WO1999016875A1 - Procede servant a effectuer le controle selectif de la presence d'une proteine de membrane et de la secretion de proteines dans des cellules eucaryotes - Google Patents

Procede servant a effectuer le controle selectif de la presence d'une proteine de membrane et de la secretion de proteines dans des cellules eucaryotes Download PDF

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WO1999016875A1
WO1999016875A1 PCT/US1998/020364 US9820364W WO9916875A1 WO 1999016875 A1 WO1999016875 A1 WO 1999016875A1 US 9820364 W US9820364 W US 9820364W WO 9916875 A1 WO9916875 A1 WO 9916875A1
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protein
spectrin
transport
golgi
atpase
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PCT/US1998/020364
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English (en)
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Jon S. Morrow
Prasad Devarajan
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Yale University
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Priority to AU10635/99A priority Critical patent/AU750395B2/en
Priority to JP2000513945A priority patent/JP2001518295A/ja
Priority to EP98953207A priority patent/EP1021539A4/fr
Priority to CA002304484A priority patent/CA2304484A1/fr
Publication of WO1999016875A1 publication Critical patent/WO1999016875A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to methods for selectively modulating the sequestration of integral membrane or secretory proteins into transport vesicles and the trafficking of such vesicles between the endoplasmic reticulum, Golgi bodies, plasma membrane and other membrane compartments.
  • the present invention based on the discovery of a new biological phenomena, provides methods and compositions for use in identifying relevant integral membrane and secretory proteins, as well as agents that inhibit the sequestration of such proteins in transport vesicles.
  • Related methods and compositions can be used to modulate the secretion and cell membrane display of various disease-related proteins. Acknowledgment of Federal Support
  • budding vesicles are encased in a closely adherent protein shell that favors their extrusion from a planar membrane compartment.
  • Three general types of coats are now recognized: Clathrin AP, COPI, and COPII. These coats are similar in many ways. They all form easily discernible electron dense layers about their respective vesicles; they are of relatively simple and uniform composition; and they assemble onto vesicles under the control of ARF like GTP -binding proteins.
  • the Golgi associated ankyrins include a novel small ankyrin (Ank GU9 ) that has been cloned and characterized (Devarajan, et ⁇ /.,1996), and larger isoforms that so far are only identified immunologically (Beck et /.,1997). Additional ankyrins also associate with other internal membrane compartments such as lysosomes (Hoock et /.,1997).
  • spectrin differs from the coatomer proteins in that it does not form geometrically precise coats and is more difficult to visualize by electron microscopy, as with other coat proteins spectrin' s association with Golgi membranes is stimulated by ARF in a PtdInsP 2 -dependent manner (Godi et /.,1997).
  • Evidence disclosed below indicates a direct and specific role for spectrin-ankyrin and other adapter proteins in mediating the transport of integral and secretory proteins. This spectrin-ankyrin-adapter protein trafficking/tethering system is collectively termed SAATS.
  • the present invention is based on our discovery of a covert intracellular processing and trafficking system that mediates the sequestration of integral membrane and secretory proteins into transport vesicles for transport from the endoplasmic reticulum to the cis-Golgi apparatus, from the cis-Golgi to the medial-Golgi apparatus, from the medial-Golgi to the trans-Golgi apparatus and from the trans-Golgi apparatus to the cell membrane or other cellular compartments.
  • General methods are presented that will allow specific transport inhibitors for a selected membrane or secreted protein to be identified, and several examples of the application of these methods are presented.
  • FIG. 1 Diagram of the spectrin/ankyrin/adapter protein trafficking/tethering system (SAATS) and one way protein transport may be blocked.
  • SAATS spectrin/ankyrin/adapter protein trafficking/tethering system
  • A general schematic by which dynactin moves a vesicle from the endoplasmic reticulum (ER) to the Golgi.
  • ER endoplasmic reticulum
  • ERGIC endoplasmic reticulum Golgi intermediate compartment
  • Centractin is an actin related protein, or ARP that binds to spectrin.
  • various proteins bind to vesicular associated spectrin directly or indirectly via adapter proteins such as ankyrin or by binding to another membrane carrier protein that is subsequently bound by SAATS (such as for secreted proteins).
  • L lumen of the ER.
  • Selected protein transport is achieved by blockage of its attachment to SAATS, such as in a cell transfected with a cDNA encoding a spectrin ⁇ I N . 5 construct. In such cells, certain integral membrane or secretory proteins are not sequestered into the vesicle for transport from the endoplasmic reticulum to the Golgi.
  • Figure 2 A series of appropriate and representative ⁇ l ⁇ * spectrin constructs useful for blocking membrane protein display and for determining suitable SAATS targets for pharmacologic agent identification. Some of the important functional domains are also depicted. Ankyrin binding occurs in repeat unit 15. The amino-terminal domain (green) bestows actin and presumably ARP binding on spectrin complexes. Membrane association domains (MAD1 and MAD2) are also shown. A third membrane association domain (MAD3) also resides somewhere between repeat units 3 and 9. Similar constructs prepared from other spectrins involved in protein trafficking ⁇ e.g., ⁇ lll spectrin) can also be prepared.
  • Figure 3 Illustration of the use of cultured Madin Darby Canine Kidney (MDCK) cells to monitor the surface expression of Na,K- ATPase by indirect immunofluorescence and immunoprecipitation.
  • MDCK Madin Darby Canine Kidney
  • the expression of amino terminal ⁇ l spectrin constructs in MDCK cells disrupts the native Golgi spectrin-ankyrin skeleton and Na,K- ATPase assembly.
  • A The intracellular distribution of wild type Golgi spectrin was monitored using Mab VIIIC7, which reacts with full length Golgi spectrin but not the shorter FLAG tagged ⁇ I N .
  • Figure 4 Illustration of alternative assays demonstrating the impairment of Na,K-ATPase trafficking from the ER induced by SAATS inhibition. Dispersal of Golgi spectrin by ⁇ I N _ 5 blocks Na,K- ATPase transport to the medial Golgi and its incorporation into a detergent stable skeletal complex.
  • A The intracellular distribution of the endogenous ⁇ l ⁇ * spectrin (determined by Mab VIIIC7, which does not react with the expressed ⁇ I N . 5 peptide), and Na,K- ATPase in MDCK cells transfected with ⁇ I N . 5 spectrin.
  • the band - 106 kDa in the wild type cells (*) is inconstant in its appearance, and may represent a cross-linked adduct of ⁇ -Na,K- ATPase with ⁇ -Na,K- ATPase or other protein, as is commonly observed after reduced SDS-PAGE in MDCK cells (Morrow et ⁇ /.,1989).
  • C Fraction of Na,K- ATPase present in the soluble and detergent insoluble pools of MDCK cells transfected with different ⁇ l spectrin constructs.
  • Fxl soluble fraction
  • Fx2 cytoskeletal fraction
  • D The results of three separate determinations of ⁇ -Na,K- ATPase extractability of wt and two transfected MDCK cell lines were quantified in-duplicate by densitometry, and all analyses averaged. Error bars represent ⁇ 1SD. Note the substantial loss of fully assembled and detergent insoluble Na,K- ATPase in the ⁇ I N _ 5 expressing cells.
  • Figure 5 Illustration of selective blockage of Na,K- ATPase and VSV-G transport, but not E-cadherin, by ⁇ IN-5. Comparison of the effect of ⁇ I N _ 5 spectrin on Golgi and the assembly of different proteins in MDCK cells. Wild-type (B,D,F,H) .and ⁇ I N _ 5 transfected cells (A,C,E,G) are shown. The localization of VSV- G protein (E,F) was measured after tr.ansient infection.
  • E-cadherin (C,D) was monitored with a Mab from Transduction labs, and also by the extent to which the precursor peptide was proteolyzed (inset, Western blot) from 135 kDa (*) to 120 kDa (EC) (a process that occurs in the trans-Golgi) (Shore and Nelson, 1991). There was no significant difference in the extent of E-cadherin processing or its level of assembly at the plasma membrane in the ⁇ I N _ 5 line vs. wt cells. Despite the disruption of Na,K- ATPase and VSV-G transport (and the wt
  • Golgi spectrin skeleton Fig. 3
  • the Golgi appears to remain largely intact as measured by the distribution of ⁇ -COP (A,B) and by the presence of normal appearing juxtanuclear Golgi structures in uranyl acetate and lead stained electron microscopy (G,H) (arrows).
  • Bar lO ⁇ in (A-F), 0.5 ⁇ in (G,H).
  • Original magnification (G,H) 63,000x.
  • Figure 6 Illustrates the rescue of Na,K- ATPase trafficking by inclusion of a specific SAATS binding domain that in this case, binds to ankyrin.
  • MDCK cells were transfected with either ⁇ I N diligent 5 as above, or a with ⁇ I N . 5 ⁇ ⁇ 5 .
  • This latter construct incorporates the ankyrin binding domain of spectrin into the ⁇ I N _ 5 peptide.
  • FIG. 7 Illustrates how to make SAATS block VSV-G transport (but not Na,K-ATPase).
  • MDCK cells infected with vesicular stomatitis virus were used to monitor the trafficking of VSV-G protein.
  • the transport of this protein is normally to the basolateral membrane, similar to that for Na,K- ATPase (as shown in the wt cells and in Figure 5).
  • This transport is blocked by SAATS inhibition with the ⁇ I N plausible 5 construct, as is Na,K- ATPase (cf. Figure 6).
  • VSV-G trafficking is not restored by the inclusion of the ankyrin binding domain of spectrin (repeats 14-15) in the construct.
  • Figure 8 Illustrates the identification of a small peptide sequence responsible for association of Na,K- ATPase with ankyrin.
  • A Schematic representation of the five cytoplasmic domains of ⁇ -Na,K- ATPase and their relationship to the ankyrin binding peptide sequences identified here. Codon positions defining each peptide are shown. Previous studies have established broad reactivity of cytoplasmic domains II and III with ankyrin, with domain II contributing most of the binding activity (Devarajan, et ⁇ /.,1994; Jord.an et al., 1995).
  • Each depicted peptide (II-IIC) was prepared as a fusion construct with SjGST, .and examined for its ability to bind at various concentrations either purified ANK1 (from human red cells) or kidney ankyrin (ANK3) derived from whole MDCK cell lysates. Results from a single experiment are shown.
  • the top panel shows Coomassie blue stained SDS-PAGE analysis of each peptide, as well as the entire MDCK cells extract applied to the affinity column to detect ANK3 binding.
  • purified erythrocyte ankyrin was used.
  • Peptide IIA sequence -SYYQEAKSSKIMESFKNMVPQQALV-, represents the minimal active sequence detected. We term this the minimal ankyrin binding domain (MAB).
  • Figure 9 Illustrates that the specific deletion of a SAATS attachment sequence in an integral membrane protein selectively blocks its transport alone.
  • MDCK cells were transiently transfected with either wild-type Na,K- ATPase to which a FLAG epitope tag had been added to its NH2 -terminus, or by a similarly FLAG-tagged mutant Na,K- ATPase in which codons 142 to 166 had been deleted. These residues correspond to the minimal ankyrin binding domain (MAB) identified in Figure 8.
  • MAB minimal ankyrin binding domain
  • the cells were also co-transfected with wt ⁇ -Na,K-ATPase, so as to assure sufficient ⁇ -Na,K- ATPase to pair with the ⁇ -Na,K- ATPase.
  • (top) Flag tagged wt-Na,K- ATPase is delivered normally to the plasma membrane
  • (bottom) Flag tagged Na,K- ATPase lacking MAB does not assemble at the plasma membrane, and eventually is targeted for degradation in lysosomes.
  • the mutant Na,K- ATPase is the only protein whose transport is disrupted.
  • Figure 10 Illustrates selective blockage of Na,K- ATPase transport in normal cells by the expression of a small peptide inhibitor.
  • Wild-type MDCK cells were transiently transfected with green fluorescent protein linked to the 25 residue MAB peptide. This is the same sequence whose deletion caused Na,K- ATPase to be retained in the ER and ultimately lysosomes in Figure 9. After approximately 1-2 days in culture, cells were fixed and stained for GFP (using anti-GFP antibodies, left panel) or for Na,K- ATPase (right panel, red).
  • Figure 11 Illustrating the three-dimensional structure of MAB, which is well suited for the rational design of small molecule agents with similar pharmacologic action as MAB.
  • This structure was determined using carrier mediated crystallization based on the active GST-MAB peptide shown in Figure 8 (Zhang et al., 1997a).
  • the basic structural motif is that of a seven residue "loop" on a "stalk" composed of antiparallel ⁇ -strands.
  • A,C,E Surface accessibility depictions showing the amphipathic surfaces formed by the "loop”.
  • B,D,F Ribbon diagrams demonstrating the back-bone contour.
  • Figure 12 Illustrating another integral membrane protein of significant medical interest that is linked to SAATS by a different ankyrin interaction.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • TNR Transmembrane-Nucleotide binding -Regulatory domain
  • CFTR and TNR are highly polarized, and unlike Na,K- ATPase, are expressed predominately in the apical domain of these polarized epithelial cells.
  • B Despite the fact that CFTR and TNR are apical proteins, they nevertheless binds components of the SAATS system, which is responsible for their trafficking. Either whole cell lysates (L) of cells extracted with RIP A buffer, or Fx 1 and Fx 2, were immunoprecipitated with irrelevant antibody control (C) or with anti-CFTR antibody (IP). The washed immunoprecipitates were then analyzed by SDS-PAGE and examined by western blotting for either ankyrin.
  • Figure 13 Illustrating both down and up-regulation of integrin display in cultured endothelial cells by SAATS.
  • SV40 transformed murine endothelial cells were transfected with ⁇ I N . 5 or ⁇ I N _ 5; ⁇ 5 , and the surface display of three different integrins was measured by flow cytometry. Note the blockage of alpha V, beta 3, and beta 1 integrins by the ⁇ I N . 5 peptide. Conversely, the ⁇ I N _ 5>15 peptide markedly enhances the surface display of alpha V and beta 1 integrin (but not beta 3 integrin). This enhanced display (as opposed to blockage) is presumably achieved by enhancing the efficiency of SAATS directed cargo loading of vesicular transport by choosing peptides or agents that enhance the binding of a given membrane protein to SAATS .
  • FIG 14 Illustrating additional examples of SAATS regulation of integrin display in cultured endothelial cells. Experiment was carried out as in Figure 13. Note that alpha-V integrin is also modulated by SAATS. Also note that in this example, ⁇ I N . 2> i 5 was used to rescue beta-1 expression. ⁇ I N (2004) 2 is an even more broadly blocking inhibitor of SAATS trafficking (see Figure 1), but in this instance, supranormal levels of beta-1 can be achieved by inclusion of repeats 14,15 into the ⁇ I N _ 2 peptide.
  • Figure 15 Illustration of PEC AM (CD31) surface modulation by SAATS inhibitory peptides employing two different epitope tags, FLAG and GFP.
  • FIG. 16 Illustrates the modulation of CD45 and TNFR-1 in T-lymphocytes by SAATS inhibitors.
  • Jurkat T-lymphocytes were transfected with the constructs indicated, including ⁇ l 1 . 15 alone. Note the marked down regulation of
  • CD45 a documented ankyrin binding protein, by constructs lacking repeat 15, as well as by the ⁇ l ⁇ 4 _ ⁇ 5 peptide itself (which lacks the constitutive Golgi targeting signal, see Fig. 2).
  • Figure 17 Illustrates the modulation of Fas and Fas-L in T-lymphocytes by SAATS. Experiments were as before. Note the changes in both Fas and Fas-L due to ⁇ I N . 5 and ⁇ I N . 5 15 peptides.
  • Figure 18 Illustrates the use of antibody or injected small peptides to modulate Na,K- ATPase in wild-type MDCK cells. Clusters of wild-typeMDCK cells were micro injected with either (A) GST alone; or (B).with the ⁇ I N . 4 peptide generated as a recombinant fusion peptide with GST, and then stained for Na,K- ATPase by indirect immunofluorescence.
  • Figure 19 Presents the full-length cDNA sequence of a novel isoform of ⁇ lll spectrin that may be an additional component of SAATS.
  • a partial cDNA clone identified as an expressed sequence (EST) was identified as a spectrin family member, extended to complete the sequence by 5 'RACE PCR amplification, and sequenced.
  • B The resulting full-length sequence is predicted to encode a novel isoform of spectrin, termed ⁇ lll (Morrow, 1997), that is similar to both ⁇ l and ⁇ ll, but shows significant differences in selected regions.
  • C Sequence similarity between the various beta-spectrins. Also shown is a phylogenetic dendritogram showing the approximate relationship of ⁇ lll spectrin to ⁇ l and ⁇ ll spectrins.
  • Figure 20 Illustrative examples of SAATS inhibitory peptide constructs, in two different vectors: pcDNA3 (FLAG tagged) and enhanced green fluorescent protein (eGFP).
  • pcDNA3 FLAG tagged
  • eGFP enhanced green fluorescent protein
  • Figure 21 Figure 4B represents a model of how Na,K-ATPases may interact with one or more ankyrin repeat units.
  • Figure 22 Modulation of surface display of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by SAATS.
  • MDCK cells were transfected with either the ⁇ I N . 5 or ⁇ I N . 5)15 constructs and then surface labeled with biotin. After labeling, cells were solubilized, and the total CFTR in each culture immunoprecipitated with anti-CFTR antibodies. The precipitates were then analyzed by SDS-PAGE, transferred to nitrocellulose, and surface CFTR detected by avidin labeling. Total CFTR was also determined in these cellular extracts by western blotting (data not shown). In all cell lines, there was minimal change in the total CFTR in the cells.
  • Figure 23 Schematic representation of the Spectrin Heterodimer. Modes of Carrying Out the Invention
  • ABGP 205 ankyrin brain (Luna and H ⁇ tt.1992) mositol t ⁇ phosphate receptor anky ⁇ n brain (Joseph and Samanta,1993) CD44 (gp85) ankyrin lymphoma (Lokeshwar and
  • CD44-l ⁇ ke l l ⁇ kD protein ankyrin endothehum (Bourguignon et al ,1992) CAMS related to Ll/neurofascin ankyrin bram (Davis and Bennett, 1993,
  • H/K-ATPase anky ⁇ n gastric cells (Smith et al ,1993) glycopho ⁇ n C protein 4 1 erythrocytes (Hemming et al ,1995) Thy-1 protein 4 1 lymphocytes (Bourguignon et al ,1986) cadhe ⁇ n ⁇ (E)-catenm kidney (Lombardo et al ,1994a) stomatm adducm RBC and others (Smard e/ n/ ,1994) bg subumts of t ⁇ me ⁇ c G-proteins direct (Pitcher et al ,1992, Touhara et al ,1994)
  • CD45 (gpl80) (tyrosine phosphatase) direct lymphocytes (Bourguignon et al ,1985,
  • A60 direct brain (axons) (Hayes e( ⁇ / ,199 5 )
  • the practice of the present invention thus generally involves the modulation of specific interactions between SAATS and the recognized cargo or carrying proteins of the ER and Golgi. Also the elucidation of particular binding sites (which are often complex and involve homologues of spectrin + adapter proteins) is described in order to identify targets for blocking specific interactions.
  • the SAATS system can discriminate between H,K- ATPase, Na,K- ATPase, CFTR and other transport molecules, or can be modulated at the level of binding domains common to various classes of proteins.
  • identifying the binding domain for CFTR, the gene product responsible for cystic fibrosis, in SAATS by techniques disclosed herein, would allow early analysis of possible transport enhancing agents that might ameliorate the clinical severity of this disorder by enhancing the delivery of mutant (but functional) CFTR to the plasma membrane.
  • Figures 12 and 22 the binding of CFTR to ankyrin components of SAATS is illustrated as well as its enhanced delivery to the membrane by the ⁇ I N _ 5 15 construct.
  • adapter proteins other than ankyrin, such as protein 4.1 homologues, adducin homologues, catenin homologues, and others are contemplated.
  • the skilled artisan can explore the role of such adapter proteins by techniques disclosed herein in order to determine their participation in the modulation of the interaction of SAATS with proteins trafficking through the ER and Golgi.
  • the action of similar pathways acting on the delivery of proteins from the medial-Golgi and trans-Golgi to the plasma membrane or other internal membrane-bound compartments is also likely, but remains to be more fully explored with techniques disclosed herein.
  • the methods outlined here offer a novel approach to controlling the activity of specific membrane proteins, be they receptors, adhesion receptors, ion channels, or transporters.
  • the approach outlined herein targets the very appearance of a protein at the cell surface or in a secretory vesicle. This strategy is somewhat similar in concept to approaches that attempt to specifically suppress the synthesis of a given protein, such as by anti-sense RNA or by specific transcriptional regulators.
  • the methods outlined herein are fundamentally different in that they: 1) do not attempt to suppress the synthesis of a given protein, only its delivery to the correct cellular or tissue compartment; 2) do not require intracellular expression of RNA, obviating many hurdles inherent in such a task; and 3) lend themselves to high-throughput in vitro screening assays, and should be amenable to regulation by small molecule effectors.
  • the methods outlined here offer a novel therapeutic approach that is complementary to and synergistic with other leading drug development strategies.
  • Transport vesicle means any small membrane bounded structure containing protein and involved in the movement of such protein from one membrane compartment to another.
  • Cell membrane means a lipid and protein containing bilayer structure bounding any cellular compartment including the surface of the cell, the so called plasma membrane.
  • Adapter protein means any protein that provides for a direct or indirect linkage to the spectrin backbone of SAATS or to any other multifunctional proteins that directly or indirectly determine the specificity of cargo or cargo protein carrier capture by SAATS.
  • an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences of the vesicular skeletal protein and associated adapter protein.
  • an agent is said to be rationally selected or rationally designed when the agent is chosen on a nonrandom basis that takes into account the sequence of the target site and/or its conformation in connection with the agent's action.
  • Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up the relevant vesicular skeletal protein or associated adapter protein.
  • a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to a fragment or relevant domain of a spectrin protein.
  • cell absent any further designation, means any eukaryotic cell population, including primary and transformed immortalized cell lines, that express spectrin, or an equivalent protein.
  • Madin Darby canine kidney (MDCK) cells can be employed in the claimed methods. Transfection of some cell lines with broadly active
  • SAATS inhibitors such as the ⁇ I N _ 5 or ⁇ I N consult 2 constructs, may lead to poor cell survival and the activation of apoptosis or lethal swelling.
  • This problem by be often circumvented by selecting less broadly active SAATS agents (eg. larger constructs, or constructs in which only small portions of spectrin, ankyrin, or one of the adapter proteins has been deleted or modified).
  • This strategy of scanning mutagenesis will minimize the global disruption cellular transport that accompanies the most severe disruptors of SAATS, and improve cell viability so that determinations can be made on less hardy cell lines.
  • This technique also has the advantage of more precisely pinpointing the locus of SAATS activity for a given protein. Transfection levels may also be modified or controlled to manage poor cell survival.
  • apoptosis inhibitors in the cell medium, such as caspase inhibitors or Fas blocking antibodies (see U.S. Patents 5,632,994, 5,656,725, 5650,491 and 5,635, 187) .
  • Cells may also be engineered to express increased levels of bcl-2 (see U.S. Patent 5,650,491). g.
  • stringent conditions refers to conditions those commonly defined and available, such as those defined by Sambrook et al ⁇ Molecular Cloning: A Laboratory Approach, Cold Spring Harbor Press, NY, 1989) or Ausubel et al. ⁇ Current Protocols in Molecular Biology, Greene Publishing Co., NY, 1995).
  • Hybridization is a function of sequence identity (homology), G+C content of the sequence, buffer salt content, sequence length and duplex melt temperature (T[m]) among other variables.
  • sequence identity identity
  • the buffer salt concentration and temperature provide useful variables for assessing sequence identity (homology) by hybridization techniques. For example, where there is at least 90 percent homology, hybridization is commonly carried out at 68° C. in a buffer salt such as 6XSCC diluted from 20XSSC. See Maniatis et al.
  • the buffer salt utilized for final Southern blot washes can be used at a low concentration, e.g., 0.1XSSC and at a relatively high temperature, e.g. 68° C, .and two sequences will form a hybrid duplex (hybridize).
  • Use of the above hybridization and washing conditions together are defined as conditions of high stringency or highly stringent conditions.
  • Moderately high stringency conditions can be utilized for hybridization where two sequences share at least about 80 percent homology.
  • hybridization is carried out using 6XSSC at a temperature of about 50-55° C.
  • a final wash salt concentration of about 1-3XSSC and at a temperature of about 60-68° C are used.
  • specific hybridization refers to conditions in which a high degree of complementarity exists between a nucleic acid comprising the sequence of at least one of the Figures and another nucleic acid.
  • complementarity will generally be at least about 75%, 80%, 85%, preferably about 90-100%, or most preferably about 95-100%.
  • the present invention relates to a method for selectively controlling the transport of integral membrane and secretory proteins by controlling their sequestration into transport vesicles that mediate protein trafficking between the ER, Golgi, plasma membrane, and other membrane compartments.
  • the present patent application expands on the discovery that specific isoforms of spectrin and various adapter proteins, such as ankyrin, associate with vesicle and Golgi membranes by multiple interactions (Devarajan et ⁇ /.,1994; Devarajan and Morrow, 1995; Devarajan and Morrow, 1996; Devarajan et al., 1996a; Devarajan et al., 1996b; Devarajan et ⁇ /.,1997; Godi et ⁇ /.,1997).
  • SAATS spectrin-ankyrin-adapter protein trafficking system
  • Inhibitors of ADP-ribosylation factor (ARF) block the binding of the PH domain of ⁇ l spectrin via a PtdInsP 2 -dependent process, implying that SAATS may also contribute to the organization and trafficking of specific phospholipids.
  • Agents modulating PtdInsP2 phospholipid display, or ARF activity directly, may also thus form the basis for a class of therapeutics.
  • An examples of such an agent would be a permeant non-hydro lyzable homologue of GTP, such as a permeant GTP- ⁇ -S.
  • Example 1 Identification of integral membrane and secretory proteins whose vesicular transport is mediated by SAATS
  • Step Al Select a model cultured cell line that expresses the membrane or secretory protein that is being evaluated, and for which an assay exists that will allow the detection of surface or secreted target proteins.
  • Examples would be immunofluorescent detection of surface protein; flow cytometry; surface labeling assays, or transport assays using pulsed 35 S methionine labeling. Assays that monitor the transport of a specific protein may also be performed by the use of GFP - labeled proteins and time lapse vital flourescent microscopy. Also envisioned is the detection of a specific transport or electrical activity associated with the surface display or secretion of the targeted protein. As a specific example, cultured Madin Darby Canine Kidney cells (MDCK) may be used to monitor the surface expression of Na,K- ATPase by indirect immuno fluorescence (See Figs 3 through 10, 18).
  • MDCK Madin Darby Canine Kidney cells
  • Step A2 Prepare using standard transfection methods of the model cell line a series of individual clonal lines or lines transiently transfected or lines infected using retroviral vectors or other virus based infection strategies, each expressing one of a complimentary and overlapping series of ⁇ l ⁇ * spectrin cDNA constructs of varying lengths under the control of a strong eukaryotic expression vector.
  • a series of suitable ⁇ l ⁇ * spectrin constructs is listed in Figure 2. For example, stable MDCK cell lines expressing the ⁇ I N _ 5 spectrin peptide (See Figs. 3 and others), which competitively displaces or substitutes for the endogenous SAATS system, as one appropriate line.
  • rDNA recombinant DNA
  • transformation of appropriate cell hosts with an rDNA (recombinant DNA) molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed.
  • electroporation and salt treatment methods are typically employed, see, for example, Cohen et al, Proc Acad Sci USA (1972) 69:2110; and Maniatis et al, Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
  • electroporation, cationic lipid or salt treatment methods are typically employed. See, for example, Graham et al, Virol (1973) 52:456; Wigler et al, Proc Natl Acad Sci USA (1979) 76: 1373-76.
  • Successfully transformed cells i.e., cells that contain an rDNA molecule of the present invention
  • cells resulting from the introduction of an rDNA of the present invention can be cloned to produce single colonies.
  • Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using a method such as that described by Southern, J Mol Biol (1975) 98:503, or Berent et al, Biotech (1985) 3:208 or the proteins produced from the cell assayed via an immunological method.
  • tags such as green fluorescent protein are employed in the construction of the recombinant DNA, the transfected cells may also be detected in vivo by the fluorescence of such molecules by cell sorting.
  • Step A3 Evaluate each clonal line or cells expressing the desired construct (in assays in which transient expression is used, e.g. Figure 9) for disruption of the surface display or secretion of the selected protein. Expression of full length ⁇ l ⁇ * spectrin (or other full length spectrins) will not disrupt SAATS. Expression of at least one of the truncated constructs shown in Figure 2 or analogous constructs for other spectrins and other vesicular skeletal proteins will act in a dominant negative way to disrupt SAATS. A comparison of the inhibitory ability of different constructs will reveal the boundaries of an inhibitory peptide or cDNA construct encoding such inhibitory peptide. For example, inhibition of surface display of Na,K- ATPase in MDCK cells by expression of the ⁇ I N . 5 spectrin construct (Fig. 3).
  • Step A4 Knowledge of the minimal inhibitory peptide can be further refined by repeating steps A2 and A3, above, using ⁇ l ⁇ * spectrin constructs or similar constructs prepared from other spectrins in which specific regions deleted in the inhibitory construct are returned to the inhibitory construct, thereby identifying the regions required to rescue surface display or secretion of the selected (targeted) protein.
  • steps A2 and A3 using ⁇ l ⁇ * spectrin constructs or similar constructs prepared from other spectrins in which specific regions deleted in the inhibitory construct are returned to the inhibitory construct, thereby identifying the regions required to rescue surface display or secretion of the selected (targeted) protein.
  • Step A5 A further refinement of the SAATS docking site for the targeted protein may be obtained by determining whether it attaches directly to spectrin at the site identified in Steps A1-A4, or whether an intermediary adapter protein is utilized. In most instances, this can be readily determined by in vitro binding or co-immunoprecipitation assays (eg Figure 3), in which the interaction of the targeted protein with spectrin and/or adapter proteins (such as ankyrin, protein 4.1 , adducin, ⁇ -catenin, see TABLE IB) is evaluated. Such methods are well known in the art as exemplified by Harlow et al. , 1988.
  • the interaction of Na,K- ATPase with the 14-15 repeat unit of spectrin is via an ankyrin Gl 19 adapter protein intermediate, as detected by co- immunoprecipitation ( Figure 3), and by direct binding assays indicating that ankyrin binds to this region of spectrin (Kennedy et al, 1991) and that Na,K- ATPase binds to ankyrin (Morrow et ⁇ /.,1989) and at a specific locus within Na,K-ATPase (Devarajan, et ⁇ /.,1994).
  • Spectrin and associated cDNA constructs Recombinant peptides are prepared as fusion proteins with glutathione-S-transferase, using the prokaryotic expression vector pGEX (Smith and Johnson, 1988; Kennedy et ⁇ /.,1991). Spectrin clones used for these are identical to sequences published for ⁇ ll spectrin (Hu et al, 1992; Chang et ⁇ /.,1993; Chang and Forget, unpublished), ⁇ l spectrin (Winkelmann et al, 1988; Winkelmann et al, 1990b; Winardi et al, 1993), and ⁇ lll spectrin as shown in figure 19.
  • the desired segment of cDNA is excised by EcoRl or other suitable restriction enzymes from pGEM 7z(-), purified from agarose gels by electroelution and subcloned into the appropriate pGEX vector, usually pGEX-2T.
  • pGEX-2T the appropriate pGEX vector
  • the polymerase chain reaction was typically used in conjunction with Pfu polymerase and selected primers to amplify regions of the spectrin clones to facilitate plasmid construction and in some cases linker-insertion mutagenesis (Sambrook et ⁇ /.,1989; Kennedy, et ⁇ /.,1991). Occasionally Taq polymerase was used to facilitate cloning of PCR products into the pCRII vector of the TA cloning kit (Invitrogen). The validity of all PCR generated constructs is confirmed by DNA sequencing.
  • PCR primers are engineered to have BamHI sites 5' and EcoRI sites 3' to ease directional subcloning into pGEX-2T.
  • Two to five additional bases were usually added at the ends of each primer to enhance their susceptibility to restriction endonucleases.
  • the amino acid boundaries for the amplification of the spectrin structural repeats are selected based upon the phasing of Drosophila ⁇ -spectrin (Winograd et ⁇ /.,1991).
  • Peptides representing the unique alternatively spliced COOH-terminal region of the ⁇ l spectrin found in brain and muscle are prepared from plasmid pMSP6 AC (Winkelmann, 1990) by digestion with Xma I. The resulting 767 bp fragment containing the alternatively spliced unique
  • spectrin ⁇ l ⁇ H C-terminus of spectrin ⁇ l ⁇ H is gel purified and subcloned into pGEX-2T at the Sma I site.
  • Other typical details of primer selection and plasmid construction are summarized as in Kennedy, et al, ⁇ 99 ⁇ ; Lombardo, et al, 1994b.
  • the various ⁇ l spectrin peptides are expressed as above from existing human ⁇ l spectrin constructs (Winkelmann et al, 1990a; Winkelmann, et al, 1990b; Weed et ⁇ /.,1996) .
  • Constructs for eukaryotic cell expression are typically prepared in the pcDNA3- vector (Invitrogen) or other comparable vector incorporating a suitable eukaryotic promoter.
  • pcDNA3 vector stable MDCK cell lines can be established by selection with G418 after lipofectamine transfection (Weed, et ⁇ /.,1996).
  • Transient experiments can also be carried out in this system or in several other systems well suited for transient assays, such as COS cells.
  • VSV-G newly confluent cells are infected for 60 min. with VSV-G in Transwell ® filters (100 ml, 108 titer virus Pimplikar et al.,1994). Infected cells are incubated for 3 hours and fixed prior to analysis.
  • Recombinant proteins are expressed in E. Coli strains HB101, DH5a or the protease deficient strain CAG-456 and purified as before (Smith and Johnson,1988; Kennedy, et al., 1991).
  • peptides were further purified by gel filtration on a 1 x 100 cm column of Sephacryl S-200 HR in the presence of 1 M NaBr, 10 mM Na 2 HPO 4 , 0.5 mM ⁇ GTA, 0.5 mM ⁇ DTA, 15 mM sodium pyrophosphate, 1 mM NaN 3 , 0.5 mM DTT, pH 8.0.
  • Bovine brain spectrin is prepared from fresh brain membranes by low ionic strength extraction of demyelinated brain membranes followed by gel filtration on Sephacryl-S-500 HR (Harris, et ⁇ /.,1985; Bennett, 1986) .
  • Antibodies and immunodetection procedures Constructs epitope labeled by the 8 aa FLAG marker (IBI Scientific) were detected with Mab M5 (IBI Scientific); ⁇ l spectrin antibodies included: Mab VIIIC7, Mab VD4, Mab IVC9, and Mab IVF8 (Harris, et ⁇ /.,1986); Pab C19 against 19 COOH-terminal residues of ⁇ l ⁇ l spectrin (gift, V. Marchesi); and Pab MUSI against region III of ⁇ l ⁇ 2 spectrin (Malchiodi-Albedi et a/., 1993).
  • Pab 10D detected ⁇ ll spectrin (Devarajan, et ⁇ /.,1996).
  • Other antibodies were: Mab ⁇ -COP, I. Melman; Pab ⁇ -cadherin, Transduction labs; Mab -Na,K-ATPase, UBI; Mab ⁇ -Na,K-ATPase, M. Caplan; Mab VSV-G, J. Rose; and Pab centractin, ⁇ . Holzbaur. Cells were grown on glass cover slips or culture dishes, washed x3 with PBS, fixed 15 min. in acetone, blocked 30 min. in goat serum, and incubated with 1° antibody in 2% BSA in PBS + 10%> goat serum at RT for 60 min.
  • spectrin Mab's were used at 1 :100; E-cadherin at 1 :5000; b-COP, 10D, C19 at 1 :200.
  • Detection used 2° antibodies conjugated to CY3 or CY2 (Vector).
  • Confluent MDCK cells were PBS washed, lysed 20 min. at 4°C by gentle rocking in IP buffer (10 mM Tris-HCl, pH 7; 150 mM NaCl; 5mM EDTA; ImM EGTA; 2 mg/ml BSA; 0.5% deoxycholate; 1% NP-40; 0.5 mM pefablock; ImM PMSF; and ImM leupeptin.
  • IP buffer 10 mM Tris-HCl, pH 7; 150 mM NaCl; 5mM EDTA; ImM EGTA; 2 mg/ml BSA; 0.5% deoxycholate; 1% NP-40; 0.5 mM pefablock; ImM PMSF; and ImM leupeptin.
  • the lysate (2 ml) was decanted and centrifuged 1 min. at 10,000xg, precleared by 60 min. incubation with 25 ml of non-immune rabbit serum with 200 ml of a 50%) Protein A Se
  • Example 2 Identification of Na,K- ATPase as an integral membrane protein participating in SAATS
  • Golgi spectrin and A ⁇ k o are localized with the Golgi, while -Na,K- ATPase is predominately distributed at the plasma membrane where it is tethered via the plasma membrane form of ankyrin to ll ⁇ ll spectrin (Morrow, et a/., 1989; Nelson and Hammerton,1989) ( Figure 3).
  • Golgi spectrin Ank G1 ]9 , and -Na,K- ATPase
  • cells were grown on glass cover slips or culture dishes, washed x3 with PBS, fixed 15 min. in acetone, blocked 30 min.
  • AnkGl 19 was monitored by the antibody "Jasmin” (Devarajan, et ⁇ /.,1996); ⁇ -Na,K- ATPase by a Mab from Upstate Biotechnology. Detection was by indirect immunofluorescence.
  • Electron microscopy was performed by fixing cell monolayers in situ for 1 hr (Karnovsky's fixative 18). Fixed monolayers were washed in 0.1 M Na-cacodylate, pH 7.4, and postfixed in 1%> OsO 4 in 0.1M s-collidine. After dehydration in graded ethanol and washing with propylene oxide, samples were embedded in Epox-812 (Ernest Fullman, Inc. N.Y.). Ultrathin sections stained with aqueous uranyl acetate and lead citrate were viewed on a Zeiss EM-910 at 80 kV.
  • IP buffer 10 mM Tris-HCl, pH 7; 150 mM NaCl; 5mM EDTA; ImM EGTA; 2 mg/ml BSA; 0.5% deoxycholate; 1% NP-40; 0.5 mM pefablock; ImM PMSF; and ImM leupeptin.
  • the lysate (2 ml) was decanted and centrifuged 1 min. at 10,000xg, precleared by 60 min. incubation with 25 ml of non-immune rabbit serum with 200 ml of a 50%> Protein A Sepharose (Pharmacia).
  • ATPase with the 14-15 repeat unit of spectrin occurs via an ankyrin Gl 19 adapter protein intermediate (see Figure 3B).
  • ⁇ l ⁇ * spectrin, AnkGl 19, centractin, and Na,K-ATPase are co-precipitated from detergent lysates by Mab VIIIC7 (lane IP).
  • Soluble ll ⁇ ll spectrin (as detected here by immunoblotting for all spectrin), the form found predominately at the plasma membrane, is also present in these detergent lysates (lane lys) but is not part of the precipitable Golgi spectrin complex. Control experiments with preimmune or irrelevant antibody did not precipitate any of these components.
  • Na,K-ATPase is blocked at the ER to medial-Golgi transition by ⁇ I N _ 5 spectrin Normally, after synthesis and core glycosylation in the ER, ⁇ -Na,K- ATPase undergoes further glycosylation in the medial Golgi and then is vectorally incorporated as an ⁇ , ⁇ -Na,K-ATPase heterodimer into a detergent insoluble ⁇ ll ⁇ ll spectrin and ankyrin cortical skeletal lattice (Nelson and Veshnock,1986; Morrow, et ⁇ /.,1989; Nelson and Hammerton,1989; Mays et ⁇ /.,1995).
  • the distribution of Na,K-ATPase in the ⁇ I N . 5 expressing cells suggested that the exit of Na,K- ATPase from the ER might be blocked
  • Fxl soluble fraction
  • Fx2 cytoskeletal fraction
  • c polypeptide with its competent ankyrin binding domain but lacking region I/MAD 1 sequences ⁇ cf. Fig 2C), only marginally impacted the assembly of Na,K-ATPase into a detergent insoluble skeleton.
  • MDCK cells were transfected with either ⁇ I N . 5 as above, or a with ⁇ I N consult 5 !5 .
  • This latter construct incorporates the ankyrin binding domain of spectrin into the ⁇ I N _ 5 peptide. Note the complete restoration of Na,K-ATPase transport, as measured in this case by its surface display and secondarily by a reduction in cell size (which is a consequence of cell swelling due to a deficiency of plasma membrane Na,K- ATPase in the ⁇ I N _ 5 transfected cell.
  • MDCK cells were transiently transfected with either wild-type Na,K- ATPase to which a FLAG epitope tag had been added to its NH2-terminus, or by a similarly FLAG-tagged mutant Na,K- ATPase in which codons 142 to 166 had been deleted. These residues correspond to the minimal ankyrin binding domain (MAB) identified in Figure 8.
  • MAB minimal ankyrin binding domain
  • the cells were also co-transfected with wt ⁇ -Na,K- ATPase, so as to assure sufficient ⁇ -Na,K- ATPase to pair with the ⁇ -Na,K- ATPase.
  • Example 3 Identification of VSV-G as an integral membrane protein participating in SAATS Infection with vesicular stomatitis virus demonstrated that VSV-G transport to the plasma membrane, which is packaged exclusively by the COPII coat ( see review Schekman and Orci,1996), like Na,K- ATPase, was blocked by the ⁇ I N _ 5 spectrin construct (Fig. 5E,F). The pattern of VSV-G staining observed suggests a block prior to the intermediate compartment. Wild-type (B,D,F,H) and ⁇ I N _ 5 transfected cells (A,C,E,G) are shown. The localization of VSV- G protein (E,F) was measured after transient infection.
  • E-cadherin (C,D) was monitored with a Mab from Transduction labs, and also by the extent to which the precursor peptide was proteolyzed (inset, Western blot) from 135 kDa (*) to 120 kDa (EC) (a process that occurs in the trans-Golgi/Shore and Nelson, 1991). There was no significant difference in the extent of E-cadherin processing or its level of assembly at the plasma membrane in the ⁇ I N . 5 line vs. wt cells. Despite the disruption of Na,K- ATPase and VSV-G transport (and the wt Golgi spectrin skeleton, Fig.
  • VSV-G protein is transported by SAATS without using ankyrin as its adapter protein, as illustrated by its failure to be rescued by return of the spectrin ankyrin binding domain (repeat 15) to the inhibitory ⁇ I N . 5 construct ( Figure 7).
  • MDCK cells infected with vesicular stomatitis virus were used to monitor the trafficking of VSV-G protein.
  • Newly confluent cells are infected for 60 min. with VSV-G in Transwell* filters (100 ml, 108 titer virus Pimplikar et ⁇ /.,1994). Infected cells are incubated for 3 hours and fixed prior to analysis.
  • Mab VSV-G (J. Rose) was used as the primary antibody in 2% BSA in PBS + 10% goat serum at room temperature for 60 minutes after blocking with goat antiserum for 30 minutes.
  • the transport of this protein is normally to the basolateral membrane, similar to that for Na,K- ATPase (as shown in the wt cells and in Figure 5).
  • This transport is blocked by SAATS inhibition with the ⁇ I N _ 5 construct, as is Na,K-ATPase (cf. Figure 6).
  • VSV-G trafficking is not restored by the inclusion of the ankyrin binding domain of spectrin (repeats 14-15) in the construct.
  • Example 4 Determination that E-cadherin sequestration is not blocked by ⁇ I N _ 5 spectrin E-cadherin, another type I basolaterally targeted membrane protein was assayed for its interactions with SAATS. Wild- type ( Figure 5D) and ⁇ I N . 5 transfected cells ( Figure 5C) were assayed for the distribution of E-cadherin.
  • E-cadherin (C,D) was monitored with a Mab from Transduction labs as the primary antibody in 2% BSA in PBS + 10%) goat serum at room temperature for 60 minutes after blocking with goat antiserum for 30 minutes., and also by the extent to which the precursor peptide was proteolyzed (inset, Western blot) from 135 kDa (*) to 120 kDa (EC) (a process that occurs in the trans-Golgi Shore and Nelson, 1991). There was no significant difference in the extent of E-cadherin processing or its level of assembly at the plasma membrane in the ⁇ I N _ 5 line vs. wt cells.
  • FIG. 13 A rapid way to evaluate the participation of a variety of integral membrane proteins in SAATS trafficking is to use flow cytometry to monitor their surface display in cells transfected with the various constructs shown in Figure 2, or related constructs. Examples of this approach are shown in Figures 13 and 14. Note, that in Figure 14, another somewhat more severe blocker of SAATS function, the ⁇ I N . 2 peptide, was used with comparable results In Figure 13, SV40 transformed murine endothelial cells were transfected with ⁇ I N . 5 or ⁇ I N . 5? ⁇ 5 , and the surface display of three different integrins was measured by flow cytometry. Note the blockage of alpha V, beta 3, and beta 1 integrins by the ⁇ I N _ 5 peptide.
  • the ⁇ I N . 5>15 peptide markedly enhances the surface display of alpha V and beta 1 integrin (but not beta 3 integrin).
  • This enhanced display is presumably achieved by enhancing the efficiency of SAATS directed cargo loading of vesicular transport by choosing peptides or agents that enhance the binding of a given membrane protein to SAATS.
  • Example 6 Identification of other proteins participating in SAATS transport in lymphocytes.
  • PEC AM CD31
  • FIG. 15 PEC AM (CD31) is shown to be ankyrin SAATS transported in lymphocytes. Cells were transfected as above, and surface display of PEC AM, a cell-cell adhesion molecule of the IgG superfamily, was monitored by flow cytometry. Note that for either construct, inclusion of the ⁇ I N . 5 sequences resulted in strong blockage of transport, indicating their involvement with SAATS.
  • CD45 is shown to be SAATS regulated with ankyrin its putative adapter, while TNFR-1 is modulated by SAATS, but does not involve ankyrin as its adapter molecule.
  • Jurkat T-lymphocytes were transfected with the constructs indicated, including ⁇ l, 4 . 15 alone. Note the marked down regulation of CD45, a documented ankyrin binding protein, by constructs lacking repeat 15, as well as by the ⁇ l, 4 _ !5 peptide itself (which lacks the constitutive Golgi targeting signal, see Fig. 2).
  • These experiments also illustrate that TNFR-1 display is upregulated (rather than blocked) by ⁇ I N . 2 or ⁇ I N . 5 , suggesting that like E-cadherin, its attachment to SAATS is mediated either directly or indirectly by sequences contained within ⁇ I N _ 2 .
  • Fas and Fas-L are shown to be SAATS dependent, but only Fas appears to utilize ankyrin.
  • Jurkat T-lymphocytes were transfected with the constructs indicated. Experiments were as before. Note the changes in both Fas and Fas-L due to ⁇ I N _ 5 and ⁇ I N _ 5>15 peptides.
  • Example 7 Selective inhibition of transport of a single protein.
  • the ankyrin binding domain sequence of ⁇ -Na,K- ATPase is highly conserved across all species of Na,K- ATPase, but is not found in any other protein (Zhang et al, 1997b). Expression of this peptide sequence alone in cells ( Figure 10) selectively blocks the transport of Na,K- ATPase, without blocking the transport of any other known protein.
  • wild-type MDCK cells were transiently transfected with green fluorescent protein linked to the 25 residue MAB peptide. This is the same sequence whose deletion caused Na,K- ATPase to be retained in the ER and ultimately lysosomes in Figure 9. After approximately 1-2 days in culture, cells were fixed and stained for GFP (using anti-GFP antibodies, left panel) or for Na,K- ATPase (right panel, red). Note that the expression of GFP alone in MDCK cells (wt) did not affect Na,K- ATPase distribution, or cell size (which is dependent on Na,K- ATPase function. However, when the GFP carried the 25 residue MAB peptide, the binding of the endogenous
  • Example 8 The cystic fibrosis transmembrane conductance regulator participates in SAATS transport.
  • Figure 12 illustrates another integral membrane protein of significant medical interest that is linked to SAATS by different ankyrin interaction.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • TNR Transmembrane-Nucleotide binding -Regulatory domain
  • CFTR and TNR are highly polarized, and unlike Na,K- ATPase, are expressed predominately in the apical domain of these polarized epithelial cells.
  • B Despite the fact that CFTR and TNR are apical proteins, they nevertheless binds components of the SAATS system, which is responsible for their trafficking. Either whole cell lysates (L) of cells extracted with RIP A buffer, or Fx 1 and Fx 2, were immunoprecipitated with irrelevant antibody control (C) or with anti-CFTR antibody (IP).
  • the washed immunoprecipitates were then analyzed by SDS-PAGE and examined by western blotting for either ANKQ,,, or ANK R .
  • both ankyrins co-immunoprecipitate with CFTR, in a fashion exactly analogous to the way Na,K- ATPase binds SAATS.
  • CFTR and Na,K- ATPase bind at distinct sites on SAATS, enabling their selective modulation.
  • Figure 22 illustrates the modulation of surface display of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by SAATS.
  • MDCK cells were transfected with either the ⁇ I N _ 5 or ⁇ I N-5jl5 constructs and then surface labeled with biotin. After labeling, cells were solubilized, and the total CFTR in each culture immunoprecipitated with anti-CFTR antibodies. The precipitates were then analyzed by SDS-PAGE, transferred to nitrocellulose, and surface CFTR detected by avidin labeling. Total CFTR was also determined in these cellular extracts by western blotting (data not shown). In all cell lines, there was minimal change in the total CFTR in the cells.
  • Example 9 Selective enhancement of integral membrane protein display by SAATS The rate of exit of integral membrane and secretory proteins from the ER and
  • Golgi appears to be at least partially kinetically regulated by their rate and efficiency of docking to SAATS. Therefore, upregulation of the surface display or secretion of a given protein, as well as its blocking, can be achieved by modulation of SAATS.
  • This is illustrated in Figures 13 to 16, where it can be noted that for those molecules utilizing ankyrin as an adapter complex, supranormal levels are achieved by augmenting their transport with the ⁇ IN-5,15 peptide, which increased ankyrin dependent transport at the expense of ankyrin independent transport ⁇ e.g., the fall in TNFR-1 with ⁇ I N . 5)15 expression, Figure 16).
  • Example 10 Determination of which vesicular skeletal proteins and associate adapter proteins are specifically involved in mediating sequestration of a given cell membrane or secretory proteins into transport vesicles.
  • the general method described herein is based on the discovery that the delivery of a given membrane protein or class of membrane proteins to the plasma membrane of eukaryotic cells may be inhibited or augmented by altering the binding of such proteins to the spectrin/ankyrin/adapter protein trafficking system (SAATS).
  • SAATS spectrin/ankyrin/adapter protein trafficking system
  • the secretion of proteins that bind to so-called cargo receptors in the endoplasmic reticulum may also be controlled by controlling the binding of such cargo receptor proteins to SAATS.
  • SAATS is a cryptic protein sorting apparatus operating to concentrate specific proteins in the endoplasmic reticulum (ER), and to facilitate their inclusion into vesicles undergoing transport or diffusion between the ER and Golgi compartments.
  • ER endoplasmic reticulum
  • a similar pathway is also active between the Golgi and the plasma membrane of many cells, but the most general use of this method of control is achieved by blocking SAATS at the ER to cis Golgi transition.
  • the method is implemented by 1) identifying to which component of the SAATS system a given integral membrane protein binds, discussed as follows; and 2) identifying a peptide, small molecule analog, antibody, or antisense oligonucleotide that will disrupt in vivo the binding interaction between the target protein and SAATS. This latter aspect of the present invention is discussed in the next section of this specification.
  • Typical of such assays would be equilibrium dialysis; fluorescent based assays in which the change in intrinsic fluorescence of a protein is monitored as a function of binding; assays based on osmolarity or light scattering; and others commonly known to practitioners of the art.
  • surface plasmon resonance such as implemented in Pharmacia's BiaCore instrument, is also well suited to detecting specific macromolecular interactions withing SAATS, often even in unpurified solutions prepared from cell or bacterial lysates.
  • radioactive labeled compounds or other sensitive detection techniques such as enhanced chemiluminescence in conjunction with standard microtiter or blot overlay techniques.
  • Genetic selection assays are also useful, such as two-hybrid assays in yeast, or phage epitope display library screening.
  • Example 11 Identification of peptides and small molecules that modulate sequestration and/or vesicle transport of integral membrane and secretory proteins
  • a target protein able to block (or augment) the interaction between a target protein and SAATS.
  • this method may be approached by either: A) identifying broad inhibitors of SAATS function that disrupt a class or multiple classes of membrane or secretary proteins, such as peptide agents or expressed recombinant genes that encode specific functional domains in SAATS, and that block the docking of classes of membrane proteins by competitive inhibition with the endogenous SAATS system; or B) following a drug design strategy that can be either rational, with inhibitory agents determined based on detailed knowledge of binding sites mediating the attachment of the target protein to SAATS, or random, based on in vitro swxogate assays of SAATS function.
  • Step Bl Determine whether protein X when mature is monomeric or homopolymeric, or whether it exists in its mature state as a heterocomplex with another membrane protein. This step may not be necessary if protein X can be shown to bind SAATS directly, but many membrane and all secretary proteins will only attach to SAATS via their interaction with another membrane protein. If this is the case, either agents must be identified that will prevent the formation of the heterocomplex, or the complex will be controlled as a unit ⁇ i.e., the display of all components of the functional heterocomplex ) will be controlled together by controlling their SAATS assembly. Standard protein biochemical and biophysical techniques can be used to make these determinations, including co-immunoprecipitation from non-ionic detergent extracts of cultured cells expressing protein X.
  • the demonstration of direct binding of ⁇ -Na,K- ATPase (as opposed to ⁇ -Na,K- ATPase) to ankyrin can be demonstrated by a variety of in vitro and in vivo techniques, including co-sedimentation, gel overlay binding assays, and surface plasmon resonance. In these assays ⁇ e.g. see Morrow et al. 1989), direct binding of ankyrin to the ⁇ -Na,K- ATPase, but not ⁇ -Na,K- ATPase can be demonstrated.
  • Step B2 Determination of the specific binding site within protein X that links it to SAATS.
  • Many conventional methods may be used to accomplish this.
  • a common one is the use of deletional mutagenesis using a series of recombinant peptides, often generated as fusion peptides linked to glutathione-S-transferase (GST).
  • GST glutathione-S-transferase
  • GST-fusion peptides representing the cytoplasmic domains of Na,K-ATPase (Devarajan et al, 1994a; Devarajan et al, 1994b).
  • Step B3 Use of conventional structure determination methods to resolve the 3-D structure of the binding domain. If a complete structure determination of protein X is not possible, a useful method for determining the relevant structure of the binding site is carrier mediated crystallization, in which the binding domain of interest is crystallized as a fusion protein with GST (for example, see Lim et ⁇ /.,1994). Subsequent determination of the structure of this complex yields the structure of the key binding site. Other structural determination methods, such as multidimensional NMR, are of course applicable as well.
  • the determination of the 3-D structure of the minimal ankyrin binding domain in ⁇ -Na,K- ATPase by crystallization of the active GST-fusion peptide would be useful, as we have recently demonstrated (Zhang, et al, 1997b). (See Figs. 4B, 4C, and 11).
  • Step B4 Use of the structure determined above to design candidate small molecules that will inhibit the binding interaction and assay of these compounds by their ability to block the in vitro interaction between the active site of protein X and its SAATS binding site.
  • the goal is to identify small molecules that augment a given SAATS interaction, so as to identify a therapeutic that enhances the display or transport or secretion of a given protein (such as would be desirable in the treatment children with cystic fibrosis, Figure 12B), molecules that augment in vitro the interaction between protein X and SAATS would be sought.
  • Step B3 alternate: Alternatively, an empirical approach identifying lead compounds based on a surrogate measures of SAATS function is possible. Since all or most proteins exit the ER compartment via a direct or indirect binding to SAATS, in vitro assays based on specific binding sites for each membrane or secretory protein of interest provide an ideal basis for high-throughput in vitro screens of SAATS function. For example, high-throughput screens in microtiter plates, seeking to identify all agents that block or diminish the binding of spectrin to AnkGl 19, or AnkGl 19 to protein X, would be one such suitable assay. Alternatively, one could prepare similar assays in which protein X or ankyrin or other SAATS component was immobilized onto an inert substrate (eg.
  • Example 12 Strategies based on regulation of spectrin binding to the Golgi complex
  • a candidate compound to modulate interaction of SAATS with selected integral membrane or secretory proteins such a compound is mixed with the selected protein and whatever vesicular skeletal protein or adapter protein is relevant. After mixing under conditions that allow association of these components, the mixture is analyzed to determine if the agent inhibited or enhanced binding of the selected protein to its SAATS binding partner.
  • Inhibitors and enhancers are thus confirmed as being able to modulate this interaction.
  • a skilled artisan can readily employ numerous art-known techniques for determining whether a particular agent modulates the binding of such selected proteins to their SAATS binding partner. Agents can be further tested for the ability to modulate binding using a cell-free assay system or a cellular assay system.
  • Example 14 provides one such method that can be used to assay for relevant activity.
  • an agent is said to inhibit SAATS binding activity when the agent reduces the binding of a selected integral membrane or secretory protein to its SAATS binding partner.
  • the preferred inhibitor will selectively inhibit such binding specifically, not affecting the interaction of any other proteins to SAATS. Further, the preferred inhibitor will reduce such binding by more than about 50%, more preferably by more than about 90%, most preferably eliminating substantially all binding of the selected protein to SAATS.
  • an agent is said to enhance SAATS binding activity when the agent increases the binding of a selected integral membrane protein to SAATS.
  • the preferred binding enhancer will increase SAATS binding activity by more than about 50%), more preferably by more than about 90%o, most preferably more than doubling the level of binding or transport of such proteins to SAATS.
  • the preferred inhibitors and enhancers will be selective for a specific species of integral membrane or secretory protein.
  • the agents of the present invention can be, as examples, peptides, small molecules, and vitamin derivatives, as well as carbohydrates. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.
  • One class of agents of the present invention are peptide agents whose amino acid sequences are chosen based on the amino acid sequence of various spectrin homologues. Small peptide agents can serve as competitive inhibitors of SAATS binding, sequestration and transport.
  • the peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art.
  • the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included.
  • agents of the present invention are antibodies immunoreactive with critical epitopes of vesicular skeletal proteins such as spectrin and associated adapter proteins such as ankyrin.
  • Antibodies are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of, e.g., spectrin, which binds the selected integral membrane or secretory.
  • Critical regions for the transport of Na,K- ATPase include the domains identified in Figure 3 or the cytoplasmic 29 residues of VSV-G.
  • Such agents can be used in competitive binding studies to identify second generation inhibitory agents.
  • Example 13 Use of agents that modulate the sequestration and/or vesicular transport of integral membrane and secretory proteins for therapeutic purposes
  • Cardiovascular Hypertension, Cholesterol and hyperhpidemia Modification of membrane channels, conditions, an ti -arrhythmic agents e g Ca channel thrombin receptors, endothelial adhesion antagonists), Thrombosis control, anti-platelet agents, receptors endothelial surface modifying agents, restenosis control Degenerative Disorders Osteoporosis, BMP regulation, calcitomn/PTH balance regulation, calcium homeostasis Dermatologic/ cosmetic Pso ⁇ asis, scleroderma, cutaneous hypersensitivity, Topical application may offer early poison ivy, therapeutic advantage Genetic Disease Cystic fibrosis (eg D508 mutation reduces transport of Enhanced binding to SAATS may
  • an ti -arrhythmic agents e g Ca channel thrombin receptors, endothelial adhesion antagonists
  • Thrombosis control e g Ca channel thrombin receptors, endothelial adhesion antagonists
  • Axonal transport modulators also Transplantation Epitope modulation, HL-A antigen display in grafts antigen presentation control, epitope
  • Diabetes autoimmiune control Type I Insulin receptor Glucose transporter and Insultin receptor modulation, glucagon regulation control Reproductive biology Sperm/ovum receptors, contraception, infertility, maternal/fetal immune disorders Endocrine Disorders Goiter, hyper hypo thyroidism, growth hormone and receptors, pituitary disorders
  • agents identified by the foregoing techniques will be dependent on their intended purpose, i.e., to enhance or inhibit the transport of a selected integral membrane protein or secretory protein, and on their chemical nature, i.e., peptide or small molecule.
  • agents that selectively increase the delivery of mutant (but functional) CFTR to the plasma membrane would allow the identification of agents that selectively increase the delivery of mutant (but functional) CFTR to the plasma membrane.
  • Appropriate agents might optimally be delivered by inhalation therapy (see U.S. Patents 5,669,376 and 5,655,516).
  • an appropriate agent can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • compositions of the present invention may contain other ingredients, such as suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action.
  • suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action.
  • suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble variants, for example, water-soluble salts.
  • suspensions of the active compounds, and as appropriate, oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension and include, for example, sodium carboxym ethyl cellulose, sorbitol, and/or dintran.
  • the suspension may also contain stabilizers.
  • Liposomes can also be used to encapsulate the agent for delivery into the cell.
  • the pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient.
  • Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release variants thereof.
  • agents of the present invention that modulate sequestration and trafficking of selected integral membrane and secretory proteins can be provided alone, or in combination with another appropriate pharmacoactive.
  • an agent of the present invention that reduces the presence of various cell surface receptors involved in the attachment of viral envelopes might be administered in combination with other anti-viral agents.
  • two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.
  • Example 14 Identification of the spectrin or adaptor protein domain(s) responsible to binding and transport of an integral membrane or secretory protein.
  • ⁇ l ⁇ spectrin contains a series of functional domains that have been shown to confer or may confer the ability to bind specific adaptor proteins and/or proteins to be transported.
  • ankyrin contains a number of repeats (13 to 24 repeat units characteristic of most ankyrins), with each ankyrin-repeat structure composed of two alpha helices and a ⁇ -hairpin loop.
  • the complexity of both the individual repeats and the number of repeats in the structure of both structural proteins such as spectrins and adaptor proteins such as ankyrin may allow for the varied ability of both molecules to bind to multiple distinct integral membrane or secretory proteins.
  • multiple repeat units create a structure in which interactions between the helices form a central core structure, while the tips of the exposed ⁇ -hairpin turns provide putative protein-protein interaction surfaces (See Figure 21).
  • the seven residue loop within MAB of ⁇ -Na,K- ATPase interacts with a specific site in ankyrin created by the tips of one or more of these ⁇ -hairpin turns. Since a multiplicity of potential binding pockets would be created by the 13 to 24 repeat units characteristic of most ankyrins, specific and unique binding sites presumably also exist for the other short peptide sequences responsible for ankyrin binding activity in other proteins.
  • adaptor proteins to bind to spectrins or integral membrane or secretory proteins to bind directly to spectrins can be assayed to identify the spectrin domain, or combinations of domains responsible for specific binding of a given adaptor protein, adaptor protein and integral membrane or secretory protein or integral membrane or secretory protein directly.
  • an adaptor protein such as ankyrin
  • ankyrin an adaptor protein such as ankyrin
  • Identification of the specific domains responsible for binding then allows for the development of peptide based molecules, or mimetopes with similar structures, to modulate or disrupt specific binding of the many protein-protein interactions involved in SAATS.
  • deletional analysis of the ankyrin repeat region is used to produce a series of fragments of the protein.
  • the fragments are then tested for their ability to bind to the integral membrane protein of interest.
  • Any means commonly available of detecting the ability of the ankyrin fragment to bind to the specific integral membrane protein can be used.
  • cell free systems can be used wherein the binding is detected by the ability of the ankyrin fragment, detected by radiolabelling, fluorescent tag, or specific antibody, to be co-immunoprecipitated by antibodies to the protein to which binding is suspected.
  • the ability of the ankyrin fragment to co-sediment with permeabilized cells or membrane preparations enriched in the proteins of interest is another method of detecting binding.
  • Other methods include binding to immobilized ligands; co-sedimentation, co-elution on gel filtration chromatography; surface plasmon resonance, fluorescent energy transfer; osmolarity; light-scattering; resonance raman spectroscopy, NMR spectroscopy, yeast two-hybrid systems; expression cloning, and phage display.
  • Other methods will also be known to the practitioner of the art.
  • ankyrin repeat units if that is the target of the assay, are often insoluble or unstable, it will often be of value to examine smaller internal sequences, such as those from the beta-hairpin loops of the ankyrin repeat structure, for binding activity. .
  • High through-put assays can also be developed to detect binding the a library of ankyrin fragments comprising all individual repeat units plus all possible combinations of the repeat units.
  • Examples of such assays would include incorporating single, two-mer, and trimer combinations of the beta-hairpin loop sequences of all known ankyrin repeat units into pseudo-libraries suitable for screening in the yeast two-hybrid or CHO two-hybrid KISS assays (Fearon et al.,1992) against the ligand of interest. Similar strategies could also be employed utilizing the same peptide sequences, generated either recombinantly or by synthetic methods, immobilized on nitrocellulose or immobilon or similar membranes, or on plastic multiwell plates, or with the sequences incorporated into bacteriophage so as to create a phage display library suitable for binding to the protein of interest. (Scott, 1992)
  • T-lymphoma transmembrane glycoprotem (gpl 80) is linked to the cytoskeletal protem, fod ⁇ n. J Cell Biol 101 :477-87.
  • Cianci C. D., Z. Zhang and J. S. Morrow 1997. Brain and muscle express a unique alternative transcript of all spectrin. submitted Cohen, C. M. and S. F. Foley. 1980. Spect ⁇ n-dependent and -mdependent association of F-actin with the erythrocyte membrane. Journal of Cell Biology 86:694-8.
  • KISS Karyoplasmic interaction selection strategy
  • Plectm and IFAP-300K are homologous proteins bmdmg to microtubule-associated proteins 1 and 2 and to the 240-k ⁇ lodalton subunit of spectrm. J Biol Chem 262:1320-1325.
  • Hyvonen M., M. J. Macias, M. Nilges, H. Oschkmat, M. Saraste and M. Wilmanns. 1995. Structure of the binding site for inositol phosphates in a PH domain. Embo J 14:4676-85.
  • the lymphoma transmembrane glycoprotem GP85 (CD44) is a novel guanme nucleofade-bindmg protem which regulates GP85 (CD44)-anky ⁇ n mteracfaon. J Biol Chem 267:22073-8.
  • the cGMP-gated cation channel of bovine rod photoreceptor cells is associated with a 240-kDa protein exhibiting immunochemical cross-reactivity with spectrm J Biol Chem 265:18690-18695.
  • N-CAM neural cell adhesion molecule
  • Beta spectrm m human skeletal muscle Tissue-specific differenfaal processmg of 3' beta spect ⁇ n pre-mRNA generates a beta spectrin isoform with a unique carboxyl terminus. J Biol Chem 265:20449-54.

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Abstract

Procédé et compositions correspondantes modulant la présence sur une membrane de surface cellulaire de protéines de membrane entières sélectionnées ou la sécrétion de protéines sécrétrices sélectionnées, ce qui consiste à augmenter ou à diminuer le transport intracellulaire de la protéine depuis le réticulum endoplasmique vers et à travers l'appareil de cis-Golgi. Des procédés associés permettent d'identifier si le transport intracellulaire d'une protéine spécifique a pour médiateur le système de trafic de protéines d'adaptation de spectrine et ankyrine (SAATS). Des procédés correspondants servent à déterminer si un composé candidat inhibe ou augmente le transport intracellulaire d'une protéine sélectionnée depuis le réticulum endoplasmique vers l'appareil de cis-Golgi par SAATS. Des agents et des procédés peuvent être mis en application dans une variété de domaines, y compris en tant qu'immunorégulateurs, inhibiteurs de transport d'ions, modulateurs vasculaires et agents chimiothérapeutiques contre le cancer.
PCT/US1998/020364 1997-09-30 1998-09-30 Procede servant a effectuer le controle selectif de la presence d'une proteine de membrane et de la secretion de proteines dans des cellules eucaryotes WO1999016875A1 (fr)

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AU10635/99A AU750395B2 (en) 1997-09-30 1998-09-30 A method for selectively controlling membrane protein display and protein secretion in eukaryotic cells
JP2000513945A JP2001518295A (ja) 1997-09-30 1998-09-30 真核細胞において膜タンパク質ディスプレイおよびタンパク質分泌を選択的に制御する方法
EP98953207A EP1021539A4 (fr) 1997-09-30 1998-09-30 Procede servant a effectuer le controle selectif de la presence d'une proteine de membrane et de la secretion de proteines dans des cellules eucaryotes
CA002304484A CA2304484A1 (fr) 1997-09-30 1998-09-30 Procede servant a effectuer le controle selectif de la presence d'une proteine de membrane et de la secretion de proteines dans des cellules eucaryotes

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002002610A2 (fr) * 2000-06-29 2002-01-10 Incyte Genomics, Inc. Molecules de circulation et de secretion
WO2003020925A1 (fr) * 2001-08-31 2003-03-13 University Of Leeds Methodes visant a augmenter la productivite de l'expression de genes heterologues
US8788213B2 (en) 2009-01-12 2014-07-22 Intrexon Corporation Laser mediated sectioning and transfer of cell colonies

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US7425426B2 (en) * 2004-03-15 2008-09-16 Cyntellect, Inc. Methods for purification of cells based on product secretion

Non-Patent Citations (5)

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Title
BECK K. A., ET AL.: "GOLGI SPECTRIN: IDENTIFICATION OF AN ERYTHROID BETA-SPECTRIN HOMOLOG ASSOCIATED WITH THE GOLGI COMPLEX.", THE JOURNAL OF CELL BIOLOGY : JCB, THE ROCKEFELLER UNIVERSITY PRESS, US, vol. 127., no. 03., 1 November 1994 (1994-11-01), US, pages 707 - 723., XP002916080, ISSN: 0021-9525, DOI: 10.1083/jcb.127.3.707 *
BECKK. A., NELSON W. J.: "THE SPECTRIN-BASED MEMBRANE SKELETON AS A MEMBRANE PROTEIN-SORTING MACHINE.", AMERICAN JOURNAL OF PHYSIOLOGY. CELL PHYSIOLOGY., AMERICAN PHYSIOLOGICAL SOCIETY., US, vol. 270., no. 05., 1 May 1996 (1996-05-01), US, pages C1263 - C1270., XP002916079, ISSN: 0363-6143 *
DEVARAJAN P, ET AL.: "NA,K-ATPASE TRANSPORT FROM ENDOPLASMIC RETICULUM TO GOLGI REQUIRES THE GOLGI SPECTRIN-ANKYRIN G119 SKELETON IN MADIN DARBY CANINE KIDNEY CELLS", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 94, no. 20, 1 September 1997 (1997-09-01), US, pages 10711 - 10716, XP002916078, ISSN: 0027-8424, DOI: 10.1073/pnas.94.20.10711 *
DEVARAJAN P., ET AL.: "IDENTIFICATION OF A SMALL CYTOPLASMIC ANKYRIN (ANKG119) IN THE KIDNEY AND MUSCLE THAT BINDS BETAISIGMA SPECTRIN AND ASSOCIATES WITH THE GOLGI APPARATUS.", THE JOURNAL OF CELL BIOLOGY : JCB, THE ROCKEFELLER UNIVERSITY PRESS, US, vol. 133., no. 04., 1 May 1996 (1996-05-01), US, pages 819 - 830., XP002916081, ISSN: 0021-9525, DOI: 10.1083/jcb.133.4.819 *
See also references of EP1021539A4 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002002610A2 (fr) * 2000-06-29 2002-01-10 Incyte Genomics, Inc. Molecules de circulation et de secretion
WO2002002610A3 (fr) * 2000-06-29 2002-09-19 Incyte Genomics Inc Molecules de circulation et de secretion
WO2003020925A1 (fr) * 2001-08-31 2003-03-13 University Of Leeds Methodes visant a augmenter la productivite de l'expression de genes heterologues
US8788213B2 (en) 2009-01-12 2014-07-22 Intrexon Corporation Laser mediated sectioning and transfer of cell colonies

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