Title:
LDL receptor signaling pathways
Kind Code:
A1


Abstract:
The invention provides methods and compositions for inducing and detecting signal transduction through LDL receptors, including specifically detecting a stress that alters a functional interaction of a low density lipoprotein (LDL) receptor binding polypeptide with an LDL receptor interaction domain, by (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 a 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 the binding polypeptide is 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.



Inventors:
Herz, Joachim (Dallas, TX, US)
Gotthardt, Michael (Dallas, TX, US)
Application Number:
11/443904
Publication Date:
09/21/2006
Filing Date:
05/30/2006
Assignee:
Board of Regents, The University of Texas System
Primary Class:
Other Classes:
435/6.13, 435/7.1
International Classes:
C12Q1/68; G01N33/53; G01N33/92
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Primary Examiner:
LI, RUIXIANG
Attorney, Agent or Firm:
RICHARD ARON OSMAN (530 Lawrence Expy # 332, Sunnyvale, CA, 94085, US)
Claims:
What is claimed is:

1. A method for identifying a candidate drug which may alter low density lipoprotein (LDL) receptor function by altering an interaction of the LDL receptor with a native intracellular LDL receptor binding polypeptide, the method comprising steps: providing a system which comprises the binding polypeptide and a corresponding binding polypeptide interaction domain of the LDL receptor, introducing a test compound into the system and detecting a resultant level of binding between the binding polypeptide and the interaction domain, wherein a difference in the level of binding as compared to that of the system without the test compound indicates that the compound is a candidate drug, wherein the binding polypeptide is a c-Jun NH2-terminal kinase (JNK) interacting protein (JIP) selected from JIP1 and JIP2.

2. A method according to claim 1, wherein the system is a two-hybrid assay.

3. A method according to claim 1, wherein the system is a biochemical pull-down assay.

4. A method according to claim 1, wherein the system is a high-throughput, in vitro fluorescent polarization assay.

5. A method according to claim 1, wherein the system is a high-throughput, in vitro solid-phase binding assay.

6. A method according to claim 1, wherein the system is a high-throughput, conformational sensor-solid-phase chemiluminescence assay.

7. A method according to claim 1, wherein the system is a cell expressing both the binding polypeptide and the interaction domain.

8. A method according to claim 1, wherein the system is an in vitro, cell-free mixture comprising a determined amount of the binding polypeptide and the interaction domain.

9. A method according to claim 1, wherein the LDL receptor is 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.

10. A method according to claim 2, wherein the LDL receptor is 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.

11. A method according to claim 3, wherein the LDL receptor is 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.

12. A method according to claim 4, wherein the LDL receptor is 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.

13. A method according to claim 5, wherein the LDL receptor is 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.

14. A method according to claim 6, wherein the LDL receptor is 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.

15. A method according to claim 7, wherein the LDL receptor is 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.

16. A method according to claim 8, wherein the LDL receptor is 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.

Description:

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of and claims priority under 35 U.S.C.§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.

The research carried out in the subject application was supported in part by grants from the National Institutes of Health (HL20948 and HL63762). The government may have rights in any patent issuing on this application.

INTRODUCTION

1. Field of the Invention

The field of the invention is methods for inducing and detecting LDL receptor signaling.

2. Background of the Invention

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

The invention provides methods and compositions for detecting and modulating, including inducing and suppressing, signal transduction through LDL receptors.

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.

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

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.

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.

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 1
LDL Receptor Binding Polypeptides
ParentalActive 90%
NameSimilar Genbank Accession Nos.SequenceSpecies
SEMCAP-1gb|AF104358.1|AF104358 (m)SEQ IDSEQ ID
gb|AF089817.1|AF089817 (r)NO: 1NOS: 1-10
gb|AF089816.1|AF089816 (h)
JIP-1gb|AF054611.1|AF054611 (m)SEQ IDSEQ ID
gb|AF109772.1|AF109772 (r)NO: 11NOS: 11-20
gb|AF074091.1|AF074091(h)
gb|AF003115.1|MMAF003115 (isoforms/splice
variants)
PSD-95dbj|D50621.1|MUSPSD95SP (m)SEQ IDSEQ ID
gb|M96853.1|RATPSD95A (r)NO: 21NOS: 21-30
gb|U83192.1|HSU83192 (h)
JIP-2gb|AF218778.1|AF218778 (h)SEQ IDSEQ ID
emb|AL021708.1|HSU56K21 (isoforms/spliceNO: 31NOS: 31-40
variants)
Talingb|AF177198.1|AF177198 (h ortholog)SEQ IDSEQ ID
NO: 41NOS: 41-50
OMP25gb|AF107295.1|AF107295 (r)SEQ IDSEQ ID
NO: 51NOS: 51-60
CAPONgb|AF037071.1|AF037071(r)SEQ IDSEQ ID
dbj|AB007933.2|AB007933 (partial h)NO: 61NOS: 61-70
PIP4, 5EST gb|AA544527.1|AA544527 (m)SEQ IDSEQ ID
kinaseNO: 71NOS: 71-80
Na channelgb|L42341.1|MUSSOCHA (m, homolog)SEQ IDSEQ ID
brain 3ref|NM_013119.1|| (r, homolog)NO: 81NOS: 81-90
dbj|AB037777.1|AB037777 (partial h, homolog)
Mint1gb|L34676.1|MUSX11P (m)SEQ IDSEQ ID
gb|AF029107.1|AF029107 (r)NO: 91NOS: 91-100
ref|NM_005503.1|| (h)
ICAP-1ref|NM_008403.1|| (m)SEQ IDSEQ ID
ref|NM_004763.1|| (h)NO: 101NOS: 101-110
APCdbj|AB012109.1|AB012109 (h)SEQ IDSEQ ID
subunit10NO: 111NOS: 111-120

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.

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.

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.

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.

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.

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 2
Immunogenic polypeptides eliciting specific rabbit polyclonal antibody:
Polypeptide-KLH conjugates immunized per protocol described above.
Polypeptide SequenceImmunogenicity
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+++

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.

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.

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.

EXAMPLES, PROTOCOLS AND EXPERIMENTAL PROCEDURES

I. High-Throughput In Vitro Fluorescence Polarization Assay

Reagents:

LDL Receptor Interaction Domain peptide (size minimized, rhodamine-labeled; final conc.=1-5 nM)

Binding Polypeptide (final conc.=100-200 nM)

Buffer: 10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH 7.6

Protocol:

1. Add 90 microliters of receptor peptide/binding polypeptide mixture to each well of a 96-well microtiter plate.

2. Add 10 microliters. of test compound per well. 3. Shake 5 min and within 5 minutes determine amount of fluorescence polarization by using a Fluorolite FPM-2 Fluorescence Polarization Microtiter System (Dynatech Laboratories, Inc).

II. Conformational Sensor—ELISA Format Assay

Buffer and Solution Preparation:

1. 10× Assay Buffer:

    • 100 ML of 1M Hepes
    • 300 mL of 5M NaCl
    • 20 mL of 1M MgCl
    • Add MQ H2O to 1 L

2. Master Mix of peptide/protein

    • Protein: Glutathione-S-transferase/binding polypeptide fusion protein: final conc=100 nM
    • LDL Receptor Interaction Domain peptide (size minimized, biotinylated; final conc.=1 uM)
    • Add Assay Buffer and H2O to bring to final volume: final buffer conc=1×

3. Antibody Mix:

    • anti-GST, rabbit (final conc.=1:10,000)
    • anti-rabbit-HRP (final conc.=1:10,000)
    • Add T-TBS to bring to final volume: final buffer conc=1×
      Procedure:

1. Make 50 mL of Master Mix (see 2 above) of appropriate peptide/polypeptide combinations (use 50 mL polypropylene tubes). Incubate for 1 hr at RT p 2. Add 95 uL of Master Mix to each well of a 96-well plate** ** Reacti-Bind Streptavidin-Coated, White Polystyrene Plates (#15118B), which have been blocked by Super-Blocking Reagent from Pierce.

3. Transfer 5 uL of each test compound (stock=60 uM) to each well of the plate

4. Incubate plate for 1 hr at RT

5. While incubating, make rabbit anti-GST antibody and anti-rabbit-HRP Antibody Mix (see 3 above). Incubate on ice for 1 hr.

6. Wash plates 3× with H2O thoroughly

7. Add 100 uL of Antibody Mix into each well of the plate

8. Incubate for 1 hr at RT

9. Wash 3× with H2O

10. Dilute Supersignal substrate (mixed Luminol and peroxide) in 1:2 H2O and then add 100 uL into each well

11. Shake 3-5 min. Read chemiluminescence.

III. High-Throughput In Vitro Binding Assay.

A. Reagents:

Neutralite Avidin: 20 μg/ml in PBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at room temperature.

Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl2, 1% glycerol, 0.5% NP-40, 50 mM b-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors.

33P LDL Receptor Interaction Domain 10× stock: 10−8-10−6M “cold” interaction domain supplemented with 200,000-250,000 cpm of labeled interaction domain (Beckman counter). Place in the 4° C. microfridge during screening.

Protease inhibitor cocktail (1000×): 10 mg Trypsin Inhibitor (BMB #109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma # B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB #917575), and 2 mM NaVO3 (Sigma # S-6508) in 10 ml of PBS.

Binding Polypeptide: 10−7-10−5 M biotinylated binding polypeptide in PBS.

B. Preparation of Assay Plates:

Coat with 120 μl of stock N-Avidin per well overnight at 4° C.

Wash 2 times with 200 μl PBS.

Block with 150 μl of blocking buffer.

Wash 2 times with 200 μl PBS.

C. Assay:

Add 40 μl assay buffer/well.

Add 10 μl compound or extract.

Add 10 μl 33P-interaction domain(20-25,000 cpm/0.1-10 pmoles/well=10−9-10−7 M final conc).

Shake at 25° C. for 15 minutes.

Incubate additional 45 minutes at 25° C.

Add 40 μM biotinylated binding polypeptide (0.1-10 pmoles/40 ul in assay buffer)

Incubate 1 hour at room temperature.

Stop the reaction by washing 4 times with 200 μM PBS.

Add 150 μM scintillation cocktail.

Count in Topcount.

D. Controls for All Assays (Located on Each Plate):

a. Non-specific binding

b. Soluble (non-biotinylated binding polypeptide) at 80% inhibition.

IV. Parental Binding Polypeptide Interactions with Natural Sequence Interaction Domains

Materials. Restriction endonucleases and other DNA modifying enzymes (T4 DNA ligase, calf intenstinal alkaline phosphatase) were purchased from Boehringer Mannheim and New England Biolabs (Beverly, Mass.). The Taq polymerase and the TAKARA LA PCR Kit were obtained from Perkin Elmer and PANVERA, respectively. Glutathione-agarose was purchased from Sigma, Protease Inhibitor Cocktail from Boehringer Mannheim. The MATCHMAKER LexA two-hybrid system used and yeast culture media were obtained from CLONTECH. Antibodies against LRP (13) and Megalin (11) have been described previously. Human LDL was iodinated using the iodine monochloride method (14).

Yeast Plasmid Construction and the Two-hybrid Assay. All LDL receptor family cytoplasmic tail LexA fusion proteins were constructed using the pLexA vector (MATCHMAKER system, CLONTECH). The cytoplasmic domains and Dab1 were amplified by PCR from human and mouse brain 1st strand cDNA or cDNA clones. PCR products were subcloned into the EcoRI and NcoI sites of pLexA for the Megalin and VLDL Receptor, and into EcoRI and BamHI sites for the other receptors. The Dab-1 cDNA (2) was transferred via EcoRI and SalI into the EcoRI and XhoI digested prey plasmid pB42AD (MATCHMAKER system, CLONTECH). All constructs were sequenced after cloning, and all bait vectors were tested for self-activation. Bait and prey vectors were cotransfected into yeast cells (leucine auxotrophic strain EGY48) using the LiAc method as described in the MATCHMAKER manual (CLONTECH). The two-hybrid assay used two reporters (LEU2 and lacZ) under the control of LexA operators. Cells were spread on selective plates (His-; Leu-; Trp-) and grown for 3 days. Single clones were grown on selective plates in patches and harvested after 2-3 days. To recover DNA from yeast, cells were resuspended in 50 μl STES (0.5M NaCl, 0.2M Tris-HCl pH 7.6, 0.01M EDTA, 1% SDS). Glass beads were added and the cells vortexed vigorously. After addition of 20 μl H2O and 60 μl phenol/chloroform cells were vortexed for 1 more minute and centrifuged (14,000× g, 5 min). The aqueous phase was collected and the DNA was precipitated with EtOH. DNA from positive clones was retransformed into DH5α and checked by DNA sequencing. Yeast mating was performed after transformation of LexA plasmids into the yeast strain YM4271 following the instructions in the MATCHMAKER manual (CLONTECH).

Generation and purification of glutathione-S-transferase (GST)-fusion proteins. GST-fusion plasmids of Dab1 and FE65 have been described previously (2). Positive clones derived from the yeast two-hybrid screen were cloned into pGEX-4T1 (Pharmacia) using the flanking EcoRI and XhoI restriction sites and verified by sequencing. Fusion proteins were expressed in BL21 or BL21 codon+bacteria (Stratagene) following induction by 1 mM isopropyl-thio-D-galactopyranoside for 5 h. Proteins were recovered by Triton lysis (phosphate buffered saline (PBS) with 1% Triton X-100, and Protease Inhibitor cocktail) and purified using glutathione-agarose beads.

In Vitro Binding Assay. Liver and kidney membrane extracts were prepared as described previously (15). Lysates were incubated with 50 μl of glutathione-agarose and 10 μg of the respective purified GST-fusion protein for 6 h at 4° C. Glutathione beads were washed rapidly three times in 150 mM NaCl, 10 mM Tris-HCl pH 7.5, 2mM each MgCl2, CaCl2, and MnCl2 for 10 min. SDS sample buffer was added to the supernatant or beads. Proteins were separated by electrophoresis on 4% (for Megalin) and 8% (for LRP) SDS-polyacrylamide gel electrophoresis under nonreducing conditions and analyzed by immunoblotting using specific antibodies (11,13) and ECL detection.

In Situ Hybridization. Templates used for in situ hybridization were amplified from mouse cDNA clones for LRP, Megalin, and ApoER2, and from the yeast-two-hybrid clones using the pB42AD2 primer in combination with primers specific for individual interacting clone (meg8, Semcap-1; meg 11, Jip-1; meg20, Jip-2). PCR products were cloned into pCR2.1-TOPO or pCRII-TOPO (Invitrogen) and sequenced. For each labeling reaction, 0.5 μg of linearized template was transcribed using T7 RNA polymerase (Ambion, Tex.) and 100 μCi of 33P-UTP (Amersham).

Time-mated wild-type female mice at 13.5 days post coitum were anaesthetized with metofane and perfused via the left ventricle with PBS followed by 4% paraformaldehyde. Whole embryos were immersed overnight in 4% paraformaldehyde at 4° C., and subsequently transferred to PBS. The tissue was placed in 70% ethanol, dehydrated through graded ethanol solutions, cleared in xylene, and infused with paraffin. Sagittal sections were cut at 5 μm intervals and mounted on Vectabond-treated slides (Vector Laboratories).

In situ hybridization was performed on adjacent sections to determine the expression pattern of ApoER2, LRP, Megalin, SEMCAP-1, JIP-1 and JIP-2. Xylene was used to remove paraffin. Sections were then rehydrated through graded ethanol solutions, refixed in 4% paraformaldehyde, digested with Pronase (20 μg/ml Pronase for 7.5 min), and acetylated (0.1 M triethanolamine-HCL [pH 7.5]/0.25% acetic anhydride for 5 min). Slides were hybridized for 12 hr at 55° C. in a solution containing 50% formamide, 0.3% dextrane sulfate, 1× Denhardt's solution, 0.5 mg/ml tRNA, and 7.5×106 cpm/ml riboprobe. After hybridization, slides were washed in 5×SSC/100 mM DTT at 65° C. for 30 min, and covered with K.5 nuclear emulsion (Ilford) before exposure at 4° C. for 21 to 35 days.

Cell Lines and Tissue Culture. Cell lines containing the wild type LDL-Receptor (TR715-19) and the LDL receptor containing a stop codon in the cytoplasmic domain at position 807 (TR807-3) have been published previously (16,17). The plasmid pcDNA3.1/ZEO DAB555 contains the mouse Dab1 cDNA under control of the CMV promoter in the pcDNA3.1/ZEO(+) vector (Invitrogen) and was used to create Dab1 overexpressing cell lines derived from TR715-19 and TR807-3, designated TR 3097 and TR 3098, respectively. Multiple clones were generated after transfection and Zeocin selection and analyzed for levels of LDLR and Dab1 expression by immunoblotting using specific antibodies and ECL detection. All cell lines were maintained in Dulbecco's minimal essential medium (DMEM) with 100 units/ml penicillin, and 100 μg/ml streptomycin sulfate, supplemented with 5% (v/v) fetal calf serum (Life Technologies, Inc., Grand Island, N.Y.).

Cellular binding and degradation assays. For uptake, binding, and degradation assays, the cells were plated in 6-well plates at a cell density of 60,000 cells/well in Medium A (1:1 mixture of Dulbecco's minimal essential medium and Ham's F-12 medium) supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin sulfate, and with 5% (v/v) fetal calf serum. On day 2, the cells were washed twice with PBS and re-fed with medium A containing 5% (v/v) newborn calf lipoprotein-deficient serum, 10 μM compactin, and 100 μM mevalonate. On day 3 the cells were washed twice with PBS and switched to medium B (Dulbecco's modified Eagle's medium (minus glutamine) containing 2 mg/ml fatty acid-free bovine serum albumin) with 125I-LDL. After 1, 3, and 5 h, the medium and the cells were harvested and surface binding, uptake, and degradation were measured as previously described (17).

Results. To identify other adaptor proteins besides Dab1 and FE65 that interact with the cytoplasmic tails of LDL receptor family members we performed a series of yeast-two-hybrid screens of various tail bait constructs against a panel of commercially available libraries (described in detail in ‘Methods’). The genes that were identified in these screens and that survived specificity testing include natural murine homologs of the parental sequences shown in Table I. Most of these genes have known functions that are related to cell adhesion, cell activation, reorganization of the cytoskeleton and neurotransmission. Dab1 has previously been shown to bind to the cytoplasmic tails of LDL receptor family members (2,3). JIP-1 and JIP-2 are scaffolding proteins for components of the Jun N-terminal Kinase (JNK) signaling pathway (18) and JIP-1 was recently shown to also interact with p190 rhoGEF (19). SEMCAP-1 is an adaptor protein that can bind to the cytoplasmic tails of membrane proteins such as SemF (20) and LDL receptor family members, but also to the cytoplasmic and membrane associated regulation of G-protein signaling (RGS) protein GAIP (21). Mintl, or X11, is another scaffold protein that interacts with the cytoplasmic tail of the amyloid precursor protein (APP) via its phosphotyrosine binding (PTB) domain, and with the presynaptic protein Munc-18 via its amino terminus (22). Munc-18 is necessary for synaptic vesicle exocytosis (23). CAPON is a PTB domain containing adaptor protein that was shown to bind neuronal nitric oxide synthase (nNOS) and is thought to dislodge nNOS from the postsynaptic density protein PSD-95, thus inactivating the enzyme (24). PSD-95, a scaffolding protein that organizes active components of the postsynaptic neurotransmission machinery such as glutamate receptors, K-channels, kinases and nNOS into functional microdomains (25) was also found to interact directly with LDL receptor family tails. Although it does not contain a PTB domain, the integrin cytoplasmic domain associated protein-1 (ICAP-1) binds to an NPxY sequence motif in the integrin tail (26,27) and thus presumably also to the NPxY motifs that are present in the cytoplasmic tails of all LDL receptor family members (2). Omp25 is an outer mitochondrial membrane protein that contains a PDZ domain and thereby interacts with the inositol phosphatase synaptojanin, a protein thought to be involved in the recycling of synaptic vesicles (28). Another protein that is homologous to phosphatidyl inositol 4,5 kinase and therefore is presumably involved in inositol metabolism was also identified in our screen. A close homologue of the cytoskeletal protein talin, the a subunit of the brain-specific sodium channel 3 and APC10, a component of the anaphase promoting complex also bound directly to LDL receptor family tails by yeast-two-hybrid interaction.

Next, we determined the binding specificity of each of the proteins that had been identified in the initial screens against a panel of bait constructs containing the whole or part of the cytoplasmic tails of the presently known LDL receptor family members. The tail sequences contained in these bait constructs are shown in Table 3.

TABLE 3
LDL receptor two-hybrid bait sequences
Bait SequenceSequence SourceSource Residues
SEQ ID NO: 121LDLR human2431-2583
SEQ ID NO: 122VLDLR mouse2477-2641
SEQ ID NO: 123LR11 human6557-6714
SEQ ID NO: 124ApoER2− mouse1925-2104
SEQ ID NO: 125ApoER2+ mouse2642-2998
SEQ ID NO: 126MegA human13457-14073
SEQ ID NO: 127Meg human13457-13748
SEQ ID NO: 128MegC human13761-14073
SEQ ID NO: 129MegB human13682-13886
SEQ ID NO: 130LRPA human13808-13921
SEQ ID NO: 131LRPB human13808-14035
SEQ ID NO: 132LRP human13808-14103

The ApoER2 tail was tested with (+) and without (−) its alternatively spliced insert. For Megalin and for LRP the complete tails and parts thereof containing a single NPxY motif were tested separately. Dab1 bound strongly to all the LDL receptor family tails, but only to one of the NPxY motifs in the LRP and Megalin tail (LRPB and MegA, respectively). LRP and Megalin have the longest cytoplasmic tails that contain several potential adaptor binding motifs and thus bound most of the proteins. In contrast, the LDL receptor and the VLDL receptor bound only Dab1 (2, 3). Interestingly, the ApoER2 tail containing the alternatively spliced insert bound the scaffold proteins JIP-1, JIP-2 and PSD-95, while the tail without this insert did not. This suggests that the alternative splicing of the ApoER2 tail has important regulatory functions and may determine the ability of ApoER2 to activate MAP kinase dependent signals.

Next, we sought to determine whether the protein interactions we had found in the yeast-two-hybrid screen could be reproduced by a different approach, such as a biochemical pull-down assay. The respective cDNA fragments were cut out from the yeast prey vector and cloned into a bacterial GST expression vector. GST fusion proteins were incubated with membrane extracts from liver (LRP) and kidney (Megalin). GST alone served as a negative control, Dab1 and FE65 served as positive controls for interactions of fusion proteins with LRP. Binding of FE65 was specific for LRP, Dab1 bound to both receptors, consistent with the two-hybrid results. All other fusion proteins tested, with the exception of the talin homologue and Mint-1, bound to both native Megalin and LRP in membrane extracts.

Our experiments so far have revealed a spectrum of cytoplasmic adaptor or scaffold proteins that can interact with the cytoplasmic tails of LDL receptor family members in vivo. In the case of Dab1, there is strong genetic and biochemical evidence that binding of this protein to the tails of the VLDL receptor and ApoER2 is absolutely required for the transmission of a critical developmental signal to migrating neurons (2,3,10). However, as we have shown, Dab1 not only interacted with these two receptors, but in fact bound tightly to the tails of all known members of the LDL receptor gene family. Whether these interactions occur only under in vitro conditions or also take place in vivo was not known. To address this question we examined the effect of Dab1 expression on the activity of the LDL receptor in cultured cells. Chinese hamster ovary (CHO) cells expressing the wild type human LDL receptor with or without its cytoplasmic tail (Chen et al., 1990) were transfected with a Dab1 expression plasmid. Stable cell lines that expressed approximately equal amounts of Dab1 were selected. All four cell lines were then tested for their ability to bind, internalize and degrade 125I labeled human LDL. Cell lines expressing the human LDL receptor without a cytoplasmic tail bound, internalized and degraded the human LDL to the same extent, whether Dab1 was present or not. In contrast, CHO cells expressing the wild type human LDL receptor bound approximately 2 times more LDL on their surface when Dab1 was present. Cellular uptake was not significantly affected by Dab1 expression, although the degradation rate was slightly reduced. These findings indicate that Dab1 binds to a site in the LDL receptor tail that overlaps with the endocytosis signal, and thereby increases the surface pool of the receptor by competing with the assembly of the endocytosis complex.

Our yeast-two-hybrid screen has revealed a number of other scaffold and adaptor proteins that function in cellular signaling cascades in which Dab1 is not yet known to be involved. Two of these proteins, JIP-1 and JIP-2, serve as scaffolding proteins for MAP kinases, and SEMCAP-1 is an adaptor protein that can also bind to the C-terminus of the GTPase activating RGS protein GAIP. Membrane-associated SEMCAP-1 colocalizes with clathrin and is primarily found in vesicles just underneath the plasma membrane, a compartment where LDL receptor family members also reside. In addition to the demonstrated role of LDL receptor family members and Dab1 in cellular tyrosine kinase signaling, recruitment of MAP kinases and interactions with RGS proteins considerably expands the functional roles of this class of cell surface receptors. We tested whether LDL receptor gene family members are expressed together with these adaptors and may thus functionally interact with them in vivo. We used in situ hybridization to compare the expression pattern of ApoER2, LRP and Megalin with that of SEMCAP-1, JIP-1 and JIP-2 during embryonic development of the brain, a time period in which these LDL receptor family members perform critical functions. At E13.5 ApoER2 was expressed throughout the brain in a pattern almost identical to that of JIP-1 and JIP-2. SEMCAP-1 also showed a largely overlapping expression pattern. Notably, JIP-1 and JIP-2 were predominantly expressed in the more superficial cortical plate, while SEMCAP-1 was mainly expressed in the subventricular zone and in the population of migrating neurons in the developing cortex. ApoER2 was present throughout the cortex. In contrast, LRP expression in the developing brain was comparatively low and restricted mainly to the choroid plexus, and Megalin was almost exclusively expressed in the ventricular zone. SEMCAP-1 and JIP-1 were expressed in the structures as LRP and Megalin.

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The foregoing descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation. All publications and patent applications cited in this specification and all references cited therein are herein incorporated by reference as if each individual publication or patent application or reference were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.