GFRalpha3 and its uses

Abstract

The present invention relates to nucleotide sequences, including expressed sequence tags (ESTs), oligonucleotide probes, polypeptides, vectors and host cells expressing, and immunoadhesions and antibodies to mammalian GFRα3, a novel α-subunit receptor of the GDNF (i.e. GFR) receptor family. It further relates to an assay for measuring activation of an α-subunit receptor by detecting tyrosine kinase receptor activation (i.e., autophosphorylation) or other activities related to ligand-induced α-subunit receptor homo-dimerization or homo-oligomerization.

Description

TECHNICAL FIELD [0001] The present invention relates generally to the identification and isolation of novel DNA and to the recombinant production of novel polypeptides which are characterized by the presence of GFRα3 sequences, an α-subunit receptor. It further relates to an assay for measuring ligand-induced activation of an α-subunit receptor by detecting autophosphorylation of a kinase domain of an α-receptor-receptor protein tyrosine kinase (rPTK) fusion using a kinase receptor activation, enzyme-linked immunosorbent assay (KIRA ELISA) or by other means to detect α-subunit homodimerization. INTRODUCTION BACKGROUND [0002] Neurotrophic factors such as insulin-like growth factors, nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, -4 / 5 and -6, ciliary neurotrophic factor, GDNF, and neurturin have been proposed as potential means for enhancing specific neuronal cell survival, for example, as a treatment for neurodegenerative diseases such as amyotrophic lateral ...

AI Technical Summary

Benefits of technology

[0016] In view of the surprising finding herein that the α-subunit receptor can dimerize upon ligand binding, and further that such dimerization can activate a kinase domain fused the α-subunit receptor, a method is provided herein to measure ligand-induced α-subunit receptor activation, i.e. homo-dimerizaiton or homo-oligomerization. In one embodiment is provided a sensitive, reliable assay that measures agonist- or ligand-induced α-subunit receptor activation, i.e., homo-dimerization or homo-oligomerization, preferably by measuring receptor protein tyrosine kinase (rPTK) autophosphorylation of a polypeptide fusion comprising the ligand binding domain of an α-subunit receptor and the intracellular catalytic domain of a receptor protein tyrosine kinase. The construct can further optionally comprise a flag epitope to facilitate trapping and detection of the activated (e.g., dimerized, phosphorylated) α-subunit receptor. The assay is desirably useful for qualitatively and quantitatively measuring α-subunit receptor activation as well as facilitating identification and characterization of potential agonists and antagonists for a selected α-subunit receptor. It is a further object of the invention to provide an assay which enables ligand-receptor interactions to be studied for any selected α-subunit receptor, and preferably a GFRα subunit receptor.

[0017] This assay must have a capacity for high throughput, that is, the ability to reliably evaluate large numbers of samples in a relatively short period of time (e.g., in one day). The assay ideally does not use radioactive materials and is also amenable to automation.

[0018] In at least one embodiment of the invention is provided a generic assay which enables a α-subunit receptor of interest to be studied, regardless of whether or not a receptor-specific capture agent having the desired characteristics is available. Furthermore, it is an object of the invention to provide an assay which substantially represents the ligand-binding activity of the α-subunit receptor in situ. This is desirable insofar as it reduces the possibility that altered interactions between the receptor and the ligand may occur as a consequence of the receptor not being membrane-bound. In one embodiment of the assay is provided a method for measuring ligand binding by detecting serine-threonine kinase phosphorylation, phosphorylation of intracellular kinases and phosphatase activity of a catalytic domain fused to the α-subunit receptor. Accordingly, the invention provides an assay for measuring activation or ligand binding of an α-subunit receptor construct chimera by detecting its homo-dimerization or homo-oligomerization by in turn measuring kinase or phosphatase activity (i.e., by autophosphorylation) of catalytic domain that is fused to the ligand-binding domain of an α-subunit receptor of interest.

[0023] (b) An analyte is then added to the wells having the adhering cells, such that the receptor construct is exposed to (or contacted with) the analyte. This assay enables identification of agonist and antagonist ligands for the α-subunit receptor of interest. In order to detect the presence of an antagonist ligand which blocks binding and / or activation of the receptor by an agonist ligand, the adhering cells are exposed to the suspected antagonist ligand first and then to the agonist ligand (or to a mixture of the agonist and antagonist) so that competitive inhibition of receptor binding and activation can be measured. Also, the assay can identify an antagonist which binds to the agonist ligand and thereby reduces or eliminates its ability to bind to, and activate, the kinase domain. To detect such an antagonist, the suspected antagonist and the agonist for the receptor are incubated together and the adhering cells are then exposed to this mixture of ligands.

Problems solved by technology

The aberrant expression or uncontrolled regulation of any one of these receptor tyrosine kinases can result in different malignancies and pathological disorders.

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