Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1997-03-04
2003-10-07
Kunz, Gary (Department: 1647)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S252300, C435S320100, C536S023500
Reexamination Certificate
active
06630345
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates generally to receptors implicated in neurotransmitter release, and particularly to a novel G-protein-coupled receptor which binds &agr;-latrotoxin (&agr;-LTx) independently of calcium (Ca
2+
) presence and is thus a regulator of neurotransmitter release which mediates the calcium-independent stimulation of neurotransmitter release by &agr;-latrotoxin.
BACKGROUND OF THE INVENTION
&agr;-Latrotoxin, a vertebrate neurotoxin of Black Widow Spider venom, is a strong stimulator of spontaneous neurotransmitter release from the nerve terminal (Clark et al.,1970
, Nature
, 225 703-705). In physiological solutions, &agr;-latrotoxin-evoked neurotransmitter release occurs by synaptic vesicle exocytosis and is accompanied by presynaptic membrane polarization and the influx of calcium ions through the channels induced by the toxin and through presynaptic calcium channels. However, an unusual characteristic of &agr;-latrotoxin's stimulatory action is that it does not require extracellular Ca
2+
, provided Mg
2+
is present in the extracellular solution and can occur even without a noticeable increase in intracellular Ca
2+
concentration. Stimulation of neurotransmitter release by &agr;-latrotoxin requires binding to its high-affinity membranae receptors. An immunofluorescence study of the neuromuscular junction indicates that the &agr;-latrotoxin receptors may not be restricted to the areas of synaptic contacts. Two types of receptors, differing in their calcium requirement of &agr;-latrotoxin-binding have been described. The calcium-dependent receptor has been identified as neurexin I&agr;, a member of a large family of multiply spliced neuron-specific proteins, the neurexins. In contrast, brain glycoprotein of the Mr 120,000 which does not belong to the neurexin family recently has been purified and proposed as a calcium-independent receptor for, and a likely mediator of, the calcium-independent stimulation of neurosecretion by &agr;-latrotoxin (Krasnoperov et al., (1996),
Biochem. Biophys. Res. Commun
., 227:868-875 and Davletov et al., (1996)
J. Biol Chem
. 271, 23239-23245).
&agr;-Latrotoxin receptors have been identified biochemically, using an iodinated radioactive derivative of the toxin by Tzeng and Siekevitz,
J. Neurochem
. (1979) 33, 263-274; Meldolesi,
J. Neurochem
. (1982) 38, 1559-1569; Scheer and Meldolesi,
EMBO J
. (1985) 4, 323-327. These receptors were of low abundance (about 300 fmol/mg of membrane protein), and their affinity to &agr;-latrotoxin was high (Kd in the range of 10
−9
-10
−10
M). Previously, the purification of a high-affinity &agr;-latrotoxin-binding protein was identified as neurexin I&agr;, a member of a large family of multiply spliced neuron-specific proteins, the neurexins was reported in Petrenko,
FEBS Lett
. (1993) 325, 81-85; Petrenko et al., (1990) EMBO J. 9, 2023-2027; and Ushkaryov et al.,
Science
(1992) 257, 50-56. The structural features and developmental profile of neurexins suggest that they perform a function in cell adhesion or recognition important in axonogenesis, see, Ushkaryov et al.,
Science
(1992) 257, 50-56; and Puschel and Betz,
J. Neurosci
. (1995) 15, 2849-2856. A hypothesis has been formulated that these highly polymorphic cell surface membrane proteins may define the specificity of synaptic connections in the brain Ushkaryov et al.,
Science
(1992) 257, 50-56.
&agr;-Latrotoxin-binding properties of purified and recombinant neurexin-I&agr; are very similar to the binding properties of the membrane-bound &agr;-latrotoxin receptors with one significant exception: neurexin I&agr; binds &agr;-latrotoxin only in the presence of calcium, whereas the brain membranes bind the toxin even in the presence of EDTA. Therefore, neurexin I&agr; may be important in calcium-dependent effects of &agr;-latrotoxin, such as degeneration of nerve terminals, but not in the stimulation of neurosecretion in calcium-independent environment. Since purified &agr;-latrotoxin can form cation channels in artificial lipid bilayers, neurexin I&agr; may facilitate its insertion into the cellular membrane by binding with &agr;-latrotoxin, resulting in calcium fluxes through the formed cation channels. However, in the absence of calcium, this mechanism would not explain robust stimulation of spontaneous neurotransmitter release by this toxin.
In view of the above, a need therefore exists to elucidate the calcium-independent activation of &agr;-latrotoxin, and to thereby further understand its activity, and possibly devise strategies for intervention that may benefit neuronal activity. It is accordingly toward the fulfillment of these needs that the present invention is directed.
SUMMARY OF THE INVENTION
In one aspect, the present invention concerns the identification of a second &agr;-latrotoxin receptor which binds &agr;-latrotoxin both in the presence or absence of calcium and is pharmacologically and structurally distinct from neurexin I&agr;, an earlier described receptor of &agr;-latrotoxin. This receptor, designated CIRL (calcium-independent receptor of &agr;-latrotoxin) belongs to the secretin receptor subfamily of G-protein coupled receptors, and, together with &agr;-latrotoxin, interacts with syntaxin, a component of the neuronal exocytotic machinery. CIRL, as a neuronal signaling receptor, is thus critically important for the calcium-independent stimulation of neurotransmitter release by &agr;-latrotoxin. A further aspect of the present invention involves the isolation, purification and characterization of the calcium-independent receptor of &agr;-latrotoxin (CIRL).
In its broadest aspect, the present invention extends to a novel neuronal receptor which is a regulator of neurotransmitter release, and thus mediates &agr;-latrotoxin (&agr;-LTx) toxicity both in the presence or absence of calcium.
In a specific example, the calcium-independent receptor of &agr;-latrotoxin (CIRL) has been identified as a G-protein-coupled receptor which contains a subunit of an apparent Mr 120,000, as determined by SDS-PAGE analysis.
In a still further aspect, the present invention extends to methods of utilizing CIRL. This receptor can be expressed and used to screen libraries of agents, or mixtures of natural origin (e.g., brain homogenates, detergent extracts, cell conditioned media or extracts, etc.) for ligands thereof which can then be utilized in various therapeutic methods.
Still further, since this receptor is enriched in the striatum of the mammalian brain, antibodies or nucleic acid probes thereto can be prepared which can then be utilized in diagnostic methods for screening for the presence of various neurological diseases characterized by the changes in receptor expression, or mutations thereto, or the presence of excess receptors. These diseases include, but are not limited to, neurological diseases such as schizophrenia, Alzheimer's disease, epilepsy, stress disorder, Huntington's disease, Parkinson's disease, as well as peripheral neuromuscular diseases such as myasthenia gravis.
In a particular embodiment, the present invention relates to all members of the herein disclosed family of calcium-independent receptors of &agr;-latrotoxin, and to genetically engineered cells which express such receptors.
The present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a calcium-independent receptor of &agr;-latrotoxin (CIRL); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the calcium-independent receptor of &agr;-latrotoxin (CIRL), having a nucleotide sequence or is complementary to a DNA sequence shown in
FIG. 2B
(SEQ ID NO:2). In a still further embodiment, the gene constructs of this invention can be utilized in gene therapy in individuals wherein the lack of, or changes in, or modifications to, this receptor causes deficits in neurotransmission.
The human and murine DNA sequences of the calcium-independent receptor of
Krasnoperov Valery G.
Petrenko Alexandre G.
Hayes Robert C.
Klauber & Jackson
Kunz Gary
New York University
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