Molecular interactions and signalling mechanisms of 5-HT4 and 5-HT7 serotonin receptors

The basis for the current project is observations in our lab indicating special properties of two serotonin receptors, 5-HT4 and 5-HT7. First, we have demonstrated that the 5-HT7 receptor behaves as if it were preassociated with G protein in the absence of ligand and reduces the signalling capacity of other receptors, possibly through preventing activated G protein from reaching adenylyl cyclase. We now propose to address the molecular determinants and mechanisms behind this effect by studying chimaeric and truncated receptors as well as through utilisation of FRET technology to study the molecular interactions between labelled receptors and G protein subunits. Second, studies of effects of several different ligands at the 5-HT7 receptor indicates that this receptor displays ligand-selective receptor conformations, and may be a suitable model receptor to study this phenomenon. One recent finding in this regard was that a subset of antagonists at the 5-HT7 receptor seem able to downregulate the receptor, a property classically ascribed to agonists, which would desensitise a receptor upon prolonged stimulation. We will follow up these findings by examining to which extent different 5-HT7 receptor antagonists are able to induce receptor endocytosis and deg radation, and through which pathways and mechanisms. Third, we have found that 5-HT4 receptors appear in the ventricle of failing rat and human hearts and mediate an inotropic response comparable in size to that elicited through beta-adrenoceptors (beta- AR), despite a much lower measurable increase in cellular cAMP. This may indicate that cAMP generated trough 5-HT4 receptor as opposed to beta-AR stimulation is much more tightly coupled to the inotropic response, possibly through different intracellular localisation/compartmentalisation. We now propose to use FRET technology with intracellular cAMP probes as well as studies of the phosphodiesterases involved and their localisation, to address this issue.