EP1554585A1

EP1554585A1 - Reporter module molecules having active ion channel activity

Info

Publication number: EP1554585A1
Application number: EP03740756A
Authority: EP
Inventors: John Colyer
Original assignee: University of Leeds
Current assignee: University of Leeds
Priority date: 2002-06-25
Filing date: 2003-06-25
Publication date: 2005-07-20
Also published as: AU2003277986A1; GB0214553D0; WO2004001423A1

Abstract

The present invention provides a reporter module molecule comprising a first target protein directly fused to or linked via a linker to a first ion channel fragment and a second target protein directly fused to or linked via a further linker to second ion channel fragment, so that when the target proteins interact the two said ion channel fragments are in close proximity and spontaneously reconstitute to form an active ion channel. The invention further provides uses of the reporter module molecule especially in studying heterodimerisation and/or homodimerisation of protein:protein interactions and in methods of identifying binding partners of a target protein from a cDNA library or ligand library.

Description

Reporter Module Molecules having Active Ion Channel Activity

The present invention relates to ion channel reporter module molecules, methods of producing such module molecules and uses therefore especially in detecting interactions between proteins such as heterodimer or homodimer formation and/or mapping interaction circuits of membrane proteins and/or for assessing therapeutic efficacy of agents on, for example, hormone or receptor binding. Further uses of the reporter module molecules include detecting protein interactions in healthy and disease states and as biosensors.

Background to the Invention

The transient interaction between proteins is a key component of most biological processes and is central to the passage of information in signal transduction cascades. In many instances, receptors oligomerise in the presence of a ligand and initiate a signalling cascade; in other examples adapter molecules, effector molecules or courier molecules interface briefly with the ligand bound receptors. Transient interactions between proteins are used throughout biological systems as a means, for example, of inhibiting enzyme function, re-tuning enzyme specificity and as a means of transferring information across physical barriers. In each case, the assembly and disassembly of competent protein complexes is dynamic and the frequency of interaction is regulated so as to control the biological process in question. Information transfer across a membrane is an important aspect of biology, which invariably involves the transient interaction between membrane proteins. Errors in this information transfer process can cause disease states (e.g.diabetes mellitis).

The code used by hormones to transfer information into the cell is complex. Signals are heterogeneous with respect to their -intensity, duration, frequency and spatial distribution. Each of these qualities conveys only part of a message and is subsequently decoded by the cell and used to fine-tune the response to the original stimulus. Our understanding of the components of a signal and its decoding is inextricably linked with the development of the enabling technology. The understanding of such processes, is best exemplified in the field of Ca²⁺-signalling. In this specific example, cell permeable Ca²⁺-sensitive dyes were developed which were able to measure intracellular Ca²⁺ ion concentrations in living cells. These dyes were non-invasive, responded to changes in Ca²⁺ instantly and delivered an optical signal, which could be quantified and imaged. This technological breakthrough precipitated huge advances in the understanding of signals sent via Ca²⁺, including descriptions of Ca²⁺-spikes, Ca²⁺-waves, Ca²⁺-sparks, the frequency, amplitude and duration of each of these and the messages, which these events impart.

Proteimprotein interactions are important for another reason in that the sequencing of entire genomes from a variety of organisms needs to be translated for a greater understanding of the molecular basis of biological "events". This translation requires an understanding of how proteins interact with one another, which proteins they each interact with, and how these interactions change with respect to time and circumstance and different cell environments. The mapping of such protein interaction circuits, particularly those involving proteins in the plasma membrane, we believe will be facilitated by the present invention which will be detailed hereinafter.

It is known from the prior art to study proteimprotein interactions by well established techniques, such as the yeast two hybrid technique (Fields & Song, 1989) or by fluorescence resonance energy transfer (FRET, Adams et al., 1991). However, these methods are limited in that they require an in depth knowledge of the target under study. Another technique known from the prior art (WO 89/01159) is the provision of artificial membranes comprising a closely packed array of self-assembling molecules, some of which comprise a receptor molecule such as an immunoglobulin conjugated to a supporting entity such as a membrane protein or lipid head group. The membranes also incorporate a plurality of gramicidin ion channels. The ion channel, gramicidin acts as the signal device to mark antibody: antigen interaction, in this way specific interactions of cell membrane bound proteins may be studied. It is also known to tether/attach such membranes to a solid inorganic surface such as gold to form electrode-membrane combinations for use as biosensors (WO97/01092). However, the membrane itself and the technique employing them have limited application in cell biology since gramicidin cannot be expressed in most cells and tethering the membrane to a gold substrate is not applicable to production and use in a live cell context.

There is therefore a need to provide alternative reporter molecules for use in studying ubiquitous proteimprotein interactions and which can deliver a substantial, readily quantified measure of the biological "event" of interest and which can be detected in real-time, imaged and which does not require an in depth knowledge of the target under study. We believe that the present invention may provide some of these advantages.

Statement of the Invention

According to a first aspect of the invention there is provided a reporter module molecule comprising a first target protein directly fused to or linked via a linker to a first ion channel fragment and a second target protein directly fused to or linked via a further linker to second ion channel fragment, so that when the target proteins interact the two said ion channels fragments are in close proximity and spontaneously reconstitute to form an active ion channel.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

It will be appreciated that in the present invention, ion channel activity is the "output" device providing the means for detecting a biological event or interaction. Thus, the ion channel activity may be measured directly by means of electrical conductivity or it may be measured by, for example and without limitation, fluorescence which occurs as a result of or consequence of ion channel activity. Reference herein to a reporter module molecule is intended to include a reporter group which utilises ion channel activity or a consequence of ion channel activity as the means for detecting or reporting a biological event or protein interaction.

According to a further aspect of the invention there is provided a reporter module molecule comprising a first target protein directly fused to or linked via a linker to an ion channel and a second target protein directly fused to or linked via a further linker to an ion channel inhibitor, so that when the target proteins interact the ion channel is inhibited.

Each target protein is fused to a component of a channel. In its simplest form, two targets could be fused to components of a channel which are able to reconstitute into a functional channel. In one embodiment, each target could be fused to an ion channel fragment. In other embodiments, a plurality of targets could each be fused to channel portions of different proportions. Thus it will be appreciated that the number of targets assembling a complex and studied by this invention could be greater than 2.

Preferably, in the instance where the target proteins are associated with a linker, they are fused to the linkers.

Preferably, the linker comprises a hydrophilic, protease resistant, flexible structure of length at least one and up to 100 residues, preferably it comprises in the region of at least one to 20 residues.

Preferably, the ion channel is a peptic ion channels, such as the M2 protein of influenza virus, mellitin, a NB protein of influenza B, a SH protein of paramyxovirus SV5, a vpu protein of HIV- 1 or mutant forms or fragments thereof.

The ion channel reporter (ICR) module molecule of the present invention is constructed using simple, naturally occurring, peptide cation channels such as and, by way of example only, the influenza M2 protem transmembrane domain. Preferably, two copies of this transmembrane domain are linked via an additional transmembrane domain to form an ion channel fragment. Other peptide structures which form ion channels, but which are not found in Nature have been described (Lear, J.D., Wasserman, Z.R., & DeGrado, W.F. (1988) Science 240, 1177-1181), and could be used in this invention.

Preferably, two such channel fragments are brought into close proximity by the interaction of at least one upstream or downstream receptor protein. Synthetic receptors and ligands based on coiled-coil structures will ideally be linked to the aforementioned channel fragment modules.

Preferably the peptide ion channel module is selected so that it possesses characteristics required by the reporter technology of the present invention. Preferably the ion channel is capable of conducting monovalent or divalent ions, and preferably it possesses a low open probability when expressed as free peptides but an enhanced open probability when such peptides are brought close to each other in space by the interaction of domains fused to each peptide.

The transmembrane domain of influenza M2 protein is an attractive candidate for the ion channel module of the present invention. A peptide of preferably, about 25 amino acids in length, based on the wild type sequence of the transmembrane domain of influenza M2 protein is known to form a proton (and Na ) channel in black lipid membranes (Duff & Ashley, 1992). The spontaneous association of monomeric peptide units to a functional tetramer forms these proton channels. This self-assembly occurs with low frequency and a short lifetime (~20ms) which is able to create an ion channel of low open probability and low unit conductance. Such a circumstance is ideal for the reporter technology of the present invention as it provides a low background signal (spontaneous channel reconstitution). It is envisaged that mutant forms of the M2 protein transmembrane domain may be even better candidates for the ion channel module of the present invention. In particular, a mutant lacking 4 residues from the transmembrane domain (M2del28-31) displays enhanced ion conductance (when expressed in oocytes, ~40-fold cf. Wild type M2) and is operative at neutral pH (Pinto et al., 1992), whereas the wild type sequence is active at acidic pH. Accordingly, in a preferred embodiment of the invention, this mutant form is the ion channel conductor.

A further advantage of the present invention is based on using all L-residue ion channels as the output device. Preferably ion channels are, in one embodiment all L- residues. All cells will be able to express the reporter group giving it universal applicability in cell biology.

Preferably, in the instance where the ion channel fragment is provided with a linker it is fused to the linker.

Preferably, at least one ion channel fragment is further associated with a green fluorescent protein (GFP) reporter. This particular embodiment provides the advantage of imaging of cells expressing the GFP-reporter so as to identify whether the reporter protein has been correctly targeted to, for example, the plasma membrane.

Alternatively at least one channel ion fragment may be further associated with an epitope so that its presence may be detected immunologically.

It will be appreciated that it will also be possible to monitor proteimprotein interaction by, for example, measuring electrophysiology parameter or by using ion specific dyes.

Preferably, the first and/or second target protein is a receptor molecule, such as and without limitation, a human growth hormone. Alternatively the first protein may comprise Nef (from HTV-1) and the second protein a host CD4 receptor, the interaction therebetween being a transient event, which permits evasion of the immune response by the HIV virus. Alternatively the proteins may be substrates for protein kinases where their association is influenced by the state of phosphorylation of at least one partner. Accordingly the present invention allows measurement of kinase and/or phosphatase activity as an indicator of proteimprotein interaction.

In another embodiment of the invention, the first protein is a G-protein coupled receptor and the second protein is a G-protein. It will be appreciated that other forms of post-translational modification of proteins occur in cells and can influence protein- protein interactions. These are described in WO 99/11774, WO 00/50631, WO 00/50635 and WO 00/50896 and represent yet further examples of target molecules of the present invention.

With regard to the particular embodiment of the invention using G-protein coupled receptors (GPCR), these are an important class of plasma membrane receptor protein which interact with ligands and transduce signals into cells. They have proven to be important targets for therapeutic intervention, and many drugs in current use modify the activity of members of this class of protein (e.g. antagonists of β-adrenergic receptors in hypertension and heart failure). The GPCRs participate in a common step which can be observed using the current invention, namely the interaction of a heterotrimeric G-protein with the GPCR, which is augmented upon activation of the GPCR. Thus by fusion of an ion channel fragment to the GPCR and the fusion of a complimentary ion channel fragment to the α-subunit of an appropriate G-protein, the reporter technology can be used to study this aspect of GPCR signal transduction. It can also be used to identify compounds that modify the transduction of signals through this pathway.

Preferably, the first and/or second target proteins of interest may be each or singly membrane bound.

In another embodiment of the invention the first and/or second target proteins of interest may each or singly are soluble proteins. In this particular embodiment, the reporter module molecule may be modified so that it preferably carries a phosphoryl group and/or another suitable group that influences its association with a binding partner. A coiled coil poypeptide can be engineered to contain a phosphorylation (or other modification) site, which influences association with a partner in a phosphorylation (or other modification) status dependent manner. In this embodiment, phosphorylation status of the reporter molecule, which is indicative of kinase and/or phosphatase activity, can be measured at a location close to a membrane.

The reporter module molecule may also be modified so that it is fused to an intermediate template, preferably the template is provided with appropriate side chains to facilitate attachment to the ion channel fragment.

Preferably the technique may be used in live cells, both in vitro and in vivo. It is envisaged that discrimination between heterodimer formation and homodimer formation will assist in mapping interactions in such cells.

Preferably the coiled-coil polypeptides having an addition site for a phosphate, ubiquitin, glycosyl or ADP-ribosyl moiety, are used in assays for monitoring or modulating protein modification.

The reporter module molecule preferably comprises a coiled-coil and at least one engineered site for the addition of at least one of the following: a phosphate, ubiquitin, glycosyl or ADP-ribosyl moiety, where the polypeptide binds to at least one binding partner in at least one of the following manners: phosphorylation, ubiquitination-, glycosylation- or ADP-ribosylation-dependent manner.

According to a further aspect of the invention there is provided use of the ICR module molecule of the present invention in the study of heterodimerisation and/or homodimerisation of growth hormone receptors in order to define temporal and spatial qualities of signal transduction. According to a yet further aspect of the invention there is provided a method of detecting proteimprotein interactions, interference with receptor binding, change in healthy and disease states and/or screening a ligand library comprising contacting a first protein linked to a first ion channel fragment under investigation with a second protein linked to a second ion channel fragment and measuring ion channel activity.

It will be appreciated that the reporter module molecule of the present invention is versatile in that activity of the ion channel may be used to measure either when there is activity of the ion channel or when there is inhibition of normal activity, hi this way the turning on or off of the ion channel activity in the presence of environmental conditions and or foreign agents maybe assessed.

According to a yet further aspect of the invention there is provided a kit for determining the proteimprotein interaction of a biological system in real time comprising the reporter module molecule as hereinbefore described and optionally including a coiled -coil comprising an engineered site for the addition of a phosphate, ubiquitin, glycosyl or ADP-ribosyl moiety, where the polypeptide binds to at least one binding partner in a phosphorylation-, ubiquitination-, glycosylation- or ADP- ribosylation-dependent manner, and packaging materials;

According to a yet further aspect of the invention there is provided a method to monitor the activity of proteimprotein interactions by monitoring the addition of at least one of: phosphate, ubiquitin, glycosyl or ADP-ribosyl moiety to at least one polypeptide.

According to a yet further aspect of the invention there is provided a method to monitor the activity of proteimprotein interactions comprising monitoring the removal of at least one of: phosphate, ubiquitin, glycosyl or ADP-ribosyl moiety from at least one polypeptide. The products and methods of the present invention can be used for monitoring and/or modulating protein :protein interaction/modification which may be involved in repair, replication, recombination and packaging, secretion and chromosome condensation, cell cycle control, cell growth, inflammation, signal transduction, immune response and malignant transformation. They can also be used in the screening of candidate bioactive agents (e.g. drugs) for efficacy or side effects, whereby the ability of such an agent to modulate the activity of a protein φrotein interaction may be indicative a a therapeutic effect or propensity toward provoking unintended side-effects to a therapeutic or other regimen in which that agent might be used.

According to a yet further aspect of the present invention there is provided compounds identified according to the screening method of the present invention. According to a yet further aspect of the invention there is provided use of a therapeutic agent identified by the screening method of the present invention in the preparation of a medicament for the treatment of hormone related and/or protein related and/or irnmunity conditions/diseases and/or 1 to control parasites or pests.

The technology of the present invention permits the imaging of receptor protein activation, which by analogy with the Ca²⁺-signalling field, will enable the decoding of signal transduction messages in new and greater depth. Furthermore, the technology of the present invention will be able to identify interacting partners for all plasma membrane proteins, and in this format will facilitate understanding of proteomic information through the construction of protein interaction circuits.

It is also envisaged that array formats of this technology of the present invention will provide high throughput platforms for the identification of new drug molecules (which inhibit or promote protein interaction) and novel platforms for the construction of, for example, biosensors. According to a yet further aspect of the invention the reporter module molecule of the present invention may be engineered so as to be a biosensor.

Preferably, the reporter module molecule may be attached to a chip or another appropriate substrate.

According to a yet further aspect of the invention there is provided the reporter module molecule as hereinbefore described incorporated into a membrane.

According to a yet further aspect of the invention there is provided the reporter module molecule as hereinbefore described incorporated into a cell.

The reporter technology of the present invention is based on the transient reconstitution of an active ion channel during the period of association of the two proteins under study. Ion channels have immense activity as they harness the electrochemical gradient of their permeant ion species which provides significant signal amplification for the reporter technology.

We propose a number of suitable ion channels M2 protein of influenza virus, mellitin, the protein of influenza B, SH protein of paramyxovirus SV5, vpu protein of HIV-1 ; and the M2 protein, or mutant or any forms thereof.

h the absence of membrane protein interaction, the ion channel fragment modules will meet infrequently, and thus channel activity will be absent or low. During interaction of the target membrane proteins, the ion channel modules will collide with increased frequency and reconstitute an active channel protein.

Measurement of channel activity is well known in the art, including electrochemical, electrophysiological and, using appropriate dyes, optical methods. The use of dyes provides a facility for imaging of ion channel activity and thus obtaining spatial information relating to protein-protein interactions in real time. The invention will now be described by way of example only with reference to the following Figures wherein:

Figure 1 schematically represents membrane protein interactions;

Figure 2 represents an ion channel fragment module;

Figure 3 represents conversion of a non-functional ion channel fragment module to a functional ion channel reporter molecule;

Figure 4 illustrates the receptor and ion channel domains of the HGG-M2A peptide;

Figure 5A represents monomeric peptides, 5B represents Ni co-ordinated peptide dimers and 5C represents an active ion channel tetramer;

Figure 6 shows HGG-M2A activity at -120 mV in the absence of nickel and;

Figure 7 shows HGG-M2A activity at -120 mV in the presence of nickel.

Detailed Description of the Invention

It will be appreciated that membrane protein interaction is detected by the transient reconstitution of an ion channel during the period of interaction of the proteins of interest (Figure 1). To achieve this, the target proteins are expressed as fusion proteins, in which the reporter module comprises a protein domain equivalent to one fragment of a simple ion channel. When the two fragments of the ion channels are in close proximity, they will spontaneously reconstitute to form an active ion channel, and this event will occur with the highest frequency when the local concentration of the ion channel fragments is greatest, i.e. when the two target proteins are in physical contact. Receptor:ligand interaction regulates ion channel activity

We can demonstrate the feasibility using in vitro ion channel analysis (black lipid membrane methodology) and whole cell expression of the system together with patch clamping and fluorescent indicator dyes (for H* and/or Na ^"). We can utilise a second model system of an artificial receptor:ligand interaction. Furthermore, when the module of Figure 1 is expressed in cells, this reporter protein must adopt the correct topology and be targeted to the correct membrane. Molecular cues which control topology and targeting of membrane proteins are known, these can be incorporated in the design to facilitate production and delivery of this reporter module molecule.

Design of the receptordigand combination:

The receptordigand interaction we have developed is a coiled-coil receptor interacting with a coiled-coil ligand. Lee et al. (1996) described a coiled-coil "template sequence" (based on the leucine zipper sequence of GCN4) and which served as a template for the docking of two shorter coiled-coil peptides. One peptide docked to the N-terminal region of the template sequence and the other peptide docked to the C-terminal fragment of the template. We used the template peptide concept as our "ligand", which is able to cross-link (non-covalently) two short coiled- coil "receptors". These receptors can each be fused to the ion channel fragment modules. We use a coiled-coil sequence other than GCN4 (used by Lee et al., 1996) so that we can adjust the affinity of interaction (length and sequence can be altered in design to fine tune binding affinity), and to ensure that we avoid unwanted hybridisation (with endogenous GCN4) if used in yeast. The ligand proposed is a coiled-coil peptide of 42 residues (6 heptads) which is able to hybridise to the receptors on each copy of the ion channel fragment which will each comprise 3 heptad long coiled-coil proteins (Figure 2a). Using the design principles outlined in O'Shea et al. (1993), the receptor coiled-coils will not dimerise with one another, but will each bind with sub-micromolar (readily tunable by altering sequence and length of peptide) affinity to the ligand. Design of the ion channel fragment module:

The ion channel fragment module comprises two copies of the peptide channel (M2 transmembrane domain, or a mutant form thereof), joined in series by a linker sequence which traverses the membrane forming an anti-parallel three helix structure (Figure 2b). The sequence of the linker transmembrane helix can either be modelled on known monomeric transmembrane proteins with the correct topology (e.g. endopeptidase 24.11, Koehne et al., 1998; glycophorin C, Cartron et al., 1993) or it could be a poly-leucine α-helix which also forms a monomeric tranmembrane domain (Zhou et al. 2000, Choma et al, 2000). The inclusion of positively charged cytoplasmic loops will aid insertion of the protein in the membrane with the desired topology. Other non-helical transmembrane ion channel sequences could be used.

The ligand can be synthesised chemically (42-mer peptide), as can the receptor- fragment channel proteins (-120 residues). Alternatively, the protein may be expressed in bacteria from artificial genes created from synthetic oligonucleotides.

The protein, purified by affinity chromatography, can be reconstituted into lipid vesicles using standard techniques and the ion channel activity analysed in black lipid membranes. We predict that the frequency of spontaneous ion channel reconstitution would be higher in this format than was seen with monomeric channel peptides.

However, we also predict that the frequency of channel opening increases substantially in the presence of the "cross-linking" ligand.

hi parallel, the receptor-fragment channel constructs may be expressed in mammalian cells. In this case, the reporter group can be further modified in two ways. A leader sequence and topology control cues may be added to the construct to ensure plasma membrane insertion of the protein and desired orientation of the protein (extracellular N-terminus). In addition the construct can incorporate a fluorescent protein at the C- terminus to facilitate the detection of the location of this protein in the cell (GreenFP, RedFP Clontech). The transient expression of this construct in HEK 293, CHO or cos cells are performed using the fluorescence of the C-terminal fluorescent protein to review protein expression

Imaging of cells expressing the GFP-reporter are used to identify whether the reporter protein has been correctly targeted to the plasma membrane. Once plasma membrane targeting of the reporter protein has been confirmed, the ion channel activity of the cells can be evaluated using the whole cell patch clamp technique. The basal activity of the reporter channel can be defined by comparison of transfected and untransfected cells, and then the ligand dependent stimulation of channel activity can be determined in both cell types. Large increases in reporter channel activity may be expected in the presence of ligand.

The reporter activity can also be observed using fluorescent dyes for intracellular pH and intracellular Na+. This has a number of advantages, firstly, fluorescence technology is more widely available than patch clamp technology; secondly, the fluorescence technology is readily adapted to high throughput methods formats of experimental conduct and thirdly that spatial heterogeneity of a signal can be detected using the optical techniques. The available evidence suggests that these M2 channels will admit both H⁺ and Na⁺ ions. Fluorescent dyes are commercially available for both of these ions (BCECF, SBFI, Molecular Probes) and these may be employed to establish the most sensitive fluorimetric end-point for this reporter activity. The fluorescent readout can be examined by fluorirnetry and also imaged by confocal microscopy to examine the temporal and spatial resolution of the reporter technology.

With reference to Figure 3 there is shown an alternative embodiment of the reporter module molecule of the present invention. In this particular embodiment, the interaction between two receptor domains (C and D) is controlled by the phosphorylation status of C and D. C and D can have identical sequence (below) and a homodimer is formed whilst the coiled coil is dephosphorylated, whereas dimer formation is prevented when at least one of the partners is phosphorylated. The sequence of C (and D) in this example is: RM QLEDQVl^RLRRKSYHLENEVAALKKLVGERAAK (SEQ ID NO:l)

Interacting protein C is phosphorylated and upon interaction with a second protein D the phosphoryl group is lost. Proteins C and D are fused to templates 5 each being provided with further side chain linkages to the ion channel fragments 1 and 2.

Reporting growth hormone receptor activation in live cells

Human growth hormone co-ordinates growth and abnormalities in the production of this hormone will lead to dwarf or giant phenotypes in the case of under- and overproduction respectively. Growth hormone is a polypeptide hormone of 191 residues which activates a plasma membrane receptor by promoting the dimerisation of the receptor (analogous to the synthetic ligand:receptor, above). This homo-dimerisation of the receptor lends itself to analysis using the reporter technology of the present invention. We can create a fusion protein between the growth hormone receptor (GHR) and the ion channel fragment module, in which the ion channel is a C- terminal extension to the GHR. The signals for targeting and orientating the construct in the plasma membrane are all contained within the GHR protein. In this example however, the ion channel fragment module requires the opposite topology to that described in previous examples.

Expression of the GHR reporter group in mammalian cells (HEK 293, CHO or cos7, as above) can confirm the plasma membrane location of the reporter protein (C- terminal GFP tag or immuno logical epitope). We can then examine the activation of the growth hormone receptor upon GH addition, using the fluorescent out-put of an ion specific dye from the reporter group. The ligand concentration dependence of receptor activation (bell shaped; Wells, 1994), the temporal and spatial response to ligand presentation will be described.

We can configure the technology of the present invention in two modes of polarity (ion channel N-terminus on the extracellular face and on the intracellular face of the plasma membrane), achieve targeting of this reporter protein to the plasma membrane and have described the temporal and spatial information which can be derived from its use.

The interaction of Nef (from HTV-l) with the host CD4 receptor is a transient event, which permits evasion of the immune response by the HTV virus. We can construct a reporter assay for this interaction using the ion channel fragment approach, developed above. The background literature indicates that there is no tendency for homo- oligomer formation of either CD4 or Nef, and thus the reporter technology in its current state of development can be used in this particular case of heterodimer observation. In other cases however, where both homodimer and heterodimer interactions were possible, the reporter technology of the present invention can be refined further to allow investigators to discriminate between homo-dimer and hetero-dimer interactions. Building this discriminating power is a yet further advantage of the present invention.

Reporters able to discriminate between heterodimer and homodimer complexes

The ability to map interaction circuits for a particular membrane protein of interest is a yet further advantage of the present invention. The association of a protein with a second copy of itself (homodimer) can trigger the ion channel reconstitution and thus prevent the identification of interactions with other partners. Creation of an ion channel fragment module which cannot reconstitute with a second channel fragment of identical sequence, but which can combine with a channel fragment module of different primary sequence to reconstitute an active channel, would permit detection of heterodimer interactions exclusively and is therefore an advantage.

The residues involved in stabilising the oligomeric structure of the homo-pentameric protein phospholamban have been identified by site directed mutagenesis (Simmerman et al., 1996). These all reside within the transmembrane domain. The size, shape and chemical property of the residues on the interface between sub-units control the oligomeric state of membrane proteins (Simmerman et al., 1996, Zhou et al., 2000, Choma et al., 2000), thus providing an opportunity to design reporters capable of discriminating between homo- and hetero- interactions. The ion channel modules can be designed such that reconstitution of identical channels is prohibited, but reconstituted of non-identical channels is permitted. Walshaw & Woolfson (U. of Sussex) have developed algorithms to identify coiled-coil motifs in known proteins. These algorithms identify coiled-coil partners in various oligomeric states (dimer, trimer, tetramer and pentamer). Specific arnino acid replacements at 'a' and 'd' positions of the canonical coiled-coil structure are likely to discriminate between oligomeric states, and these will be used to construct a tetrameric (two half channel) reporter molecule which is only able to assemble as a hetero-oligomer.

EXAMPLE 1; HGG-M2A peptide

The peptide sequence NH₂ -H G G S S D P L V V A A S I I G I L A L IL W I L D R L - COO^" (SEQ ID NO:2) is divided into receptor and ion channel domains as shown in Figure 4. The ion channel sequence is a modified form of influenza virus M2 protein transmembrane domain in which a histidine residue has been altered to alanine (A in SEQ ID 2). Wild type sequence displays ion channel activity at acidic pH, this mutant sequence displays ion channel activity at neutral pH. The receptor module (HGG) is a metal binding feature.

The tri-peptide HGG has been shown to bind to Ni²⁺ and Cu²⁺ in a 2:1 complex between pH 6.5 and 7.5 (Zhang Y., Akilesh S. & Wilcox D.E. (2000). Inorg. Chem. 39: 3057-3064). Therefore, detection of the metal ions will be through an increased occurrence of channel formation, which will result in increased channel activity. The postulated mode of action is depicted in Figures 5A-C. The process, with regard to Figure 5A, shows as a first step that monomeric peptides (M) will move freely in a bilayer and will transiently interact to give an active ion channel (tetrameric). As a second step, as depicted in Figure 5B the free momomeric peptides in the presence of nickel will form aimers (D). One nickel molecule will co-ordinate two peptide molecules. Finally, as depicted in Figure 5C, two nickel co-ordinated dimers will interact to form an active ion channel tetramer (T). Co-ordination of two monomeric peptides by nickel essentially removes step 1 because of the transient interaction of four independent monomers has been removed due to the co-ordination of two monomers.

A black lipid membrane (also known as a planar lipid membrane/bilayer) was formed by 'painting' (Mueller P., Rudin D.O., Tien H.T. & Wescott W.C. (1963). J. Phys. Chem. 67: 534 - 537) a lipid solution across a 200 μm aperture in a polycarbonate cup. The lipid phophatidylethanolamine (also known as cephalin) was dried down from chloroform/methanol dissolved stocks under nitrogen and redispersed in n- decane to a final concentration of 50 mg/ml. This lipid solution was used to prime the aperture and to form the bilayer. A stable bilayer had a capacitance between 80 - 200 pF, and a current of +/- 0.5 A at an applied potential of +/- 60 V. The electrolyte solution used was 25 mM HEPES at pH 7. 1 μg of peptide, dissolved in methanol, was added to the cis chamber (the chamber in which the bilayer was formed) and allowed to stir for ~ 30 minutes (final peptide concentration = 0.68 μM). Ion channel activity was recorded at -120 mV on to DAT tape (Figure 6). Nickel was then added to the cis chamber and allowed to stir for 10 minutes. Ion channel activity was again recorded at -120 mV on to DAT tape (Figure 7). Data was sampled at 500 ms intervals after filtering at 50 Hz.

We have been able to demonstrate HGG-M2A ion channel activity at -120 mV in the presence nickel (Figure 7). Accordingly we believe that HGG-M2A is a reporter module molecule with a potential to allow the study of heterodimerisation and/or homodimerisation of proteimprotein interactions so as to define temporal and spatial qualities of signal transduction and may find further utility as a screening agent for identifying suitable therapeutics which alter ion channel activity.

Claims

1. A reporter module molecule comprising a first target protein directly fused to or linked via a linker to a first ion channel fragment and a second target protein directly fused to or linked via a further linker to second ion channel fragment, so that when the target proteins interact the two said ion channel fragments are in close proximity and spontaneously reconstitute to form an active ion channel.

2. A reporter module molecule comprising a first target protein directly fused to or linked via a linker to an ion channel and a second target protein directly fused to or linked via a further linker to an ion channel inhibitor, so that when the target proteins interact ion channel is inhibited.

3. A reporter module molecule according to either preceding claim comprising more than two target proteins each directly fused to or linked via a linker to an ion channel fragment so that when the target proteins interact one or more active ion channel is formed or inhibited.

4. A reporter module molecule according to any preceding claim wherein the target proteins are fused to the linkers.

5. A reporter module molecule according to any preceding claim wherein the linker comprises a hydrophilic peptide of at least one and up to 100 residues in length.

6. A reporter module molecule according to claim 5 wherein the hydrophilic peptide of at least one and up to 20 residues in length.

7. A reporter module molecule according to any preceding claim wherein the ion channel is a peptic ion channel.

8. A reporter module molecule according to any preceding claim wherein the ion channel is selected from the group comprising an M2 protein transmembrane domain of influenza virus, a mutant lacking 4 residues from the transmembrane domain (M2del28-31), mellitin, an NB protein of influenza B, an SH protein of paramyxovirus SV5, a vpu protein of HTV-1 or mutant forms or fragments thereof.

9. A reporter module molecule according to claim 8 wherein two copies of the M2 protein transmembrane domain are linked via an additional transmembrane domain to form an ion channel fragment.

10. A reporter module molecule according to any preceding claim wherein two ion channel fragments are brought into close proximity by interaction of at least one upstream or downstream receptor protein.

11. A reporter module molecule according to any preceding claim wherein said ion channel fragments are linked to synthetic receptors and/or ligands based on coiled-coil structures.

12. A reporter module molecule according to any preceding claim wherein the ion channel fragments comprise all L-residue ion channels.

13. A reporter module molecule according to any preceding claim wherein each ion channel fragment is fused to a linker.

14. A reporter module molecule according to any preceding claim wherein at least one ion channel fragment is further associated with either a green fluorescent protein (GFP) reporter so that its presence may be detected visibly or an epitope so that its presence may be detected immunologically.

15. A reporter module molecule according to any preceding claim wherein the first and or second target protein is a receptor molecule.

16. A reporter module molecule according to claim 15 wherein the receptor molecule is human growth hormone.

17. A reporter module molecule according to any one of claims 1 to 14 wherein the first protein is derived from HIV-1 and the second protein is a host CD4 receptor.

18. A reporter module molecule according to any one of claims 1 to 14 wherein the first protein is a G-protein coupled receptor and the second protein is a G-protein.

19. A reporter module molecule according to any preceding claim wherein the first and/or second target proteins of interest are both or singly membrane bound.

20. A reporter module molecule according to any of claims 1 to 18 wherein the first and/or second target proteins of interest are both or singly soluble proteins.

21. A reporter module molecule according to claim 19 or 20 wherein the molecule is modified to have at least one engineered site for the addition of at least one of the following: a phosphate, ubiquitin, glycosyl or ADP-ribosyl moiety, where the polypeptide binds to at least one binding partner in at least one of the following manners: phosphorylation, ubiquitination-, glycosylation- or ADP-ribosylation- dependent manner.

22. A reporter module molecule according to claim 21 wherein a coiled coil polypeptide is phosphorylated.

23. A reporter module molecule according to any one of claims 20 to 22 wherein the molecule is fused to an intermediate template provided with appropriate side chains to facilitate attachment to the ion channel fragment.

24. Use of the reporter module molecule according to any preceding claim in the study of heterodimerisation and/or homodimerisation of proteimprotein interactions so as to define temporal and spatial qualities of signal transduction.

25. A method of detecting proteimprotein interactions, interference with receptor binding, change in healthy and disease states and/or screening a ligand library comprising contacting a first protein linked to a first ion channel fragment under investigation with a second protein linked to a second ion channel fragment and measuring ion channel activity or inhibition.

26. A method to monitor the activity of proteimprotein interactions by monitoring the addition of at least one of: phosphate, ubiquitin, glycosyl or ADP-ribosyl moiety to at least one protein.

27. A method to monitor the activity of proteimprotein interactions comprising monitoring the removal of at least one of: phosphate, ubiquitin, glycosyl or ADP- ribosyl moiety from at least one protein.

28. A method of identifying binding partners of a target protein from a cDNA library or ligand library comprising measuring co-expression of a target protein fused to a reporter module molecule component so as to identify library members which interact with the target protein under investigation and optionally manipulating conditions in order to measure effects on proteimprotein interactions.

29. A method according to any one of claims 25 to 28 further including any one or more of the features of claims 1 to 23.

30. Compounds identified according to the methods of any one of claims 25 to 29.

31. A kit for determining proteimprotein interaction of a biological system in real time comprising the reporter module molecule according to any one of claims 1 to 23 and packaging materials.

32. Use of a therapeutic agent identified by the methods of any one of claims 25 to 29 in the preparation of a medicament for the treatment of hormone related and/or protein related and/or immunity conditions/diseases and/or to control parasites or pests infections.

33. Use of a therapeutic agent identified by the methods of any one of claims 25 to 29 in the preparation of a medicament for the treatment of human growth hormone related disease or HTV infection or G-protein associated diseases.

34. A biosensor comprising the reporter module molecule according to any one of claims 1 to 23.

35. A biosensor according to claim 34 wherein the reporter module molecule is attached to a chip or another appropriate substrate.

36. A membrane incorporating the reporter module molecule according to any one of claims 1 to 23.

37. A cell incorporating the reporter module molecule according to any one of claims 1 to 23.