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Ldl receptor signaling pathwaysRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidLdl receptor signaling pathways description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060211039, Ldl receptor signaling pathways. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is a divisional application of and claims priority under 35 U.S.C..sctn.120 to Ser. No. 10/211,962 filed Aug. 01, 2002, having the same title and inventors, now U.S. Pat. No. 7,056,688; which claims priority to Ser. No. 09/562,737, filed May 01, 2000, having the same title and inventors, now U.S. Pat. No. 6,428,967; which are incorporated herein by reference. INTRODUCTION [0003] 1. Field of the Invention [0004] The field of the invention is methods for inducing and detecting LDL receptor signaling. [0005] 2. Background of the Invention [0006] The members of the low density lipoprotein (LDL) receptor gene family bind a broad spectrum of extracellular ligands. Traditionally, they had been regarded as mere cargo receptors that promote the endocytosis and lysosomal delivery of these ligands. However, recent genetic experiments in mice have revealed critical functions for LDL receptor family members in the transmission of extracellular signals and the activation of intracellular tyrosine kinases. This process regulates neuronal migration and is crucial for brain development. Signaling through these receptors requires the interaction of their cytoplasmic tails with the intracellular adaptor proteins Disabled-1 (Dab1) and FE65 (2,3). Here, we disclose an extended set of cytoplasmic polypeptides that can participate in signal transmission by the LDL receptor gene family. Most of these novel polypeptides are adaptor or scaffold polypeptides that contain PID or PDZ domains and function in the regulation of cellular kinases, including tyrosine kinases, serine/threonine kinases (e.g. microtubule associated protein (MAP) kinases) and lipid kinases (e.g. PI kinases), cell adhesion, vesicle trafficking, or neurotransmission. We also show that binding of Dab1 competes with receptor internalization indicating a mechanism by which signaling through this class of receptors might be regulated. SUMMARY OF THE INVENTION [0007] The invention provides methods and compositions for detecting and modulating, including inducing and suppressing, signal transduction through LDL receptors. [0008] In a particular embodiment, the methods involve specifically detecting a stress that alters a functional interaction of an LDL receptor binding polypeptide with an LDL receptor interaction domain, the method comprising steps (a) introducing a predetermined stress into a system which provides a physical interaction of an LDL receptor binding polypeptide with an LDL receptor intracellular binding polypeptide interaction domain, whereby the system provides a stress-biased interaction of the binding polypeptide and the interaction domain, wherein the absence of the stress, the system provides an unbiased interaction of the binding polypeptide and the interaction domain; and (b) detecting the stress-biased interaction of the binding polypeptide and the interaction domain, wherein a difference between the stress-biased and unbiased interactions indicates that the stress alters the interaction of the binding polypeptide and the interaction domain. The binding polypeptide is independently selected from SEMCAP-1, JIP-1, PSD-95, JIP-2, Talin, OMP25, CAPON, PIP4,5 kinase, Na channel brain 3, Mint1, ICAP-1 and APC subunit10; and the receptor may be selected from very low density lipoprotein receptor (VLDLR), apolipoprotein E receptor-2 (ApoER2), low density lipoprotein receptor (LDLR), low density lipoprotein receptor related protein (LRP), MEGF7 and Megalin. In particular embodiments, the binding polypeptides and receptors are of natural sequence, preferably human sequence. In particular embodiments, the system is a cell expressing both the binding polypeptide and the interaction domain or an in vitro, cell-free mixture comprising a determined amount of the binding polypeptide and the interaction domain; exemplary systems include two-hybrid, biochemical pull-down, fluorescent polarization and solid phase binding assays. [0009] The compositions of the invention, useful in the subject methods, include the subject binding polypeptides and mixtures consisting essentially of an LDL receptor binding polypeptide and an LDL receptor interaction domain, wherein the receptor may be independently selected from VLDLR, ApoER2, LDLR, LRP, MEGF7 and Megalin. Other aspects of the invention include nucleic acids encoding the disclosed binding polypeptides, antibodies which specifically bind them, and methods of use. DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION [0010] The invention provides methods and compositions for inducing and detecting signal transduction through LDL receptors. In a particular embodiment, the methods involve specifically detecting a stress that alters a functional interaction of a low density lipoprotein (LDL) receptor binding polypeptide with an LDL receptor interaction domain. [0011] The binding polypeptide is independently selected from SEMCAP-1, JIP-1, PSD-95, JIP-2, Talin, OMP25, CAPON, PIP4,5 kinase, Na channel brain 3, Mint1, ICAP-1 and APC subunit 10. These names are used generically to refer to polypeptides which comprise, or have sequence similarity to, the corresponding disclosed parental sequences, wherein the sequence similarity is at least 50%, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, most preferably 100%, and specifically bind a specifically disclosed LDL receptor cytoplasmic domain (tail), as measured in one or more of the disclosed interaction assays. The polypeptides comprise, and the similarity or identity extends over at least 10, preferably at least 15, more preferably at least 25, more preferably at least 35, more preferably at least 50 contiguous residues and most preferably over the entire polypeptide sequence. [0012] For disclosed polymeric genuses, "percent (%) sequence identity over a specified window size W" with respect to parental sequences is defined as the percentage of residues in any window of W residues in the candidate sequence that are identical with the residues in the parent sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. The % identity values are generated by WU-BLAST-2.0 a19 obtained from Altschul et al., J. Mol. Biol., 215: 403-410(1990); http://blast.wustl.edu/blast/README.html. WU-BLAST-2.0a19 which uses several search parameters, all of which are set to the default values. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. Hence, a % sequence identity value is determined by the number of matching identical residues divided by the window size W for which the percent identity is reported. Exemplary species are readily generated by mutating the corresponding parental sequences and confirming LDL receptor interaction domain binding. For example, SEMCAP1 polypeptides defined by SEQ ID NOS:2-10 exemplify an active (demonstrating LDL receptor interaction domain binding) 90% genus around parental sequence SEQ ID NO: 1 (see table 1). TABLE-US-00001 TABLE 1 LDL Receptor Binding Polypeptides Parental Active 90% Name Similar Genbank Accession Nos. Sequence Species SEMCAP-1 gb|AF104358.1|AF104358 (m) SEQ ID SEQ ID gb|AF089817.1|AF089817 (r) NO: 1 NOS: 1-10 gb|AF089816.1|AF089816 (h) JIP-1 gb|AF054611.1|AF054611 (m) SEQ ID SEQ ID gb|AF109772.1|AF109772 (r) NO: 11 NOS: 11-20 gb|AF074091.1|AF074091(h) gb|AF003115.1|MMAF003115 (isoforms/splice variants) PSD-95 dbj|D50621.1|MUSPSD95SP (m) SEQ ID SEQ ID gb|M96853.1|RATPSD95A (r) NO: 21 NOS: 21-30 gb|U83192.1|HSU83192 (h) JIP-2 gb|AF218778.1|AF218778 (h) SEQ ID SEQ ID emb|AL021708.1|HSU56K21 (isoforms/splice NO: 31 NOS: 31-40 variants) Talin gb|AF177198.1|AF177198 (h ortholog) SEQ ID SEQ ID NO: 41 NOS: 41-50 OMP25 gb|AF107295.1|AF107295 (r) SEQ ID SEQ ID NO: 51 NOS: 51-60 CAPON gb|AF037071.1|AF037071(r) SEQ ID SEQ ID dbj|AB007933.2|AB007933 (partial h) NO: 61 NOS: 61-70 PIP4, 5 EST gb|AA544527.1|AA544527 (m) SEQ ID SEQ ID kinase NO: 71 NOS: 71-80 Na channel gb|L42341.1|MUSSOCHA (m, homolog) SEQ ID SEQ ID brain 3 ref|NM_013119.1|| (r, homolog) NO: 81 NOS: 81-90 dbj|AB037777.1|AB037777 (partial h, homolog) Mint1 gb|L34676.1|MUSX11P (m) SEQ ID SEQ ID gb|AF029107.1|AF029107 (r) NO: 91 NOS: 91-100 ref|NM_005503.1|| (h) ICAP-1 ref|NM_008403.1|| (m) SEQ ID SEQ ID ref|NM_004763.1|| (h) NO: 101 NOS: 101-110 APC dbj|AB012109.1|AB012109 (h) SEQ ID SEQ ID subunit10 NO: 111 NOS: 111-120 [0013] The LDL receptor interaction domain comprises or has sequence similarity to a natural LDL receptor cytoplasmic tail portion, wherein the sequence similarity is at least 50%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95% and most preferably 100%, wherein the domain is sufficient to provide a specific binding target for the binding polypeptide, comparable to that provided by the corresponding cytoplasmic tail portion of the receptor, as measured in one or more of the disclosed interaction assays. The domains comprise, and the similarity or identity extends over at least 10, preferably at least 15, more preferably at least 25, more preferably at least 35, more preferably at least 50 contiguous residues and most preferably over the entire domain sequence. The receptor is preferably selected from a natural very low density lipoprotein receptor (VLDLR, such as human, mouse and chicken sequences), apolipoprotein E receptor-2 (ApoER2, such as human and mouse sequences), low density lipoprotein receptor (LDLR, such as human, mouse, rat, xenopus, hamster, rabbit, bovine and pig sequences), low density lipoprotein receptor related protein (LRP, such as human, mouse and chicken sequences), MEGF7 (such as human and rat sequences) and Megalin (such as human, rat, C. elegans and Drosophila sequences), which are known in the art and accessible from public genetic depositories such as Genbank. Unless noted otherwise by context, the term LDL receptor, as used herein, is used generically to refer to members of the LDL receptor gene family. [0014] In one embodiment, the method comprises the steps of (a) introducing a predetermined stress into a system which provides a physical interaction of an LDL receptor binding polypeptide with an LDL receptor intracellular binding polypeptide interaction domain, whereby the system provides a stress-biased interaction of the binding polypeptide and the interaction domain, wherein the absence of the stress, the system provides an unbiased interaction of the binding polypeptide and the interaction domain; and (b) detecting the stress-biased interaction of the binding polypeptide and the interaction domain, wherein a difference between the stress-biased and unbiased interactions indicates that the stress alters the interaction of the binding polypeptide and the interaction domain. [0015] A wide variety of systems may be used in the methods. For example, in particular embodiments, the system is a cell or animal expressing both the binding polypeptide and the interaction domain or an in vitro, cell-free mixture comprising a determined amount of the binding polypeptide and the interaction domain; exemplary systems include two-hybrid, biochemical pull-down, fluorescent polarization and solid phase binding assays. Similarly, a wide variety of stresses may be assayed or evaluated, including chemical agents, such as candidate drugs, toxins, contaminants, etc.; radiation such as ultraviolet rays and x-rays; infection such as viral or bacterial infection including cellular transformation, etc. [0016] The particular method used to detect the interaction of the binding polypeptide and the interaction domain will depend on the nature of the assay, so long as the interaction is specifically detected. For example, depending on if and how the binding polypeptide and/or the interaction domains are labeled, the interaction readout may be measured by changes in fluorescence, optical density, gel shifts, radiation, etc. In a particular embodiment, the system provides a tau phosphorylation readout for the binding of the LDL receptor binding polypeptide and the LDL receptor interaction domain. [0017] The compositions of the invention, useful in the subject methods, include the subject binding polypeptides and mixtures comprising predetermined amounts of a disclosed low density lipoprotein (LDL) receptor binding polypeptide and a disclosed LDL receptor interaction domain, particularly wherein one, preferably both of these components are isolated and mixtures consisting essentially of both components, i.e. wherein other components of the mixture (except for an assayed stress) do not significantly influence the interaction of these two components. Other aspects of the invention include nucleic acids encoding the disclosed binding polypeptides, antibodies which specifically bind them, and methods of use. [0018] Subject polypeptides consisting of the disclosed parental sequences or fragments thereof are isolated, i.e. unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, preferably at least about 5%, more preferably at least about 50% by weight of total polypeptide present in a given sample, and a pure polypeptide constitutes at least about 90%, and preferably at least about 99% by weight of the total polypeptide in a given sample, as are preferred subject polypeptides comprising other than parental sequence. In addition the subject polypeptides consisting of the disclosed parental sequences or fragments thereof have corresponding-polypeptide-specific antibody binding, elicitation or binding or elicitation inhibitory activity, e.g. elicit specific antibody in a heterologous host, etc. In a particular embodiment, the subject polypeptide fragments provide specific antigens and/or immunogens, especially when coupled to carrier proteins. For example, peptides are covalently coupled to keyhole limpet antigen (KLH) and the conjugate is emulsified in Freunds complete adjuvant. Laboratory rabbits are immunized according to conventional protocol and bled. The presence of specific antibodies is assayed by solid phase immunosorbant assays using immobilized corresponding polypeptide, see, e.g. Table 2. TABLE-US-00002 TABLE 2 Immunogenic polypeptides eliciting specific rabbit polyclonal antibody: Polypeptide-KLH conjugates immunized per protocol described above. Polypeptide Sequence Immunogenicity SEQ ID NO: 71, residues 7-16 +++ SEQ ID NO: 71, residues 16-25 +++ SEQ ID NO: 71, residues 23-31 +++ SEQ ID NO: 71, residues 29-38 +++ SEQ ID NO: 71, residues 39-46 +++ SEQ ID NO: 71, residues 44-51 +++ SEQ ID NO: 71, residues 51-60 +++ SEQ ID NO: 71, residues 55-62 +++ SEQ ID NO: 71, residues 58-66 +++ SEQ ID NO: 71, residues 69-78 +++ SEQ ID NO: 71, residues 78-88 +++ SEQ ID NO: 71, residues 87-96 +++ SEQ ID NO: 71, residues 99-108 +++ SEQ ID NO: 71, residues 104-114 +++ SEQ ID NO: 71, residues 116-128 +++ SEQ ID NO: 71, residues 130-138 +++ SEQ ID NO: 71, residues 141-149 +++ SEQ ID NO: 71, residues 150-159 +++ SEQ ID NO: 71, residues 159-168 +++ [0019] In addition to direct synthesis, the subject polypeptides can also be expressed in cell and cell-free systems (e.g. Jermutus L, et al., Curr Opin Biotechnol. October 1998; 9(5):534-48) from encoding polynucleotides, such as the corresponding parent polynucleotides or naturally-encoding polynucleotides isolated with degenerate oligonucleotide primers and probes generated from the subject polypeptide sequences ("GCG" software, Genetics Computer Group, Inc, Madison Wis.) or polynucleotides optimized for selected expression systems made by back-translating the subject polypeptides according to computer algorithms (e.g. Holler et al. (1993) Gene 136, 323-328; Martin et al. (1995) Gene 154, 150-166). Hence, the polypeptides may be synthesized, produced by recombinant technology, or purified from cells. A wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY) or that are otherwise known in the art. [0020] The invention provides binding agents specific to the subject polypeptides, methods of identifying and making such agents, and their use. For example, specific binding agents are useful in a variety of diagnostic and industrial applications and include somatically recombined polypeptide receptors like specific antibodies or T-cell antigen receptors (see, e.g Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory), intracellular binding agents identified with assays such as one-, two- and three-hybrid screens, non-natural intracellular binding agents identified in screens of chemical libraries such as described below, etc. Accordingly, the invention provides complementarity determining region (CDR) sequences and libraries of such sequences. Generally, the CDR polypeptides are expressed and used as the binding domain of an immunoglobulin or fragment thereof. [0021] The invention provides polynucleotides encoding the disclosed polypeptides, which polynucleotides may be joined to other components such as labels or other polynucleotide sequences (i.e. they may be part of larger sequences) and are of synthetic/non-natural sequences and/or are isolated, i.e. unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, preferably at least about 5% by weight of total nucleic acid present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome. Recombinant polynucleotides comprising natural sequence contain such sequence at a terminus, immediately flanked by (i.e. contiguous with) a sequence other than that which it is joined to on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, more preferably fewer than 500 bases, most preferably fewer than 100 bases, which is at a terminus or is immediately flanked by a sequence other than that which it is joined to on a natural chromosome. Continue reading about Ldl receptor signaling pathways... Full patent description for Ldl receptor signaling pathways Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Ldl receptor signaling pathways patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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