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.

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The mechanism by which agonist binding triggers pore opening in ligand-gated ion channels is poorly understood. Here, we used unnatural amino acid mutagenesis to introduce subtle changes to the side chains of tyrosine residues (Tyr141, Tyr143, Tyr153, and Tyr234), which dominate the 5-HT 3 receptor binding site. Heterologous expression in oocytes, combined with radioligand binding data and a model of 5-HT (serotonin) computationally docked into the binding site, has allowed us to determine which of these residues are responsible for binding and/or gating. We have shown that Tyr 143 forms a hydrogen bond that is essential for receptor gating but does not affect binding, whereas a hydrogen bond formed by Tyr153 is involved in both binding and gating of the receptor. The aromatic group of Tyr234 is essential for binding and gating, whereas its hydroxyl does not affect binding but plays a steric role in receptor gating. Tyr141 is not involved in agonist binding or receptor gating but is important for antagonist interactions. These data, combined with a new model of the nonliganded 5-HT 3 receptor, lead to a mechanistic explanation of the interactions that initiate the conformational change leading to channel opening. Thus, we suggest that agonist entry into the binding pocket may displace Tyr143 and Tyr153 and results in their forming new hydrogen bonds. These bonds may form part of the network of bond rearrangements that trigger the conformational change leading to channel opening. Similar rearrangements may initiate gating in all Cys-loop receptors.

The ligand binding site of Cys-loop receptors is dominated by aromatic amino acids. In GABAC receptors, these are predominantly tyrosine residues, with a number of other aromatic residues located in or close to the binding pocket. Here we examine the roles of these residues using substitution with both natural and unnatural amino acids followed by functional characterization. Tyr198 (loop B) has previously been shown to form a cation−π interaction with GABA; the current data indicate that none of the other aromatic residues form such an interaction, although the data indicate that both Tyr102 and Phe138 may contribute to stabilization of the positively charged amine of GABA. Tyr247 (loop C) was very sensitive to substitution and, combined with data from a model of the receptor, suggest a π–π interaction with Tyr241 (loop C); here again functional data show aromaticity is important. In addition the hydroxyl group of Tyr241 is important, supporting the presence of a hydrogen bond with Arg104 suggested by the model. At position Tyr102 (loop D) size and aromaticity are important; this residue may play a role in receptor gating and/or ligand binding. The data also suggest that Tyr167, Tyr200, and Tyr208 have a structural role while Tyr106, Trp246, and Tyr251 are not critical. Comparison of the agonist binding site “aromatic box” across the superfamily of Cys-loop receptors reveals some interesting parallels and divergences.