Netrin receptors

Details Netrin receptors Netrin receptors

1999-05-07

2001-08-21

Allen, Marianne P. (Department: 1631)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C530S350000

Reexamination Certificate

active

06277585

ABSTRACT:

FILED OF THE INVENTION
The field of this invention is proteins which regulate vertebrate cell guidance.
BACKGROUND
In the developing nervous system, migrating cells and axons are guided to their targets by cues in the extracellular environment. The netrins are a family of phylogenetically-conserved guidance cues that can function as diffusible attractants and repellents for different classes of cells and axons
1-10
. Recent studies in vertebrates, insects and nematodes have implicated members of the DCC subfamily of the immunoglobulin (Ig) superfamily as receptors involved in migrations toward netrin sources
6, 11-13
. The mechanisms that direct migrations away from netrin sources (presumed repulsions) are less well understood. In
Caenorhabditis elegans
, loss of unc-5 (which encodes the transmembrane protein UNC-5
14
) function causes defects in these migrations
15, 16
, and ectopic expression of unc-5 in some neurons can redirect their axons away from a netrin source
17
. However, the relationship between UNC-5 and the netrins has not been defined. We disclose herein vertebrate homologues of the C. elegans UNC-5, which define a novel subfamily of the Ig superfamnily, and whose mRNAs show prominent expression in various classes of differentiating neurons and we disclose that these vertebrate UNC-5 homologues are vertebrate netrin-binding proteins.
SUMMARY OF THE INVENTION
The invention provides methods and compositions relating to vertebrate UNC-5 proteins, related nucleic acids, and protein domains thereof having vertebrate UNC-5-specific activity. The proteins may be produced recombinantly from transfected host cells from the subject vertebrate UNC-5 encoding nucleic acids or purified from vertebrate cells. The invention provides isolated vertebrate unc-5 hybridization probes and primers capable of specifically hybridizing with the disclosed vertebrate unc-5 genes, vertebrate UNC-5-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g. genetic hybridization screens for vertebrate unc-5 transcripts), therapy (e.g. gene therapy to modulate vertebrate unc-5 gene expression) and in the biopharmaceutical industry (e.g. as immunogens, reagents for modulating cell guidance, reagents for screening chemical libraries for lead pharmacological agents, etc.).
DETAILED DESCRIPTION OF THE INVENTION
The nucleotide sequences of natural unc5h-1 cDNAs from rat and human are shown as SEQ ID NOS:1 and 2, respectively; and the conceptual translates are shown as SEQ ID NOS:5 and 6, respectively. The nucleotide sequences of natural unc5h-2 cDNAs from rat and human are shown as SEQ ID NOS:3 and 4, respectively; and the conceptual translates are shown as SEQ ID NOS:7 and 8, respectively. The vertebrate UNC-5 proteins of the invention include incomplete translates of SEQ ID NOS:1, 2, 3 and 4 and deletion mutants of SEQ ID NOS:5, 6, 7 and 8, which translates and deletion mutants have vertebrate UNC-5-specific amino acid sequence and assay-discernable vertebrate UNC-5-specific binding specificity or function. Such active vertebrate UNC-5 deletion mutants, vertebrate UNC-5 peptides or protein domains comprise at least about 8, preferably at least about 12, more preferably at least about 24 consecutive residues of SEQ ID NO:5, 6, 7 or 8. For examples, vertebrate UNC-5 protein domains identified below are shown to provide protein-binding domains which are identified in and find use, inter alia, in solid-phase binding assays as described below.
Vertebrate UNC-5-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals (e.g. gene therapy, transgenics, etc.), etc. Binding assays encompass any assay where the molecular interaction of a vertebrate UNC-5 protein with a binding target is evaluated. The binding target may be a natural extracellular binding target such as a netrin protein, or other regulator that directly modulates vertebrate UNC-5 activity or its localization; or non-natural binding target such a specific immune protein such as an antibody, or an vertebrate UNC-5 specific agent such as those identified in screening assays such as described below. Vertebrate UNC-5-binding specificity may assayed by binding equilibrium constants (usually at least about 10
7
M
−1
, preferably at least about 10
8
M
−1
, more preferably at least about 10
9
M
−1
), by the ability of the subject protein to function as negative mutants in vertebrate UNC-5-expressing cells, to elicit vertebrate UNC-5 specific antibody in a heterologous mammalian host (e.g. a rodent or rabbit), etc. In any event, the vertebrate UNC-5 binding specificity of the subject vertebrate UNC-5 proteins necessarily distinguishes C. elegans UNC-5.
The claimed vertebrate UNC-5 proteins are isolated or pure: an “isolated” protein is unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, and more preferably at least about 5% by weight of the total protein in a given sample and a pure protein constitutes at least about 90%, and preferably at least about 99% by weight of the total protein in a given sample. The vertebrate UNC-5 proteins and protein domains may be synthesized, produced by recombinant technology, or purified from mammalian, preferably human 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 natural and non-natural vertebrate UNC-5-specific binding agents, methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. For example, vertebrate UNC-5-specific agents are useful in a variety of diagnostic and therapeutic applications. Vertebrate UNC-5-specific binding agents include vertebrate UNC-5-specific ligands, such as netrins, and somatically recombined protein receptors like specific antibodies or T-cell antigen receptors (see, e.g. Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory) and other natural binding agents identified with assays such as one-, two- and three-hybrid screens, non-natural binding agents identified in screens of chemical libraries such as described below, etc. For diagnostic uses, the binding agents are frequently labeled, such as with fluorescent, radioactive, chemiluminescent, or other easily detectable molecules, either conjugated directly to the binding agent or conjugated to a probe specific for the binding agent. Agents of particular interest modulate vertebrate UNC-5 function, e.g. vertebrate UNC-5-dependent cell guidance; for example, isolated cells, whole tissues, or individuals may be treated with a vertebrate UNC-5 binding agent to activate, inhibit, or alter vertebrate UNC-5-dependent cell guidance or function.
The invention provides UNC-5 related nucleic acids, which find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, diagnostic nucleic acids, etc.; use in detecting the presence of unc-5 genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional unc-5 homologs and UNC-5 structural analogs. The subject nucleic acids are of synthetic
on-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

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