptor signalling and neurotransmitter release. In response to the receptor agonist CGS21680, the cells produced cyclic AMP (cAMP), and when depolarised, they released increased amounts of noradrenaline. An A 2A -receptor antagonist, XAC, as well as an inhibitor of cAMPdependent protein kinase A (PKA), H89, depressed agonistdependent release. In the presence of XAC or H89, noradrenaline release was found to be below basal values. This suggested that release facilitation also owes to constitutive receptor activity. We demonstrate that even in the absence of an agonist, the native A 2A -receptor stimulated cAMP production, leading to the activation of PKA and enhanced noradrenaline release. Ancillary, non-cAMP-dependent effects of the receptor (i.e. phosphorylation of CREB, of Rabphilin3A) were refractory to constitutive activation. PKA-dependent facilitation of noradrenaline release was recapitulated with membrane-permeable 8-Br-cAMP; in addition to facilitation, 8-Br-cAMP caused marked inhibition of release, an effect not observed upon receptor activation. Inhibition by receptor-independent cAMP was likely due to suppression of voltagedependent calcium current (VDCC) and increased activity of Src-family kinases. Receptor-mediated release facilitation was reproduced in the presence of tetrodotoxin (blocking action potentials); hence, the signalling occurred at the active zone comprising release sites. Our findings thus support (1) presynaptic localisation of the A 2A -receptor and (2) suggest that compartmentalised pathways transmit cAMP signalling in order to facilitate depolarisation-dependent neurotransmitter release.

The principle of "fluorescence resonance energy transfer" (FRET) has been exploited in cell biology to demonstrate the interaction of two proteins in living cells. FRET is highly specific and sensitive and allows for the online recording of events that modulate the interaction. We here demonstrate that FRET-based sensor molecules represent an alternative in monitoring receptor-dependent signalling. We have used a sensor for the second messenger cAMP to assess cellular formation of cAMP and to follow receptor activity and its desensitization with sufficient temporal resolution. Compared to standard biochemical means of determining cAMP, which require a large number of cells, the sensor records transient events localized in single cells. We show in primary neurons that cAMP formation triggered by the A 2A adenosine receptor occurs in a manner similar to that observed in established models from cell culture. Another way to apply FRET in signal transduction research is in determining the conformational change of signalling proteins. One example is SAP102 (synapse associated protein of 102 kDa) and its binding partner mPINS (mammalian partner of inscuteable). Both proteins are known to form intramolecular bonds and therefore exist in an "open" and "closed" conformation which presumably reflects different activity states in a signalling pathway. For instance, binding of mPINS to the heterotrimeric G protein α subunit Gα i leads to an open conformation. With the use of constructs, where the conformationally active protein regions are tagged with FRET-partner fluorophores, we show that a G protein-coupled receptor may impinge on the folding of these proteins. This may provide a tool to scan for receptors capable of activating an effector as specific as mPINS.

Differentiation into a nerve cell-like phenotype and growth arrest of SH-SY5Y neuroblastoma cells went along with increased cAMP formation. Both receptor-dependent as well as direct activation of adenylyl cyclase by forskolin were enhanced by at least twenty-fold. Since cAMP controls many processes in nerve cell function and development we have investigated the causal factors and mechanism of sensitization in SH-SY5Y cells. The degree of sensitization depended on pre-incubation of the cells with retinoic acid; however, maximizing the extent of sensitization required the withdrawal of serum from the culture medium. This was necessary for the cells to secrete endogenous substances into the culture supernatant. Because sensitization was blocked by inhibitors of gene transcription we surmised that the autocrine factors were relevant for sensitization and were generated by de novo protein synthesis. A gene expression screen revealed several factor candidates (including dkk1, EphB2, NPY, VEGFB); our preliminary data indicated that a combination of these may be needed to induce full sensitization. Our data further suggest that sensitization was not due to up-regulation of stimulatory G proteins or adenylyl cyclase. Rather, the immediate cause may be clustering of the catalyst and its activator G s . This interpretation is consistent with the effect caused by altering the membrane lipid composition which enhanced and reduced cAMP formation in undifferentiated and differentiated cells, respectively.

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