WO2002046219A2 - Proteines d'activation de rho-gtpase et procedes correspondants - Google Patents

Proteines d'activation de rho-gtpase et procedes correspondants Download PDF

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WO2002046219A2
WO2002046219A2 PCT/EP2001/013948 EP0113948W WO0246219A2 WO 2002046219 A2 WO2002046219 A2 WO 2002046219A2 EP 0113948 W EP0113948 W EP 0113948W WO 0246219 A2 WO0246219 A2 WO 0246219A2
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polypeptide
rich
nucleic acid
cip4
amino acid
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WO2002046219A3 (fr
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Pontus Aspenstrom
Ninna Richnau
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Ludwig Institute For Cancer Research
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • C07K14/4706—Guanosine triphosphatase activating protein, GAP
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to nucleic acids, polypeptides, homologues, antibodies, and assay and other methods relating to and based on the identification of novel Rho-GTPase activating protein (GAP) which interacts with CIP4 and plays a role in modulating processes involving the eukaryotic cell cytoskeleton.
  • GAP Rho-GTPase activating protein
  • the cytoskeleton is a major determinant for eukaryotic cell function.
  • the cytoskeleton is formed by three distinct filament systems; the microfilament system, the intermediate filament system and the microtubule system, which act in concert to orchestrate processes such as cell locomotion, changes in cell morphology and intracellular transport (1,2).
  • Cytoskeletal elements, in particular the microfilament system are under a constant reconstruction in response to external stimuli.
  • Actin monomers which polymerise into asymmetric filaments, form the core of the microfilament system.
  • a large number of actin-binding proteins assist to organise actin filaments in a variety of supramolecular structures .
  • Rho GTPases A family of signalling intermediates, the Rho GTPases, have been shown to be pivotal regulators of the microfilament system and thereby of the morphogenetic and motile properties of mammalian cells.
  • Rho GTPases The potential of the Rho GTPases to function as signalling switches resides in their ability to cycle between active, GTP-bound states, and inactive, GDP-bound states. This cycling is orchestrated by guanine nucleotide exchange factors (GEFs) , GTPase-activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs) . GEFs stimulate the replacement of GDP by GTP, whereas GAPs stimulate the intrinsic GTP hydrolysis of the GTPase (5-7) . GDIs act by blocking GDP- dissociation and, in resting cells, the Rho GTPases are thought to reside in an inactive complex with GDI proteins (7,8) .
  • GEFs guanine nucleotide exchange factors
  • GAPs GTPase-activating proteins
  • GDIs guanine nucleotide dissociation inhibitors
  • Rho family consists of at least 16 distinct members which can be further divided into six subgroups; Cdc42 (Cdc42, TC10, TCL, Chp) , Rac (Racl-3, RhoG) , Rho (Rho A-C) , Rnd (Rndl- 2, RhoE) , RhoD and RhoH (9,10).
  • Rho GTPases have been shown to regulate several vital cellular processes in addition to cytoskeletal rearrangements .
  • Rac and Cdc42 participate in transcriptional control via the JNK/SAPK and p38 MAPK signalling cascades, ' Rho has a role in SRF-regulated gene transcription and all three contribute to transcriptional activation via the NF- ⁇ B signalling pathway (4,10-15) .
  • the RhoGTPases are also participants in signalling leading to cell cycle entry and apoptosis (4,13) .
  • Rho GTPases The identification of binding partners for the Rho GTPases has resulted in insights into the mechanisms by which these proteins mobilise the microfilament system (4,14,15) .
  • WASP iskott-Aldrich syndrome protein
  • WASP Wiskott-Aldrich syndrome (19,20).
  • WASP is a multidomain adapter protein, which contains a phosphoinositide-binding domain, a CRIB (Cdc42/Rac interactive binding) domain that specifically binds Cdc42 (21) , and an extended proline-rich domain that binds SH3 domain-containing proteins such as Nek, Src and Btk/Tec (18) .
  • the members of the WASP family bind directly to actin and to the so-called Arp2/3 complex.
  • This multi-subunit protein complex has an essential role in regulating actin polymerisation in cells. It is suggested that Arp2/3 is needed both for the formation of new actin filaments, and for the binding to the sides of actin filaments, thereby forming branched actin filament networks (16-18) . .
  • Cdc42-binding proteins have been found to affect the organisation of the actin cytoskeleton, although less is known about the mechanisms by which they act. A common denominator for most of these proteins is the presence of a CRIB domain (21) .
  • MSE55 has been shown to induce filopodia-like protrusions when overexpressed in fibroblasts (22)
  • MSE55-related Borg Binds of Rho GTPases
  • CIP4 The Cdc42-interacting protein 4 (CIP4) , however, binds Cdc42 via a domain motif unrelated to the CRIB domain (25) .
  • CIP4 binds Cdc42 via a domain motif unrelated to the CRIB domain (25) .
  • Over-expression of CIP4 in fibroblasts leads to disappearance of filamentous actin bundles in these cells.
  • simultaneous expression of activated Cdc42 and CIP4 results in a relocalisation of the uniformly distributed CIP4 into peripheral and dorsal clusters or villi-like structures, which might represent precursors of filopodia (25) .
  • RhoGAP domain-containing protein that specifically interacted with the SH3 domain of CIP4 using a yeast two-hybrid screen. This protein has been named RICH-1 for RhoGAP Interacting with CIP4 Homologues.
  • RhoGAP domains of RICH-1 were shown to be homologous to a protein of unknown function, KIAA0672. We have proposed that this protein should be renamed RICH-2.
  • the RhoGAP domain of RICH-1 was furthermore shown to be similar to the analogous domain of the Abl-binding protein 3BP-1 (26) .
  • the high degree of similarity between the proteins suggested to the inventors that RICH-1, RICH-2 and 3BP-1 form a closely related family of RhoGAPs .
  • RhoGAP domains of RICH-1 and RICH-2 specifically activate the GTP hydrolysis of Racl and Cdc42, but not of RhoA.
  • RhoGAP domain of RICH-1 abrogated PDGF- BB-induced membrane ruffles but not the serum-induced stress-fibres, providing further indication that RICH-1 is a Rac- and Cdc42-specific GAP.
  • nucleic acid molecule which has a nucleotide sequence encoding a polypeptide which includes the amino acid sequence shown in Figure 1 or Figure 2.
  • the coding sequence may be that shown in Figure 3 or - Figure 4 or it may be a mutant, variant, derivative or allele of one of the sequences shown.
  • the sequence may differ from that shown by a change which is one or more of addition, insertion, deletion and substitution of one or more nucleotides of the sequence shown. Changes to a nucleotide sequence may result in an amino acid change at the protein level, or not, as determined by the genetic code .
  • nucleic acid according to the present invention may include a sequence different from the sequence shown in Figure 3 or Figure 4 yet encode a polypeptide with the same amino acid sequence .
  • the amino acid sequence shown in Figure 1 consists of 803 residues (RICH-1)
  • RICH-IB alternatively spliced variant shown in Figure 2
  • the term RICH-1 as used in this document includes RICH-IB.
  • the encoded polypeptide may comprise an amino acid sequence which differs by one or more amino acid residues from the amino acid sequence shown in Figure 1 or Figure 2.
  • Nucleic acid encoding a polypeptide which is an amino acid sequence mutant, variant, derivative or allele of the sequence shown in Figure 1 is further provided by the present invention. Such polypeptides are discussed below. Nucleic acid encoding such a polypeptide may show at the nucleotide sequence and/or encoded amino acid level greater than about 60% homology with the coding sequence shown in Figure 3 or Figure 4 and/or the amino acid sequence shown in Figure 1 or Figure 2, greater than about 70% homology, greater than about 80% homology, greater than about 90% homology or greater than about 95% homology.
  • amino acid "homology” this may be understood to be similarity (according to the established principles of amino acid similarity, e.g. as determined using the algorithm GAP (Genetics Computer Group, Madison, WI) or identity.
  • Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al . (1990) J " . Mol . Biol .
  • Suitable conditions include, e.g. for detection of sequences that are about 80-90% identical suitable conditions include hybridization overnight at 42°C in 0.25M Na 2 HP0 4 , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55°C in O.lX SSC, 0.1% SDS.
  • suitable conditions include hybridization overnight at 65°C in 0.25M Na 2 HP0 4 , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60°C in O.lX SSC, 0.1% SDS.
  • a variant form of a nucleic acid molecule may contain one or more insertions, deletions, substitutions and/or additions of one or more nucleotides compared with the wild-type sequence (such as shown in Figure 3 or Figure 4) which may or may not disrupt the gene function. Differences at the nucleic acid level are not necessarily reflected by a difference in the amino acid sequence of the encoded polypeptide. However, a mutation or other difference in a gene may result in a frame-shift or stop codon, which could seriously affect the nature of the polypeptide produced (if any) , or a point mutation or gross mutational change to the encoded polypeptide, including insertion, deletion, substitution and/or addition of one or more amino acids or regions in the polypeptide.
  • a mutation in a promoter sequence or other regulatory region may prevent or reduce expression from the gene or affect the processing or stability of the mRNA transcript. For instance, a sequence alteration may affect splicing of mRNA.
  • polypeptides encoded by nucleotides of the present invention specifically activate, enhance or increase the GTP hydrolysis of Racl and Cdc42.
  • Nucleic acid of the present invention may encode a fusion or hybrid protein which comprises a peptide or polypeptide as described herein, for example a polypeptide having a sequence shown in Figure 3 or Figure 4, and at least one heterogeneous (i.e. foreign, non- RICH) amino acid, preferably ' at least 10, at least 20, at least 50 or at least 100 amino acids.
  • the heterogeneous amino acids may form a functional domain, for example an antigen binding domain, which may have biological activity, for example binding activity, such as an immunoglobulin antigen binding domain, or enzymatic activity, such as ⁇ -galactosidase, glutathione S- transferase or horseradish peroxidase.
  • a suitable nucleic acid according to this aspect of the present invention may comprise a nucleic sequence as shown in Figure 1 or Figure 2 or a variant thereof, as described above.
  • nucleic acid according to the present invention is provided as an isolate, in isolated and/or purified form, or free or substantially free of material with which it is naturally associated, such as free or substantially free of nucleic acid flanking the gene in the human genome, except possibly one or more regulatory sequence (s) for expression.
  • Nucleic acid may be wholly or partially synthetic and may include genomic DNA, cDNA or RNA.
  • the coding sequence shown herein is a DNA sequence. Where nucleic acid according to the invention includes RNA, reference to the sequence shown should be construed as encompassing reference to the RNA equivalent, with U substituted for T.
  • Nucleic acid may be provided as part of a replicable vector, and also provided by the present invention are a vector including nucleic acid as set out above, particularly any expression vector from which the encoded polypeptide can be expressed under appropriate conditions, and a host cell containing any such vector or nucleic acid.
  • An expression vector in this context is a nucleic acid molecule including nucleic acid encoding a polypeptide of interest and appropriate regulatory sequences for expression of the polypeptide, in an in vi tro expression system, e.g. reticulocyte lysate, or in vivo, e.g. in eukaryotic cells such as COS or CHO cells or in prokaryotic cells such as E. coli . This is discussed further below.
  • the nucleic acid sequence provided in accordance with the present invention is useful for identifying nucleic acid of interest (and which may be according to the present ⁇ invention) in a test sample, for example, homologues of the RICH-1 nucleotide sequence.
  • the present invention provides a method of obtaining nucleic acid of interest, the method including hybridisation of a probe having the sequence shown in Figure 3 or Figure 4 , or a complementary sequence, to target nucleic acid. Hybridisation is generally followed by identification of successful hybridisation and isolation of nucleic acid which has hybridised to the probe, which may involve one or more steps of PCR. It will not usually be necessary to use a probe with the complete sequence shown in any of these figures.
  • Shorter fragments may also be used, particularly fragments encoding the RICH-1 domains at residues 1 to 208, 266 to 409, 557-570, 633 to 642, 676 to 687 and 727 to 738 of the sequence shown in Fig 1.
  • Nucleic acid according to the present invention is obtainable using one or more oligonucleotide probes or primers designed to hybridise with one or more fragments of the nucleic acid sequence shown in any of the figures, particularly fragments of relatively rare sequence, based on codon usage or statistical analysis.
  • a primer designed to hybridise with a fragment of the nucleic acid sequence shown in any of the figures may be used in conjunction with one or more oligonucleotides designed to hybridise to a sequence in a cloning vector within which target nucleic acid has been cloned, or in so-called "RACE" (rapid amplification of cDNA ends) in which cDNA's in a library are ligated to an oligonucleotide linker and PCR is performed using a primer which hybridises with a sequence shown and a primer which hybridises to the oligonucleotide linker.
  • RACE rapid amplification of cDNA ends
  • Nucleic acid isolated and/or purified from one or more cells may be probed under conditions for selective hybridisation and/or subjected to a specific nucleic acid amplification reaction such as the polymerase chain reaction (PCR) (reviewed for instance in "PCR protocols; A Guide to Methods and Applications", Eds. Innis et al, 1990, Academic Press, New York, Mullis et al, Cold Spring Harbor Symp. Quant.
  • PCR polymerase chain reaction
  • PCR comprises steps of denaturation of template nucleic acid (if double- stranded) , annealing of primer to target, and polymerisation.
  • the nucleic acid probed or used as template in the amplification reaction may be genomic DNA, cDNA or RNA.
  • Other specific nucleic acid amplification techniques include strand displacement activation, the QB replicase system, the repair chain reaction, the ligase chain reaction and ligation activated transcription.
  • PCR is used herein in contexts where other nucleic acid amplification techniques may be applied by those skilled in the art. Unless the context requires otherwise, reference to PCR should be taken to cover use of any suitable nucleic amplification reaction available in the art.
  • cloning it may be necessary for one or more gene fragments to be ligated to generate a full- length coding sequence. Also, where a full-length encoding nucleic acid molecule has not been obtained, a smaller molecule representing part of the full molecule, may be used to obtain full-length clones. Inserts may be prepared from partial cDNA clones and used to screen cDNA libraries. The full-length clones isolated may be sub- cloned into expression vectors and activity assayed by transfection into suitable host cells, e.g. with a reporter plasmid.
  • a method may include hybridisation of one or more (e.g. two) probes or primers to target nucleic acid. Where the nucleic acid is double-stranded DNA, hybridisation will generally be preceded by denaturation to produce single- stranded DNA.
  • the hybridisation may be as part of a PCR procedure, or as part of a probing procedure not involving PCR.
  • An example procedure would be a combination of PCR and low stringency hybridisation.
  • a screening procedure chosen from the many available to those skilled in the art, is used to identify successful hybridisation events and isolated hybridised nucleic acid.
  • Binding of a probe to target nucleic acid may be measured using any of a variety of techniques at the disposal of those skilled in the art.
  • probes may be radioactively, fluorescently or enzymatically labelled.
  • Other methods not employing labelling of probe include examination of restriction fragment length polymorphisms, amplification using PCR, RN'ase cleavage and allele specific oligonucleotide probing.
  • Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined.
  • DNA for probing may be prepared from RNA preparations from cells.
  • Preliminary experiments may be performed by hybridising various probes under low stringency conditions to Southern blots of DNA digested with restriction enzymes. Suitable conditions would be achieved when a large number of hybridising fragments were obtained while the background hybridisation was low. Using these conditions nucleic acid libraries, e.g. cDNA libraries representative of expressed sequences, may be searched. Those skilled in the art are well able to employ suitable conditions of the desired stringency for selective hybridisation, taking into account factors such as oligonucleotide length and base composition, temperature and so on.
  • oligonucleotide probes or primers may be designed, taking into account the degeneracy of the genetic code, and, where appropriate, codon usage of the organism from the candidate nucleic acid is derived.
  • An oligonucleotide for use. in nucleic acid amplification may have about- 10 or fewer codons (e.g. 6, 7 or 8), i.e. be about 30 or fewer nucleotides in length (e.g. 18, 21 or 24) .
  • Generally specific primers are upwards of 14 nucleotides in length, but need not be than 18-20.
  • primers for use processes such as PCR.
  • Various techniques for synthesizing oligonucleotide primers are well-known in the art, including phosphotriester and phosphodiester synthesis methods .
  • Preferred nucleic acid sequences suitable for use in the design of probes or PCR primers may include conserved sequences (completely, substantially or partly) encoding the RhoGAP, endophilin-like or proline rich domains as described herein.
  • a further aspect of the present invention provides an oligonucleotide or polynucleotide fragment of the nucleotide sequence shown in any of the figures herein providing nucleic acid according to the present invention, or a complementary sequence, in particular for use in a method of obtaining and/or screening nucleic acid.
  • a sequence may differ from any of the sequences shown by addition, substitution, insertion or deletion of one or more nucleotides, but preferably without abolition of ability to hybridise selectively with nucleic acid in accordance with the present invention, that is wherein the degree of similarity of the oligonucleotide or polynucleotide with one of the sequences given is sufficiently high.
  • oligonucleotides according to the present invention that are fragments of any of the sequences shown, are at least about 10 nucleotides in length, more preferably at least about 15 nucleotides in length, more preferably at least about 20 nucleotides in length. Such fragments themselves individually represent aspects of the present invention. Fragments and other oligonucleotides may be used as primers or probes as discussed.
  • oligonucleotides are anti-sense oligonucleotide sequences based on the nucleic acid sequences described herein.
  • Anti-sense oligonucleotides may be designed to hybridise to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of polypeptide encoded by a given DNA sequence (e.g. either native polypeptide or a mutant form thereof), so that its expression is reduce or prevented altogether.
  • Anti-sense techniques may be used to target a coding sequence, a control sequence of a gene, e.g. in the 5' flanking sequence, whereby the antisense oligonucleotides can interfere with control, sequences.
  • Anti-sense oligonucleotides may be DNA or RNA and may be of around 14-23 nucleotides, particularly around 15-18 nucleotides, in length. The construction of antisense sequences and their use is described in Peyman and Ulman, Chemical Reviews, 90:543-584, (1990), and Crooke, Ann. Rev. Pharmacol. Toxicol., 32:329-376, (1992).
  • any of the sequences disclosed in the figures herein may be used to construct a probe for use in identification and isolation of a promoter from a genomic library containing a genomic RICH gene. Techniques and conditions for such probing are well known in the art and are discussed elsewhere herein.
  • restriction enzyme or nucleases may be used to digest a nucleic acid molecule, followed by an appropriate assay (for example using a reporter gene such as luciferase) to determine the sequence required.
  • a further aspect of the present invention provides a nucleic acid molecule as described herein operably linked to a promoter or other regulatory sequence.
  • promoter is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA) .
  • “Operably linked” means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
  • DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.
  • a further aspect of the present invention provides a polypeptide which has the amino acid sequence shown in Figure 1 or Figure 2, which may be in isolated and/or purified form, free or substantially free of material with which it is naturally associated, such as other polypeptides or such as human polypeptides other than that for which the amino acid sequence is shown in Figure 1 or Figure 2, or (for example if produced by expression in a prokaryotic cell) lacking in native glycosylation, e.g. unglycosylated.
  • Polypeptides which are amino acid sequence variants, alleles, derivatives or mutants are also provided by the present invention.
  • a polypeptide which is a variant, allele, derivative or mutant may have an amino acid sequence which differs from that given in a figure herein by one or more of addition, substitution, deletion and insertion of one or more amino acids.
  • Preferred such polypeptides have RICH-1 function, that is to say have one or more of the following properties : immunological cross-reactivity with an antibody reactive the polypeptide for which the sequence is given in a figure herein sharing an epitope with the polypeptide for which the amino acid sequence is shown in a figure herein (as determined for example by immunological cross-reactivity between the two polypeptides) ; a biological activity which is inhibited by an antibody raised against the polypeptide whose sequence is shown in a figure herein; activity of stimulating, increasing or enhancing hydrolysis of GTP by Rho-GTPase (GAP) , thereby switching the Rho-GTPase to an inactive state.
  • GAP Rho-GTPase
  • Rho-GTPase substrates may include Cdc42 (Ace No:P21181) and Racl (Ace No: NP_008839.2) and exclude RhoA. Alteration of sequence may change the nature and/or level of activity and/or stability of the RICH polypeptide.
  • a polypeptide which is an amino acid sequence variant, allele, derivative or mutant of the amino acid sequence shown in a figure herein may comprise an amino acid sequence which shares greater than about 35% sequence identity with the sequence shown, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90% or greater than about 95%.
  • the sequence may share greater than about 60% similarity, greater than about 70% similarity, greater than about 80% similarity or greater than about 90% similarity with the amino acid sequence shown in figure 1 or figure 2.
  • Amino acid sequence variants may include RICH-2.
  • Amino acid similarity is generally defined with reference to the algorithm GAP (Genetics Computer Group, Madison, WI) as noted above, or the TBLASTN program, of Altschul et al. (1990) J. Mol. Biol. 215: 403-10. Similarity allows for "conservative variation", i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine .
  • Particular amino acid sequence variants may differ from that shown in a figure herein by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 20-30, 30-50, 50-100, 100-150,. or more than 150 amino acids .
  • Sequence comparison may be made over the full-length of the relevant sequence shown herein, or may more preferably be over a contiguous sequence of about or greater than about 20, 25, 30, 33, 40, 50, 67, 133, 167, 200, 233, 267, 300, 333, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, or more amino acids or nucleotide triplets, compared with the relevant amino acid sequence or nucleotide sequence as the case may be.
  • the present invention also includes peptides which comprise or consist of fragments of a polypeptide of the invention.
  • Peptides can also be generated wholly or partly by chemical synthesis.
  • the compounds of the present invention can be readily prepared according to well- established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for example, in J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984) , in M. Bodanzsky and A.
  • Bodanzsky The Practice of Peptide Synthesis, Springer Verlag, New York (1984) ; and Applied Biosystems 430A Users Manual, ABI Inc., Foster City, California
  • they may be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof.
  • the present invention also includes active portions, fragments, derivatives and functional mimetics of the RICH-1, RICH-2 and RICH-IB polypeptides.
  • An "active portion" of a polypeptide means a peptide which is less than said full length polypeptide, but which retains a biological activity of the RICH-1 or RICH-IB polypeptide, for example a Rho-GAP activity.
  • an active portion of a RICH-1 polypeptide may include residues 266 to 409 of the sequence of Figure 1.
  • Other RICH-1 active portions may include the endophilin-like N terminal domain
  • Such an active fragment may be included as part of a fusion protein, e.g. including a binding portion for a different ligand which may confer on the molecule a different binding specificity.
  • Corresponding active portions of the RICH-2 polypeptide may include the endophilin-like N terminal domain (residues 1 to 211 of the KIAA0672 sequence with database Ace No: AB014572) , the RhoGAP domain (residues 265 to 412) , and the proline-rich C terminal domain (residues 733-744) .
  • Corresponding active portion of RICH-IB may include the endophilin-like domain (residues 1 to 208 of Fig 208) .
  • a "fragment" of a polypeptide generally means a stretch of amino acid residues of at least about five contiguous amino acids, often at least about seven contiguous amino acids, typically at least about nine contiguous amino acids, more preferably at least about 13 contiguous amino acids, and, more preferably, at least about 20 to 30 or more contiguous amino acids. Fragments of the RICH-1 polypeptide sequence may include antigenic determinants or epitopes useful for raising antibodies to a portion of the amino acid sequence. Alanine scans are commonly used to find and refine peptide motifs within polypeptides, this involving the systematic replacement of each residue • in turn with the amino acid alanine, followed by an assessment of biological activity.
  • Preferred fragments of RICH-1 include those which contain any of the following amino acid sequences as shown in figure 1 or in figures 6, 7 or 8 : endophilin-like N terminal domain (residues 1 to 208 in Fig 1) , the RhoGAP domain (residues 266 to 409 in Fig 1) , and the proline- rich C terminal domains (residues 557-570, 633 to 642, 676 to 687 and 727 to 738 in Fig 1) , which may be used for instance in raising or isolating antibodies.
  • Variant and derivative peptides, peptides which have an amino acid sequence which differs from one of these sequences by way of addition, insertion, deletion or substitution of one or more amino acids are also provided by the present invention, generally with the proviso that the variant or derivative peptide is bound by an antibody or other specific binding member which binds one of the peptides whose sequence is shown.
  • a peptide which is a variant or derivative of one of the shown peptides may compete with the shown peptide for binding to a specific binding member, such as an antibody or antigen-binding fragment thereof .
  • additional amino acids are included in a polypeptide or peptide as described above, these may be heterologous or foreign to the polypeptide or peptide of the invention.
  • a polypeptide or peptide may thus be included within a larger fusion, chimeric or hybrid protein, particularly where the peptide is fused or joined to a non-RICH (i.e. non-RICH-1, RICH-IB or RICH-2) sequence, i.e. a heterologous or foreign sequence.
  • a heterologous sequence may comprise a heterologous polypeptide or protein domain thereof.
  • a fusion protein may for example incorporate a tag such as six histidine residues at either the N-terminus or C-terminus to allow for purification or isolation, or a Gal4 DNA binding or activation domain for use in two hybrid assays, as described herein.
  • a suitable peptide within a fusion, chimeric or hybrid protein may be about 20, 25, 30 or 35 amino acids in length, or may be about 50, 100, 150, 200, 250,300, 350 or 400 amino acids in length.
  • Suitable polypeptides for inclusion within a fusion or hybrid protein include the sequences shown in Figure 3 and Figure 4 or a domain or fragment thereof, for example an endophilin-like domain, a RhoGAP domain or a proline rich domain, as shown in Figure 5 and described herein.
  • Suitable heterologous sequences which may be included within a fusion, chimeric or hybrid protein may be about 5, 10, 20, 25, 30 or 35 amino acids in length, or may be about 50, 100, 150, 200, 250, 300, 350 or 400 amino acids in length.
  • Suitable sequences include polypeptides, protein domains and peptides. Such a sequence may have a biological activity such as an enzymatic or binding activity which is distinct from that of the RICH polypeptide .
  • a "derivative" of a polypeptide or a fragment thereof may include a polypeptide modified by varying the amino acid sequence of the protein, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself. Such derivatives of the natural amino acid sequence may involve one or more of insertion, addition, deletion or substitution of one or more amino acids, which may be without fundamentally altering the qualitative nature of biological activity of the wild type polypeptide. Also encompassed within the scope of the present invention are functional mimetics of active fragments of the RICH polypeptides provided (including alleles, . mutants, derivatives and variants).
  • the term "functional mimetic” means a substance which may not contain an active portion of the relevant amino acid sequence, and probably is not a peptide at all, but which retains in qualitative terms a biological activity of natural RICH polypeptide ' .
  • the design and screening of candidate mimetics is described in detail below.
  • Polypeptides according to the present invention may be subjected to post-translational modifications. Such modifications may occur in the cell which naturally produces the polypeptide, in a heterogeneous system in which the polypeptide is expressed, or in vi tro . Post- translational modification of a polypeptide of the invention may thus differ from that of the natural polypeptide. Many forms of post-translational modification are known, including acetylation, acylation, sulfation, glycosylation, phosphorylation, palmitoylation, ubiquitination, ADP-ribosylation, hydroxylation, glucosylphosphatidylinositide addition, oxidation and reduction.
  • a polypeptide of the invention may, for example, be acetylated, acylated, sulfated, glycosylated, phosphorylated, palmitoylated, ubiquitinylated, ADP- ribosylated, glucosylphosphatidylinositylated, oxidised or reduced, i.e. a polypeptide may comprise one or more acetyl-, a.cyl-, sulfyl-, glycosyl-, phospho-, palmitoyl-, ubiquitin-, ADP-ribosyl- , or glucosylphosphatidylinositide groups .
  • a polypeptide according to the present invention may be isolated and/or purified (e.g. using an antibody) for instance after production by expression from encoding nucleic acid (for which see below) .
  • a polypeptide may be provided free or substantially free from contaminants with which it is naturally associated (if it is a naturally-occurring polypeptide) .
  • a polypeptide may be provided free or substantially free of other polypeptides .
  • Polypeptides according to the present invention may be generated wholly or partly by chemical synthesis.
  • the isolated and/or purified polypeptide may be used in formulation of a composition, which may include at least one additional component, for example a pharmaceutical composition including a pharmaceutically acceptable excipient, vehicle or carrier.
  • a composition including a polypeptide according to the invention may be used in prophylactic and/or therapeutic treatment as discussed below.
  • a polypeptide, peptide, allele, mutant, derivative or variant according to the present invention may be used as an immunogen or otherwise in obtaining specific antibodies.
  • Antibodies are useful in purification and other manipulation of polypeptides and peptides and. therapeutic contexts. This is discussed further below.
  • a polypeptide according to the present invention may be used in screening for molecules which affect or modulate its activity or function, e.g. its interactions with CIP4 and/or its effect on Cdc42 and/or Rac activity through the stimulation of GTP hydrolysis.
  • Such molecules may interact with any region of the RICH-1 or RICH-2 protein sequence or with the SH3 region of CIP4 which binds to RICH-1, and may be useful in a therapeutic (including prophylactic) context.
  • Substances identified as modulators of the interactions described herein are extremely useful in the modulation of a range of cytoskeletal functions and they provide basis for design and investigation of therapeutics for in vivo use. Furthermore, they may be useful in any of a number of conditions associated with the abnormal functioning of the cytoskeleton or microfilament system, including tumour cell metastasis, lamellipodia formation and conditions associated with aberrant RhoGTPase activity. As noted elsewhere, RICH-1 and fragments thereof may also be useful in combating any of these conditions and disorders.
  • the present invention relates to screening and assay methods and means, and substances identified thereby.
  • Such assay methods may be for substances which interact with or bind a polypeptide of the invention and/or modulate one or more of its activities .
  • RICH-1 or RICH-2 polypeptide or peptide (particularly a fragment of a polypeptide of the invention as disclosed) , and/or encoding nucleic acid therefor, in screening or searching for and/or obtaining/identifying a substance, e.g. peptide or chemical compound, which interacts and/or binds with the RICH-1 or -2 polypeptide or peptide and/or interferes with the interaction between RICH-1 and CIP4 and/or modulates the RhoGAP activity of RICH-1 or RICH-2 and is therefore a candidate modulator of the function or activity of one or more RhoGTPases, including Cdc42 and Rac . .
  • a substance e.g. peptide or chemical compound
  • a method according to one aspect of the invention includes providing a polypeptide or peptide of the invention and bringing it into contact with a substance, which contact may result in binding between the polypeptide or peptide and the substance. Binding may be determined by any of a number of techniques available in the art, both qualitative and quantitative.
  • a method of screening for a substance which modulates the binding activity of a RICH-1 polypeptide may include contacting one or more test substances with the RICH-1 polypeptide in a suitable reaction medium, testing the binding activity of the treated polypeptide and comparing that activity with the binding activity of the RICH-1 polypeptide in comparable reaction medium untreated with the test substance or substances . A difference in binding activity between the treated and untreated polypeptides is indicative of a modulating effect of the relevant test substance or substances.
  • One aspect of the present invention provides an assay method which includes:
  • a substance which interacts with a RICH-1 polypeptide or peptide of the invention may be isolated and/or purified, manufactured and/or used to modulate its activity as discussed.
  • a further aspect of the present invention provides an assay method for identifying molecules which modulate the activity of RICH-1 or RICH-2, including: (i) bringing into contact a RICH-1 or RICH-2 polypeptide and a putative binding molecule or other test substance; and (ii) determining the RhoGAP activity of the RICH-1 or RICH-2 polypeptide.
  • RhoGAP may be determined by measuring the GTPase activity of a RhoGTPase, which may,- for example, be Cdc42 or Rac but not Rho, in the presence and absence of RICH-1 or RICH-2. GTPase activity may be conveniently measured as described herein by preloading the RhoGTPase with [ ⁇ - 32P]GTP before incubation with RhoGAP - and measuring the amount of GTP bound to the GTPase after incubation by scintillation counting or other convenient means. Other methods of determining RhoGAP activity are known to a skilled person.
  • the present invention also is concerned with provision of assays for substances which inhibit interaction between a polypeptide of the invention and CIP4 and/or a RhoGTPase.
  • RhoGTPases suitable for use according to this aspect of the invention include Cdc42 and Rac, but not Rho.
  • a further aspect of the present invention provides an assay method for screening for a substance which modulates the binding of RICH-1 or RICH-2 and CIP4, including:
  • a further aspect of the present invention provides an assay method for screening for a substance which modulates the interaction of RICH-1 or RICH-2 and Cdc42 and/or Rac, including:
  • Interaction between the RICH polypeptide and Cdc42 or Rac may be determined by measuring the GTPase activity of said Cdc42 or Rac. Activity may be determined in the presence and absence of the RICH polypeptide as described above.
  • RICH-1 polypeptide or a RICH-2 polypeptide as described herein are suitable for use in the present assay methods, including a fragment, mutant, variant or derivative of RICH-1 or RICH-2 which is able to bind CIP4 and/or modulate the activity of a RhoGTPase.
  • CIP4 polypeptide may include FBP17 (Also called KIAA0554; Ace NO: AB011126) , syndapin (Also called pacin; Ace No: X85124) or other CIP4 related polypeptide, in addition to CIP4 (Ace No :AJ000414) .
  • An assay may be carried out under conditions in which in the absence of the test substance being an inhibitor, the RICH polypeptide binds to the CIP4 polypeptide.
  • RhoGTPase includes members of the Cdc42 subgroup such as Cdc42, TC10, TCL, Chp and members of the Rac subgroup such as Racl-3 and RhoG. As mentioned above, it is not necessary to use the entire proteins for assays of the invention which test for binding between two molecules . Fragments may be generated and used in any suitable way known to those of skill in the art. Suitable ways of generating fragments include,- but are not limited to, recombinant expression of a fragment from encoding DNA. Such fragments may be generated by taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA.
  • the portion may then be operably linked to a suitable promoter in a standard commercially available expression system.
  • Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers. Small fragments (e.g. up to about 20 or 30 amino acids) may also be generated using peptide synthesis methods which are well known in the art.
  • the interaction between the polypeptides may be studied in vi tro by labelling one with a detectable label -and bringing it into contact with the other which has been immobilised on a solid support.
  • Suitable detectable labels include 35 S-methionine which may be incorporated into recombinantly produced peptides and polypeptides.
  • Recombinantly produced peptides and polypeptides may also be expressed as a fusion protein containing an epitope which can be labelled with an antibody.
  • Fusion proteins may be generated that incorporate six histidine residues at either the N-terminus or C-terminus of the recombinant protein.
  • a histidine tag may be used for purification of the protein by using commercially available columns which contain a metal ion, either nickel or cobalt (Clontech, Palo Alto, CA, USA) . These tags also serve for detecting the protein using commercially available monoclonal antibodies directed against the six histidine residues (Clontech, Palo Alto, CA, USA) .
  • the protein which is immobilized on a solid support may be immobilized using an antibody against that protein bound to a solid support or via other technologies which are known per se .
  • a preferred in vi tro interaction may utilise a fusion protein including glutathione-S- transferase (GST) . This may be immobilized on glutathione agarose beads.
  • GST glutathione-S- transferase
  • a test compound can be assayed by determining its ability to diminish the amount of labelled peptide or polypeptide which binds to the immobilized GST-fusion polypeptide. This may be determined by fractionating the glutathione-agarose beads by SDS-polyacrylamide gel electrophoresis .
  • the beads may be rinsed to remove unbound protein and the amount of protein which has bound can be determined by counting the amount of label present in, for example, a suitable scintillation counter.
  • An assay according to the present invention may also take the form of an in vivo assay.
  • the in vivo assay may be performed in a cell line such as a yeast strain in which the relevant polypeptides or peptides are expressed from one or more vectors introduced into the cell .
  • another aspect of the present invention is a substance obtainable using an assay method as described herein.
  • Such a substance may include polypeptide, antibody, peptide, nucleic acid molecule, small molecule or other pharmaceutically useful compound.
  • Combinatorial library technology provides an efficient way of testing a potentially vast number of different substances for ability to modulate activity of a polypeptide.
  • test substances Prior to, or as well as, being screened for modulation of activity, test substances may be screened for ability to interact with the polypeptide, e.g. in a yeast two-hybrid system (which requires that both the polypeptide and the test substance can be expressed in yeast from encoding nucleic acid) . This may be used as a coarse screen prior to testing a substance for actual ability to modulate activity of the polypeptide.
  • test substance or compound which may be added to an assay ; of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.01 to 100 nM concentrations of putative inhibitor compound may be used, for example- from 0.1 to 10 nM. Greater concentrations may be used when a peptide is the test substance.
  • Compounds which may be used may be natural or synthetic chemical compounds used in drug screening programmes . Extracts of plants which contain several characterised or uncharacterised components may also be used. • A further class of putative inhibitor compounds can be derived from the RICH-1, RICH-IB or RICH-2 polypeptides or from the interacting regions of CIP4 -related polypeptides. Peptide fragments of from 5 to 40 amino acids, for example from 6 to 10 amino acids from the region of the relevant polypeptide responsible for interaction, may be tested for their ability to disrupt such interaction.
  • candidate inhibitor compounds may be based on modelling the 3 -dimensional structure of a polypeptide or peptide fragment and using rational drug design to provide potential inhibitor compounds with particular molecular shape, size and charge characteristics.
  • the substance may be investigated further. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals.
  • the present invention extends in various aspects not only to a substance identified as a modulator of polypeptide activity, in accordance with what is disclosed herein, but also a pharmaceutical composition, medicament, drug or other composition comprising such a substance, a method comprising administration of such a composition to a patient, e.g. for treatment (which may include preventative treatment) of a condition related to a cytoskeletal, particularly a microfilament , abnormality or malfunction, such as tumour cell metastasis or lamellipodia formation, use of such a substance in manufacture of a composition for administration, e.g. for treatment of a condition related to a cytoskeletal abnormality or malfunction, and a method of making a pharmaceutical composition comprising admixing such a substance with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.
  • a substance identified using as a modulator of polypeptide or promoter- function may be peptide or non- peptide in nature.
  • Non-peptide "small molecules" are often preferred for many in vivo pharmaceutical uses.
  • a mimetic or mimick of the substance may be designed for pharmaceutical use.
  • the designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g.
  • peptides are not well suited as active agents for oral .compositions as they tend to be quickly degraded by proteases in the alimentary canal .
  • Mimetic design, synthesis and testing may be used to avoid randomly screening large number of. molecules for a target property.
  • the pharmacophore Once the pharmacophore has been found, its structure is modelled to according its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data - from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process .
  • data - from a range of sources e.g. spectroscopic techniques, X-ray diffraction data and NMR.
  • Computational analysis, similarity mapping which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms
  • other techniques can be used in this modelling process .
  • the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this the design of the mimetic.
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template ' molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it . Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing. Mimetics of substances identified as having ability to modulate RICH polypeptide activity using a screening method as disclosed herein are included within the scope of the present invention.
  • a polypeptide, peptide or substance able to modulate activity of a RICH polypeptide according to the present invention may be provided in a kit, e.g. sealed in a suitable container which protects its contents from the external environment .
  • a kit may include instructions for use.
  • a convenient way of producing a polypeptide according to the present invention is to express nucleic acid encoding it, by use of the nucleic acid in an expression system. Accordingly, the present invention also encompasses a method of making a polypeptide (as disclosed) , the method including expression from nucleic acid encoding the polypeptide (generally nucleic acid according to the invention) . This may conveniently be achieved by growing a host cell in culture, containing such a vector, under appropriate conditions which cause or allow expression of the polypeptide. Polypeptides may also be expressed in in vi tro systems, such as reticulocyte lysate.
  • Methods may include modifying the polypeptide thus produced, for example by acetylation, acylation, sulfation, glycosylation, phosphorylation, palmitoylation, ubiquitination, ADP-ribosylation, hydroxylation, glucosylphosphatidylinositide addition, oxidation or reduction.
  • modification occurs within the host cell following expression of the encoding nucleic acid.
  • the polypeptide may be modified in vi tro following expression.
  • Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable - host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others.
  • a common, preferred bacterial host is E. coli .
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, . terminator fragments, polyadenylation sequences , enhancer sequences , marker genes and other sequences as appropriate.
  • Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al . , 1989, Cold Spring Harbor Laboratory Press .
  • a further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein.
  • the nucleic acid of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques .
  • the nucleic acid may be on an extra-chromosomal vector within the cell.
  • a still further aspect provides a method which includes introducing the nucleic acid into a host cell.
  • the introduction which may (particularly for in vitro introduction) be generally referred to without limitation as "transformation”, may employ any available technique.
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, .liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus .
  • suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.
  • Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well known in the art.
  • the introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) under conditions for expression of the gene, so that the encoded polypeptide is produced. If the polypeptide is expressed coupled to an appropriate signal leader peptide it may be secreted from the cell into the culture medium.
  • a polypeptide may be isolated and/or purified from the host cell and/or culture medium, as the case may be, and subsequently used as desired, e.g. in the formulation of a composition which may include one or more additional components, such as a pharmaceutical composition which includes one or more pharmaceutically acceptable excipients, vehicles or carriers (e.g. see below) .
  • a host cell containing nucleic acid according to the present invention e.g. as a result of introduction of the nucleic acid into the cell or into an ancestor of the cell and/or genetic alteration of the sequence endogenous to the cell or ancestor (which introduction or alteration may take place in vivo or ex vivo)
  • a host cell containing nucleic acid according to the present invention may be comprised (e.g. in the soma) within an organism which is an animal, particularly a mammal, which may be non-human, such as rabbit, guinea pig, rat, mouse or other rodent, cat, dog, pig sheep, goat, cattle or horse, or which is a bird, such as a chicken.
  • Genetically modified or transgenic animals or birds comprising such a cell are also provided as further aspects of the present invention. Such transgenic animals may be useful as models of a condition.
  • host cells may be used as a nucleic acid factory to replicate the nucleic acid of interest in order to generate large amounts of it. Multiple copies of nucleic acid of interest may be made within a cell when coupled to an amplifiable gene such as dihyrofolate reductase (DHFR) , as is well known.
  • DHFR dihyrofolate reductase
  • Host cells transformed with nucleic acid of interest, or which are descended from host cells into which nucleic acid was introduced may be cultured under suitable conditions, e.g. in a fermentor, taken from the culture and subjected to processing to purify the nucleic acid. Following purification, the nucleic acid or one or more fragments thereof may be used as desired.
  • RICH-1 polypeptide also enables, for the first time, the production of antibodies able to bind specifically to this molecule.
  • a further aspect of the present invention provides an antibody able to bind specifically to the polypeptide whose sequence is given in a figure herein.
  • Such an antibody may be specific in the sense of being able to distinguish between the polypeptide it is able to bind and other human polypeptides for which it has no or substantially no binding affinity (e.g. a binding affinity of about lOOOx less) .
  • Specific antibodies bind an epitope on the molecule which is either not present or is not accessible on other molecules.
  • Antibodies according to the present invention may be specific for the wild-type polypeptide.
  • Antibodies according to the invention may be specific for a particular mutant, variant, allele or derivative polypeptide as between that molecule and the wild-type polypeptide, so as to be useful in diagnostic and prognostic methods as discussed below. Antibodies are also useful in purifying the polypeptide or polypeptides to which they bind, e.g. following production by recombinant expression from encoding nucleic acid.
  • Preferred antibodies according to the invention are isolated, in the sense of being free from contaminants such as antibodies able to bind other polypeptides and/or free of serum components .
  • Monoclonal antibodies are preferred for some purposes, though polyclonal antibodies are within the scope of the present invention.
  • Antibodies may be obtained using techniques which are standard in the art . Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof.
  • Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest.
  • an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047.
  • Suitable peptides for use in immunising an animal and/or isolating anti-RICH antibody include peptides containing sequence from the endothilin-like domain, the RhoGAP domain and the proline rich domain of the full length sequence shown in Figure 1.
  • Antibodies according to the present invention may be modified in a number of ways. Indeed, the term “antibody” covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including synthetic molecules and molecules whose shape mimicks that of an antibody enabling it to bind an antigen or epitope.
  • the reactivities of antibodies on a sample may be determined by any appropriate means. Tagging with individual reporter molecules is one possibility.
  • the reporter molecules may directly or indirectly generate detectable, and preferably measurable, signals.
  • the linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non- covalently. Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule.
  • fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red.
  • Suitable chromogenic dyes include diaminobenzidine.
  • Other reporters include macromolecular colloidal particles or particulate material such- as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded.
  • These molecules may be enzymes which catalyse reactions that develop or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed.
  • Antibodies according to the present invention may be used in screening for the presence of a polypeptide, for example in a test sample containing cells or cell lysate as discussed, and may be used in purifying and/or isolating a polypeptide according to the present invention, for instance following production of the polypeptide by expression from encoding nucleic acid therefor. Antibodies may modulate the activity of the polypeptide to which they bind and so may be useful in a therapeutic context (which may include prophylaxis) to modulate the activity of Rho-GTPase.
  • the present invention also provides a substance as ' described herein for use in a pharmaceutical composition for the treatment of a cytoskeletal abnormality or malfunction in an individual .
  • administration is preferably in a "prophylactically effective amount” or a "therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual .
  • the actual amount administered, and rate and time-course of administration will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors .
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • compositions according to the present invention may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous .
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • Targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons; for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
  • an agent may be produced in target cells by expression from an encoding gene introduced into the cells, e.g. in a viral vector (see below) .
  • the vector may be targeted to the specific cells to be treated, or it may contain regulatory elements which are switched on more or less selectively by the target cells.
  • Viral vectors may be targeted using specific binding molecules, such as a sugar, glycolipid or protein such as an antibody or binding fragment thereof.
  • Nucleic acid may be targeted by means of linkage to a protein ligand (such as an antibody or binding fragment thereof) via polylysine, with the ligand being specific for a receptor present on the surface of the target cells.
  • An agent may be administered in a precursor form, for conversion to an active form by an activating agent produced in, or targeted to, the cells to be treated.
  • compositions as described herein may be used for anti-sense regulation of gene expression, e.g. targeting an antisense nucleic acid molecule to cells in which a mutant form of the gene is expressed, the aim being to reduce production of the mutant gene product.
  • Other approaches to specific down-regulation of genes are well known, including the use of ribozymes designed to cleave specific nucleic acid sequences. Ribozymes are nucleic acid molecules, actually RNA, which specifically cleave single-stranded RNA, such as mRNA, at defined sequences, and their specificity can- be engineered.
  • Hammerhead ribozymes may be preferred because they recognise base sequences of about 11-18 bases in length, and so have greater specificity than ribozymes of the - Tetrahymena type which recognise sequences of about 4 bases in length, though the latter type of ribozymes are useful in certain circumstances .
  • References on the use of ribozymes include Marschall, et al . Cellular and
  • FIG 1 shows the amino acid sequence of RICH-1.
  • the RhoGAP catalytic domain is underlined and the proline- rich regions are in bold.
  • FIG. 2 shows the amino acid sequence of RICH-IB
  • Figure 3 shows the RICH-1 nucleotide sequence which encodes the amino acid sequence shown in Figure 1.
  • Figure 4 Shows the RICH-IB nucleotide sequence which encodes the amino acid sequence shown in Figure 2.
  • FIG. 5 shows a schematic representation of RICH-1, RICH- IB and the related proteins RICH-2 (KIAA0672) and 3BP-1.
  • Figure 6 shows a sequence alignment using the DNAssist program of the RhoGAP domains of RICH-1, RICH-2, 3BP-1, p50 RhoGAP , ABR (Active Bcr-related) , pl90 RI_OGAP ( isofor ) . Identical amino acid residues are indicated in bold and the critical arginine is marked (*).
  • Figure 7 shows a sequence alignment by using the DNAssist program of the N-terminal domains of RICH-1, RICH-2 and members of the endophilin family called GRB2-likel, GRB2- like2 and EEN-B2-L4 (accession numbers NM_003025.1, NM_003026.1 and AF036272.1). The identical residues are indicated in bold.
  • Figure 8 shows a sequence alignment of the proline-rich regions in RICH-1, RICH-2, 3BP-1, p50 RhoGAP , Bcr (break- point cluster region) and p85 (p85 ⁇ . subunit of the phosphoinositide 3 '-kinase) with a SH3 -binding motif consensus sequence.
  • X nonconserved residue
  • p proline prefered
  • ⁇ hydrophobic residue
  • P conserved proline.
  • Figure 9 shows the in vi tro GTPase activation of Cdc42, Racl and RhoA stimulated by the RhoGAP domains of RICH-1, RICH-2 and p50 Rh ° GAP , and the intrinsic GTPase activity of the GTPases .
  • Onehundred percent correspond to the initial input of 32 P.
  • Each measurement represents the mean of three readings .
  • Saccharomyces cerevisae strain Y190 (genotype; MATa, gal4 -542 , gal80-538 , his3 , trpl-301, ade2 -101 , ura3 -52 , leu2-3 , 112 , URA3 : :GALl-LacZ, Lys2 : :GALl-HIS3cyh r ) was transformed with a cDNA encoding the SH3 domain of CIP4 fused to the GAL4 DNA-binding domain (GAL4DB) in the pYTH6 vector.
  • GAL4DB GAL4 DNA-binding domain
  • This GAL4DB-CIP4 SH3 -expressing yeast strain was used to screen a cDNA library from EBV- transformed human B-cells fused to the GAL4 activation domain (GAL4AD) in the pACT vector as described before (25,27) .
  • PCR polymerase chain reaction
  • RICH-1 that encoded a RhoGAP domain-containing protein.
  • the presence of a splice variant encoding an insert of an extra 82 bp was detected in human B-cells as well as in HeLa cells.
  • This splice variant encoded a protein with an open reading frame of 803 amino acid residues.
  • RICH-IB The nucleotide sequences of these proteins are shown in Figures 4 and 3 respectively.
  • RICH-1, RICH-IB and RICH-1 RhoGAP domain were generated as BamHI-EcoRI PCR fragments and inserted into the pRK5myc vector.
  • the C-terminal domain of RICH-1 (amino acids 384-803) was subcloned into the HindiII and EcoRI sites of the pEGFP-C3 vector.
  • the SH3 domains of CIP4, FBP17 and syndapin were subcloned into the pGEX-2T vectors .
  • DNA sequencing on a Perkin Elmer Genetic Analyzer 310 confirmed the fidelity of the nucleotide sequence. The DNA work followed standard procedure (28) .
  • a probe representing a DNA fragment encoding amino acids 384-803 of RICH-1 was labelled with [ 32 P] -CTP employing the rediprime labelling kit (Amersham Pharmacia Biotech) and hybridised to a hybridisation-ready Northern blot (Human Multiple Tissue Northern Blot, Clontech) according to the ExpressHyb (Clontech) protocol provided by the manufacturer .
  • Glutathione-S transferase (GST) fusion-proteins of Racl, Cdc42 (brain isoform) , and RhoA were expressed in Escherichia coli , purified on glutathion-Sepharose beads (Amersham Pharmacia Biotech) and isolated from GST fusion-proteins by thrombin cleavage as described previously (25,30).
  • the bacteria were lysed in a buffer containing 50 mM Tris-HCl pH 7.5, 5 mM MgC ⁇ 2 , 50 mM NaCl, 10% glycerol, 0.1 % Triton X-100, 1 mM PMSF, 1/100 volume of aprotinin (Trasylol, Beyer) and 1 mM dithiothreitol (DTT) .
  • the proteins were thereafter eluted from the glutathion-Sepharose beads with 5 mM reduced glutathione, desalted on PD10 pre-packed chromatography columns (Amersham Pharmacia Biotech) , equilibrated in 20 mM Tris-HCl pH 7.5, 10 % glycerol and 1 mM DTT, and thereafter concentrated using Centricon-10 (Millipore) .
  • Protein concentrations were measured using the Bradford assay.
  • GST fusion-proteins of SH3 domains were prepared as above but were retained on the glutathion-Sepharose beads .
  • GAP assay The procedure essentially followed the procedure by Self and Hall (31) . Briefly, 0.1 ⁇ g of recombinant wild-type Rac, Rho or Cdc42 was preloaded with 10 ⁇ Ci [ ⁇ - 32 P] GTP (Amersham Pharmacia Biotech) in 20 ⁇ l of 20 mM Tris-HCl pH 7.5, 25 mM NaCl, 5mM EDTA and 0.1 mM DTT. The mixture was incubated for 10 minutes at 30 °C, after which the reaction was terminated by adding 5 ⁇ l 0.1 M MgCl 2 and the resulting [ ⁇ - 32 P] GTP-loaded GTPase solutions were stored on ice.
  • GAP assays For the GAP assays, equimolar amounts of the GTPases and the GST-GAP domains were used. Three ⁇ l of the [ ⁇ - 32 P] GTP- loaded GTPase was added to a 30 ⁇ l mixture of 20 mM Tris- HCl pH 7.5, 1 mM non-radioactive GTP, 0.87 mg/ml bovine serum albumin, 0.1 mM DTT, with either GST-RhoGAP domains of RICH-1, RICH-2 or p50 Rh °TM .
  • the mixture was incubated at 30 °C and 5 ⁇ l aliquots were removed after 0, 3, 6, 9 and 15 minutes and the reaction was stopped by the addition of 1 ml ice-cold buffer A (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 5 mM MgCl 2 ) .
  • the samples were collected on nitrocellulose filters, washed with 10 ml ice cold buffer A and the portion of [ ⁇ - 32 P] GTP remaining bound to - the GTPases was determined by scintillation counting.
  • Cos-1, Swiss 3T3 and NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % FCS .and penicillin/streptomycin at 37 °C in an atmosphere of 5% C0 2 .
  • Porcine aortic endothelial cells stably expressing the PDGF ' ⁇ .receptor (clone PAE/PDGFR ⁇ .) were cultured in Ham's F12 medium supplemented as above.
  • Cos-1 cells were transfected according to the DEAE-Dextran method (32) .
  • Transfected cells were harvested 48 hours post- transfection, washed once in ice-cold PBS and lysed on ice in NP-40 buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol,. 1 % NP-40,.1 mM PMSF, 1 % aprotinin (Trasylol, Beyer) and 1 mM DTT) .
  • Lysed cells were collected in Eppendorf tubes and centrifuged for 15 minutes. The resulting supernatants were subjected to immunoprecipitation experiments or GST pull-down assays.
  • Western blots were detected by the BM chemiluminescence blotting substrate (Boehringer Mannheim) .
  • BM chemiluminescence blotting substrate Boehringer Mannheim
  • supernatants from lysed RICH-1-transfected Cos-1 were incubated with GST fusion-proteins of SH3 domains from a panel of proteins (Src, Crk, N-Grb2, C- Grb2, p85, n-Src, Lck, Nek, Abl , Abl W118A, syndapin, FBP17, CIP4, Myr3 , spectrin, cortactin, endophilin, BTK) and the presence of RICH-1 bound to the GST.10 fusion- protein was determined by Western blotting.
  • Antisera were raised against peptides representing amino acid residues 53-67 (antiserum N) and 779-792 (antiserum C) , of human RICH-1. These antisera, as well as mouse anti-Myc (9E10, Santa Cruz), mouse anti-HA (12CA5, Boehringer Mannheim) , TRITC (tetramethyl rhodamine isothiocyanate) - and FITC (fluorescein isothiocyanate) - conjugated anti-rabbit and anti-mouse antibodies (DAKO) were used to determine the subcellular localisation of CIP4 and RICH-1 mutants. Filamentous actin was visualized by FITC-conjugated phalloidin (Sigma) .
  • the cells were grown on coverslips and fixed in 2% paraformaldehyde in PBS for 20 min. The cells were washed with PBS and permeabilised in 0.2% Triton X-100 in PBS for 5 minutes. The cells were thereafter washed again and incubated in the presence of 10 mM glycine in PBS for 1 hour.
  • RICH-1 exhibited extensive similarity to a protein with the annotation KIAA0672, identified by the Kazusa DNA Research Institute. Due to the similarity between the two proteins, we suggest that KIAA0672 should be renamed RICH-2. These two proteins also displayed similarity to the c-Abl interacting protein 3BP-1, indicating that the three proteins form a closely related family of RhoGAP proteins .
  • RhoGAP domains of RICH- 1, RICH-2 and 3BP-1 demonstrated that they all contain the conserved arginine-finger, which is present in this type of domain (Fig.6) (6,7).
  • RICH-1, RICH-2 and 3BP-1 each contain one proline-rich motif whereas RICH-1 contains four proline-rich sequences (Fig.8).
  • the spacing between the individual proline-rich motives indicates that RICH-1 can bind multiple SH3 domain-containing proteins (6,36). Tissue distribution of RICH-1
  • a commercial human multiple tissue northern blot was hybridised with a probe derived from RICH-1.
  • a transcript of approximately 4 kb was present in all tissues analysed (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas) , indicating that RICH-1 is ubiquitously expressed; however, the expression was particularly high in heart and placenta. It is not likely that the transcripts of RICH-1 and RICH-IB were fully resolved on this blot, since they differ only by the presence of the 82 bp insert in RICH-1 transcripts. However, a tendency of a doublet was visible in some tissues, suggesting the presence of both splice variants.
  • RICH-1 bound to several of the SH3 domains; however, particularly strong binding was found between RICH-1 and the SH3 domains of CIP4, FBP17 and syndapin (these latter proteins are related to CIP4) .
  • Cos-1 cells were transfected with EGFP- RICH-1: 384-803 and either the full-length HA epitope- tagged CIP4 or a CIP4 mutant lacking the SH3 domain.
  • EGFP: RICH-1: 384-803 was detected by Western blotting using antiserum C, whereas CIP4 and CIP ⁇ SH3 were detected by HA antibody.
  • RICH-1 bound only to the intact CIP4, emphasising the importance of the SH3 domain of CIP4 for mediating the interaction to RICH-1.
  • RhoGAP activity of RICH-1 and RICH-2 GST fusion-proteins of RICH-1 (amino acid residues 221- 489) and RICH-2 (amino acid residues 217-469) were incubated with Racl, Cdc42 or RhoA preloaded with [ ⁇ -
  • RhoGAP domains .of RICH-1 and RICH-2 stimulated GTP hydrolysis of both Racl and Cdc42, but not of RhoA, which was refractory to the activities of the RhoGAPs .
  • p50 Rhot!AP was effective against all three GTPases.
  • RICH-2 stimulated GTP hydrolysis of Racl and Cdc42 as efficiently as p50 RhoGAP and it appeared to be a more effective GAP than RICH-1 (Fig.9) . The reason for this difference is not clear and may reflect differences in- either the catalytic activity of the proteins or in the stability of the GST fusion-proteins.
  • Subcellular localisation of RICH-1 and RICH-IB Swiss 3T3 fibroblasts and PAE/PDGFR ⁇ cells were transiently transfected with expression plasmids encoding Myc-tagged RICH-1, RICH1-B or the RhoGAP domain of RICH- 1.
  • the subcellular localisation of the proteins in transfected cells was visualised by an antibody against the Myc epitope .
  • RICH-1 displayed a uniform cytoplasmic distribution, however a tendency of the protein to accumulate in peripheral lamellae was visible. No obvious effect on the organisation of the actin filaments could be noticed.
  • the RhoGAP domain was distributed evenly in the cells, including the nucleus. The nuclear localisation could be a result of the lack of domain structures that could assist in retaining the protein in the cytoplasm.
  • Subcellular localisation of Myc-epitope tagged RICH-1 and RICH-1 RhoGAP domain was determined . in transiently transfected PAE/PDGFR ⁇ cells by detecting Myc-specific antibody followed by TRITC-conjugated anti-mouse antibody. Filamentous actin was visualised by using FITC- conjugated phalloidin.
  • RICH-IB transfected in Swiss 3T3 cells localised to vesicular structures dispersed in the cytoplasm. This pattern of localisation was unique for the RICH-IB splice variant, and provides indication that that the protein has cellular functions distinct from RICH-1.
  • RICH-1 RhoGAP domain The ability of the RICH-1 RhoGAP domain to negatively regulate Racl- and Cdc42-dependent actin rearrangement was tested by transfecting full-length RICH-1 or the RhoGAP domain into PAE/PDGFR ⁇ cells or Swiss 3T3 fibroblasts .
  • PAE/PDGFR ⁇ cells transiently transfected with Myc-RICH-1 or Myc-GAP domain were serum-starved for 12 hours and then stimulated with 100 ng/ml PDGF-BB for 10 minutes.
  • PDGF-BB has been shown to induce the formation of lamellipodia (37) which is dependent on the activities of both Cdc42 and Racl (3,4) .
  • the cells expressing full- length RICH-1 or the RhoGAP domain were unable to form lamellipodia, in contrast to the surrounding non- transfected cells, which displayed lamellipodia. This provided indication that RICH-1 effectively inactivated the endogenous Cdc42 and Racl in these cells.
  • RICH-1 co-localises with CIP4 in vivo Cdc42 has been shown to relocate CIP4 from a uniform cytoplasmic distribution into peripheral and dorsal clusters on fibroblasts (25) .
  • NIH 3T3 fibroblasts were transfected with combinations of constitutively active Cdc42 (Myc-L61Cdc42) , CIP4 (HA- CIP4) and either the full-length (Myc-RICH-1) or the C- terminal portion (EGFP-RICH-1 :384-803) of RICH-1 and thereafter assayed for the localisation of CIP4 and RICH- 1.
  • CIP4 was detected by an antibody against the HA-tag, whereas RICH-1 and RICH-1 : 384-803 were detected by the antiserum N and antiserum C, respectively.
  • constitutively active Cdc42 induced a relocalisation of CIP4, these clusters also contained RICH-1.
  • constitutively active Cdc42, CIP4 and the C-terminal portion of RICH-1, CIP4 and RICH-1 coincided to a larger extent than CIP4 and the full-length RICH-1. This difference might be caused by the absence of endophilin-like and RhoGAP domains in this deletion mutant, which may be important for the localisation of RICH-1 to specific cellular compartments.
  • RICH-1 or RICH-1 : 384-803 did not visibly alter the formation of the dorsal CIP4 clusters, but the CIP4 at the cell margin disappeared in cells expressing RICH-1 : 384-803.
  • the data shows that CIP4 and RICH-1 can interact inside cells and that the proteins affect the sub-cellular localisation of each other.
  • RICH-1 is a ubiquitously expressed protein and exists in at least two different splice variants, RICH-1 and RICH- IB.
  • the shorter splice variant is the predominant splice variant in neuronal tissue, whereas both RICH-1 and RICH- IB are expressed in B-cells and HeLa cells.
  • RICH-1 contained a domain with homology to endophilins (34) and that this domain is the only constituent in RICH-IB provide indication that this domain has a cellular function.
  • LPAAT lysophosphatidic acid acyl transferase
  • RICH-IB When ectopically expressed in fibroblasts, RICH-IB was present exclusively in vesicular structures, as opposed to RICH-1, which displayed a more uniform cytoplasmic localisation. This provides indication that RICH-IB has a role in vesicle formation and/or in membrane trafficking. RICH-IB may display an endophilin-like enzymatic activity and participate in endocytosis or exocytosis.
  • RhoGAP domains of RICH-1 and RICH-2. were able to catalyse GTP hydrolysis on Racl and Cdc42, but not on RhoA.
  • RICH-1 was furthermore shown to function as a Cdc42 and Rac-specific GAP in vivo, since cells expressing either full-length RICH-1 or the RhoGAP domain were ' unable to form membrane ruffles, a phenomenon, which requires the activities of Cdc42 and Racl.
  • the Rho-dependent serum induced stress fibres remained unaffected.
  • Overexpression either by means of ectopic expression or microinjection of RhoGAP proteins, has been a general,, and successful, method to evaluate RhoGAP activities under physiological conditions.
  • RhoGAP RhoGAP primarily for Cdc42, and to a lesser extent for Rac and Rho, in vi tro .
  • RhoGAP primarily for Cdc42, and to a lesser extent for Rac and Rho, in vi tro .
  • PDGF-induced membrane ruffling 423 .
  • the in vi tro specificity does not necessarily over-lap with the specificity in vivo .
  • RhoGAP domain-containing proteins can furthermore function as effectors, transducing additional signals downstream of Rho GTPases.
  • Work on the Rac GAP n- Chimaerin showed that microinjection of the protein in fibroblasts and neuroblastoma cells induced a membrane ruffling activity (44) . This effect was dependent on the - ability of n-Chimaerin to bind to RhoGTPases, but not by the GAP activity of the protein (44) .
  • RICH-1 was found to interact specifically with the SH3 domain of CIP4.
  • CIP4 was originally found in a yeast two- hybrid system screen for Cdc42-interacting proteins (25) .
  • overexpression of CIP4 in Swiss 3T3 fibroblasts caused a loss of actin filament bundles.
  • the concomitant expression of CIP4 and activated mutants of Cdc42 resulted in a relocalisation of CIP4 into clusters at the cell periphery and the dorsal side of the cells.
  • Rhitopically expressed RICH-1 was also found to localise to the CIP4-containing clusters, however, the function of RICH-1 in these structures is currently unknown.
  • the SH3 domain of CIP4 has been shown to bind to WASP and this interaction has been proposed to be important for a CIP4-induced relocalisation of WASP to the microtubule system (45) .
  • CIP4 and CIP4-related proteins participate in signalling pathways that regulate cytoskeletal reorganisations and intracellular transport.
  • RICH-1 and RICH-2 are important components of these signalling pathways.

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Abstract

L'invention concerne l'identification d'une nouvelle protéine d'activation de Rho-GTPase (GAP) exerçant une interaction avec CIP4 et jouant un rôle dans les processus de modulation impliquant le cytosquelette des cellules eucaryotes. Elle concerne également des acides nucléiques, des polypeptides, des homologues, des anticorps, ainsi que des essais et d'autres procédés apparentés.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002061088A2 (fr) * 2001-01-29 2002-08-08 Pe Corporation (Ny) Proteines de type ras isolees de l'homme, molecules d'acide nucleique codant ces proteines et utilisation de ces dernieres
WO2017091807A1 (fr) * 2015-11-25 2017-06-01 University Of Miami Inhibiteurs de peptide pour calcineurine

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ASPENSTR\M P: "The Rho GTPases have multiple effects on the actin cytoskeleton. " EXPERIMENTAL CELL RESEARCH., vol. 246, no. 1, 10 January 1999 (1999-01-10), pages 20-25, XP002208713 *
ASPENSTROEM P ET AL: "YEAST TWO-HYBRID SYSTEM TO DETECT PROTEIN-PROTEIN INTERACTIONS WITH RHO GTPASES" METHODS IN ENZYMOLOGY, ACADEMIC PRESS INC, SAN DIEGO, CA, US, vol. 256, no. PART B, 1995, pages 228-241, XP002070375 ISSN: 0076-6879 *
DATABASE EMBL [Online] 18 October 2000 (2000-10-18) ISOGAI ET AL.: "Homo sapiens cDNA clone:NT2RP4001849" retrieved from EMBL Database accession no. AU133334 XP002208715 *
DATABASE EMBL [Online] weakly similar to SH3-BINDING PROTEIN 3BP-1., 29 September 2000 (2000-09-29) ISOGAI T., OTSUKI T.: " Homo sapiens cDNA FLJ13219 fis, clone NT2RP4001849" retrieved from EMBL Database accession no. AK023281 XP002208714 *
HARADA AYAKO ET AL: "Nadrin, a novel neuron-specific GTPase-activating protein involved in regulated exocytosis" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 275, no. 47, 24 November 2000 (2000-11-24), pages 36885-36891, XP002178115 ISSN: 0021-9258 *
ISHIKAWA K ET AL: "PREDICTION OF THE CODING SEQUENCES OF UNIDENTIFIED HUMAN GENES X. THE COMPLETE SEQUENCES OF 100 NEW CDNA CLONES FROM BRAIN WHICH CAN CODE FOR LARGE PROTEINS IN VITRO" DNA RESEARCH, UNIVERSAL ACADEMY PRESS, JP, vol. 5, 1998, pages 169-176, XP002929570 ISSN: 1340-2838 cited in the application *
JOHANSSON ANN-SOFI ET AL: "The mammalian homologue of the Caenorhabditis elegans polarity protein PAR-6 is a binding partner for the Rho GTPases Cdc42 and Rac1." JOURNAL OF CELL SCIENCE, vol. 113, no. 18, September 2000 (2000-09), pages 3267-3275, XP002208711 ISSN: 0021-9533 *
RICHNAU NINNA ET AL: "RICH, a Rho GTPase-activating protein domain-containing protein involved in signaling by Cdc42 and Rac1." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 276, no. 37, 14 September 2001 (2001-09-14), pages 35060-35070, XP002208712 ISSN: 0021-9258 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002061088A2 (fr) * 2001-01-29 2002-08-08 Pe Corporation (Ny) Proteines de type ras isolees de l'homme, molecules d'acide nucleique codant ces proteines et utilisation de ces dernieres
WO2002061088A3 (fr) * 2001-01-29 2003-06-12 Pe Corp Ny Proteines de type ras isolees de l'homme, molecules d'acide nucleique codant ces proteines et utilisation de ces dernieres
WO2017091807A1 (fr) * 2015-11-25 2017-06-01 University Of Miami Inhibiteurs de peptide pour calcineurine

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