MethodsMutagenesis and preparation of cRNA and Oocytes -Mutant 5-HT 3A receptor subunits were developed using pcDNA3.1 (Invitrogen, Abingdon, U.K.) containing the complete coding sequence for the 5-HT 3A(b) subunit from mouse neuroblastoma N1E-115 cells as previously described 1 . For nonsense suppression the proline codon at 308 was replaced by TAG as previously described 2 . Wild type and mutant receptor subunit coding sequences were then subcloned into pGEMHE. This was linearized with Nhe1 (New England Biolabs) and cRNA synthesised using the T7 mMESSAGE mMACHINE kit (Ambion). Oocytes from Xenopus laevis were prepared and maintained as described previously 2 .Synthesis of tRNA and dCA-amino acids-Unnatural amino acids were chemically synthesised as nitroveratryloxycarbonyl (NVOC) protected cyanomethyl esters and coupled to the dinucleotide dCA, which was then enzymatically ligated to 74-mer THG73 tRNA CUA as detailed previously 3 . Immediately prior to co-injection with mRNA, tRNA-aa was deprotected by photolysis. Typically 5 ng mRNA and 25 ng tRNA-aa were injected into Stage V-VI oocytes in a total volume of 50 nl. For control experiments, mRNA was injected 1) in the absence of tRNA and 2) with the THG73 74-mer tRNA.Experiments were preformed 18-36 h post injection.Characterisation of mutant receptors-5-HT-induced currents were recorded from individual oocytes using two-voltage electrode clamp with either a GeneClamp 500 amplifier or an OpusXpress system (Axon Instruments, Inc., Union City, CA). All experiments were performed at 22-25 ºC. Serotonin (creatinine sulphate complex,

A series of tryptophan analogues has been introduced into the binding site regions of two ion channels, the ligand-gated nicotinic acetylcholine and serotonin 5-HT(3A) receptors, using unnatural amino acid mutagenesis and heterologous expression in Xenopus oocytes. A cation-pi interaction between serotonin and Trp183 of the serotonin channel 5-HT(3A)R is identified for the first time, precisely locating the ligand-binding site of this receptor. The energetic contribution of the observed cation-pi interaction between a tryptophan and the primary ammonium ion of serotonin is estimated to be approximately 4 kcal/mol, while the comparable interaction with the quaternary ammonium of acetylcholine is approximately 2 kcal/mol. The binding mode of nicotine to the nicotinic receptor of mouse muscle is examined by the same technique and found to differ significantly from that of the natural agonist, acetylcholine.

A-kinase anchoring proteins (AKAPs) are signaling scaffolds that contribute to various aspects of cAMP signaling. They do this by tethering protein kinase-A to specific subcellular sites, thereby focusing its activity toward relevant substrates. Recently the structural basis for these proteinprotein interactions has been elucidated by x-ray crystallography. Recent reports have identified AKAPs that bind to adenylyl cyclases to regulate cAMP synthesis and that sequester phosphodiesterases to break down this second messenger locally. Another emerging aspect of AKAP function is their role in integrating cAMP signaling with other signaling pathways. For example, molecular and genetic approaches have been used to show that the neuronal anchoring protein WAVE1 integrates signaling from PKA and Cdk5 to regulate actin polymerization and cytoskeletal events. Signaling scaffoldsOver the past twenty years, a hallmark achievement in cell biology has been the elucidation of the fundamental role that protein-protein interactions play in cellular signaling. Indeed, the recent large-scale genomics and proteomics projects have shown that after a certain point the evolution of complex metazoans is driven not by the creation of entirely new genes but rather by the combinatorial shuffling of modular protein-protein interaction domains [1,2]. Among different signaling pathways, this shuffling of modular domains drives the creation of new connectivities and regulatory networks [2]. Prime examples of this strategy are the numerous scaffolding and adaptor proteins that function in the assembly of multi-protein signaling complexes [3,4]. These signaling scaffolds serve as platforms for the integration and simultaneous dissemination of multiple signals. By sequestering a signaling enzyme to a specific subcellular environment, these proteins ensure that upon activation the enzyme is near its relevant targets. Thus scaffolds contribute to the spatiotemporal resolution of cellular signaling and are a key means by which a common signaling pathway can serve many different functions.One family of scaffolding proteins are the A-kinase anchoring proteins (AKAPs), which anchor protein kinase A (PKA) to specific subcellular locations [5,6]. AKAPs are a wellstudied family of signaling scaffolds and because of the range of their interactions serve as a good model for these systems. As PKA is the primary effector of the second messenger 3′5′-cyclic-adenosinemonophosphate (cAMP), AKAPs play an important role in the targeting and regulation of PKA-mediated phosphorylation events. An equally important role of AKAPs is their capacity to form multi-protein complexes that integrate cAMP signaling with other pathways and signaling events. In this review we focus on recent advances in the study of AKAPs. In terms of AKAP function, our discussion of these The AKAP/PKA complexAKAPs make up a structurally diverse protein family with >50 members. Functionally, these proteins share three common features: first, they contain a PKA-anchoring domain; second...

GABA(C) (rho) receptors are members of the Cys-loop superfamily of neurotransmitter receptors, which includes nicotinic acetylcholine (nACh), 5-HT(3), and glycine receptors. As in other members of this family, the agonist binding site of GABA(C) receptors is rich in aromatic amino acids, but while other receptors bind agonist through a cation-pi interaction to a tryptophan, the GABA(C) binding site has tyrosine at the aligning positions. Incorporating a series of tyrosine derivatives at position 198 using unnatural amino acid mutagenesis reveals a clear correlation between the cation-pi binding ability of the side chain and EC(50) for receptor activation, thus demonstrating a cation-pi interaction between a tyrosine side chain and a neurotransmitter. Comparisons among four homologous receptors show variations in cation-pi binding energies that reflect the nature of the cationic center of the agonist.