Cell Surface Receptors and Signalling
Analgesic cone shell toxins targeting sodium channels
RS Norton, S Yao, BJ Smith in collaboration with M Zhang, L Azam, D Yoshikami, BM Olivera, G Bulaj (University of Utah, UT USA) Pub ref: 162
Disulphide-rich peptide neurotoxins from venomous cone snails have considerable therapeutic potential. Although several analgesic conotoxins have already reached clinical trials, there is an ongoing need for the discovery and development of novel non-opioid analgesics, such as subtype-selective sodium channel blockers. The µ-conotoxins bind to site 1 on voltage-gated sodium channels, which is located on the extracellular surface of the pore-forming α-subunit and also binds the guanidinium alkaloids tetrodotoxin and saxitoxin. Of the nine α-subunits cloned from mammals, six bind tetrodotoxin with high affinity, and three, NaV1.5, NaV1.8, and NaV1.9, are classified as tetrodotoxin-resistant. µ-conotoxin KIIIA has potent analgesic activity in a mouse pain model. It was found to irreversibly block the voltage-gated sodium channel NaV1.2. Further engineering of µ-KIIIA may provide subtype-selective pharmacological compounds for mammalian neuronal sodium channels and potential therapeutics for the treatment of pain.
Another µ-conotoxin, µ-SIIIA, also exhibits potent analgesic activity in mice. We have determined its structure in aqueous solution and characterised its backbone dynamics by NMR and its functional properties electrophysiologically. µ-SIIIA is a potent blocker of NaV1.2, but it also blocks NaV1.4 and NaV1.6 with submicromolar potency. The selectivity profile of µ-SIIIA, including poor activity against the cardiac sodium channel subtype NaV1.5, is similar to that of µ-KIIIA, suggesting that the common C-terminal regions of both are critical for blocking neuronal NaV1.2. This is the first comprehensive structural and functional characterisation of an analgesic µ-conotoxin that targets neuronal subtypes of mammalian sodium channels.
µ-SIIIA structure. Left hand view shows the backbone in light grey and side chains of several residues highlighted. Those residues with side chains highlighted are also labelled. The three disulfide bonds (residues 3-13, 4-19 and 8-20) are shown in yellow. Right hand view shows the molecular surface in the same orientation and using the same sidechain colours.
Structure and signalling in the EGFR and gp130 families of receptors
C Luo, Y Xu, TK Lam, MJ Pocock, N Kershaw, TPJ Garret in collaboration with J-G Zhang, SE Nicholson, NA Nicola (Cancer and Haematology Division), F Walker, TG Johns, AM Scott, EC Nice, AW Burgess (Ludwig Institute)
Much is known about the functions of intra- and extracellular portions of cell-surface receptors that signal growth and development. However, it is still not clear how they transmit signals across the cell membrane. Preserving the signalling fidelity of these systems is essential to prevent diseases such as cancer for Epidermal Growth Factor Receptors, and multiple sclerosis or a variety of skin complaints in the case gp130 and Leukemia Inhibitory Factor Receptor. We are using structural methods and receptor mutations to understand the role of ligand-binding and juxtamembrane regions in controlling both the maintenance of the off state and transmission of the external signal. Ways of modifying either of these states can then be used in the design of new inhibitors of cell signalling.
Structural characterisation of K+ channel gating
OB Clarke, A Caputo and JM Gulbis
The architecture of the potassium selective pore is conserved in all potassium (K+) channels. While single channel recordings can readily discern when a channel is in a conducting or non-conducting state, at present the same does not apply to crystal structures of K+ channels. There is at present no cogent means for demarcation of physiological state, or for pinpointing non-native structure arising from removing a channel from the natural environment of a bilayer. We are primarily concerned with laying a foundation for interpretation of K+ channel structure by defining a set of consistent molecular indicators of physiological state.
Insulin and type 1 insulin-like growth factor receptor
JGT Menting, MB Margetts, CW Ward, MC Lawrence in collaboration with J Trewhella (University of Sydney), A Whitten (University of Sydney & ANSTO) Pub ref: 130, 193, 222
In the healthy individual the insulin and type 1 insulin-like growth factor receptor mediate the signalling events associated with glucose homeostasis and normal cell growth. However, their aberrant signalling plays a role in both cancer development and progression. Surprisingly, no atomic level information exists of the way these receptors bind ligand and transfer signal. We are seeking to obtain three-dimensional structures of these receptors in complex with ligand and to understand the signalling mechanism. Insights from our structure of the unliganded insulin receptor ectodomain are now being exploited to achieve these goals, with the potential to open up new therapeutic opportunities for cancer as well as diabetes.
SOCS proteins interrupt the signalling cascade by promoting ubiquitination
JJ Babon, JK Sabo, T Laktyushin, S Yao, RS Norton in collaboration with J-G Zhang, SE Nicholson, NA Nicola (Cancer and Haematology Division) Pub ref: 5
Cells communicate with one another by chemical messengers called cytokines, which play important roles in haemopoiesis, immunity and inflammation. The SOCS (Suppressors Of Cytokine Signalling) family of proteins is responsible for switching off this cytokine signal. SOCS proteins can act by inducing the degradation of signalling intermediates via an association with E3 ubiquitin ligases. We have identified the molecules involved in the SOCS-induced ubiquitination process and characterised their interaction. Using SOCS3 as a model system, we were able to demonstrate that it specifically promotes substrate ubiquitination. A detailed structure/function analysis of this process will provide insight into this important biological process.