Peter Prior

Primary structure and alternative splice variants of gephyrin, a putative glycine receptor-tubulin linker protein

A 93 kd polypeptide associated with the mammalian inhibitory glycine receptor (GlyR) is localized at central synapses and binds with high affinity to polymerized tubulin. This protein, named gephyrin (from the Greek gamma epsilon phi upsilon rho alpha, bridge), is thought to anchor the GlyR to subsynaptic microtubules. Here we report its primary structure deduced from cDNA and show that corresponding transcripts are found in all rat tissues examined. In brain, at least five different gephyrin mRNAs are generated by alternative splicing. Expression of gephyrin cDNAs in 293 kidney cells yields polypeptides reactive with a gephyrin-specific antibody, which coprecipitate with polymerized tubulin. Thus, gephyrin may define a novel type of microtubule-associated protein involved in membrane protein-cytoskeleton interactions.

Cloning and expression of the 58 kd β subunit of the inhibitory glycine receptor

Abstract The inhibitory glycine receptor (GIyR) mediates postsynaptic inhibition in spinal cord and other regions of the CNS. Purified mammalian GIyR contains two membrane-spanning subunits of 48 kd (a) and 58 kd (β) plus a 93 kd receptor-associated cytoplasmic protein. Here, the primary structure of the (β) subunit was deduced from cDNAs isolated from rat spinal cord and brain cDNA libraries. The predicted amino acid sequence exhibits 47% identity to the previously characterized rat a, polypeptide. Northern blot analysis revealed high levels of (β) subunit transcripts in postnatal spinal cord, cerebellum, and cortex. Nuclear injection into Xenopus oocytes of a β) subunit cDNA engineered for efficient expression generated weak glycine-activated chloride currents that were insensitive to the classic GIyR antagonist, strychnine. Our data indicate β differential expression of GIyR α and β) subunits in the rat nervous system and support a structural role of the (β) polypeptide in the native receptor complex.

Constitutive expression of heat shock protein 90 (HSP90) in neurons of the rat brain

Immunoblot analysis, immunocytochemistry and immuno-electron microscopy were employed to study the expression of HSP90 protein in the adult rat brain, using a specific polyclonal antiserum. Immunoblot analysis demonstrated equal levels of HSP90 in microdissected extracts from hippocampus, cortex, striatum and cerebellum. Immunocytochemistry and immuno-electron microscopy provided evidence that HSP90 is markedly expressed throughout all neuronal subpopulations of the CNS but not in non-neuronal cells except ependyma and choroid plexus. At the ultrastructural level, HSP90 immunoreactivity was predominantly found in perikarya but to a lesser extent also in dendrites and nuclei. The constitutive expression of HSP90 in widespread neuronal cell populations suggests a functional role in the physiological molecular program of CNS neurons.

Neuraxin, a novel putative structural protein of the rat central nervous system that is immunologically related to microtubule-associated protein 5.

During screening of a rat spinal cord lambda gt11 cDNA library with poly‐ and monoclonal antibodies against the postsynaptic glycine receptor a cDNA was isolated which covers an open reading frame encoding a protein of calculated mol. wt 94 kd. Sequence analysis identified a novel type of neuron‐specific protein (named neuraxin) which is characterized by an unusual amino acid composition, 12 central heptadecarepeats and putative protein and/or membrane interaction sites. The gene encoding neuraxin appears to be unique in the haploid rat genome and conserved in higher vertebrates. Northern blot and in situ hybridization revealed neuraxin mRNA to be expressed throughout the rodent central nervous system (CNS). In spinal cord, neuraxin transcripts were abundant in motoneurons which also expressed glycine receptor subunit mRNA. A bacterial fusion protein containing approximately 90% of the neuraxin sequence was found to specifically bind tubulin. Polyclonal neuraxin antibodies cross‐reacted with microtubule‐associated protein 5 (MAP5), and a monoclonal antibody against MAP5 recognized the neuraxin fusion construct. Based on these data we suggest that neuraxin is related to MAP5 and may be implicated in neuronal membrane‐microtubule interactions.

Structure and expression of inhibitory glycine receptors.

Signal transmission at chemical synapses involves specific receptors that transduce neurotransmitter binding into alterations of membrane potential. Receptors containing integral ion channels mediate rapid (in the ≤ msec range) transduction events, whereas receptors activating G-protein coupled channels operate at slower time scales (in the msec to sec range). At resting membrane potential, excitation is generated by cation influx, but inhibition of neuronal firing results from increased chloride permeability.

How to build a glycinergic postsynaptic membrane

Summary The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein found at many synapses of the mammalian central nervous system. During development, distinct isoforms of the GlyR are generated by the sequential expression of different α subunit variants. The appearance of adult-type GlyRs in spinal cord is accompanied by the accumulation of a 93×103Mr, receptor-associated peripheral membrane protein. The latter has been localized at the cytoplasmic face of glycinergic postsynaptic membranes and is thought to anchor GlyRs beneath glycinergic nerve terminals. The 93×103Mr protein binds with high affinity to polymerized tubulin, suggesting that it functions as a receptor–microtubule linking component. Our data suggest that the interaction of developmentally regulated receptor isoforms with specialized microtubule–associated proteins represents a crucial step in the assembly of postsynaptic receptor matrices.

Neuraxin corresponds to a C-terminal fragment of microtubule-associated protein 5 (MAP5)

From cloned DNA, neuraxin has been identified as a tubulin binding protein of predicted molecular weight of 94 kDa. The deduced sequence of the rat protein exhibits high homology to the C‐terminal region of mouse microtubule‐associated protein 5 (MAP5). Here, we show that different neuraxin antibodies recognize MAP5, but fail to detect a protein of 94 kDa, in subcellular and microtubular fractions of the rat central nervous system. Furthermore, tubulin binding by neuraxin was found to be dependent on taxol. These data are consistent with neuraxin corresponding to a C‐terminal fragment of MAP5 that contains a low‐affinity tubulin binding site.

The Postsynaptic Glycine Receptor — A Member of the Neurotransmitter-Gated Channel Protein Family

Postsynaptic inhibition in the central nervous system is mediated primarily by two pharmacologically distinguishable integral membrane proteins, the receptors for the amino acid neurotransmitters glycine and γ-aminobutyric acid. Whereas the γ-aminobutyric acid receptor (GABAAR) is distributed throughout higher brain regions, the glycine receptor (GlyR) predominates in spinal cord and brain stem (Barnard et al.,1987; Betz and Becker, 1988). Both proteins contain an intrinsic chloride channel which opens after binding of the respective agonists to the receptor. Thereby permeation of small anions through the postsynaptic membrane is permitted to antagonize postsynaptic depolarization. Chloride flux through the GlyR is blocked after binding of the plant alkaloid strychnine, resulting in overexcitation of the motor system, i.e. muscular convulsions (Barnard et al., 1987; Betz and Becker, 1988). In our laboratory, immobilized 2-aminostrychnine, a biologically active derivative of this antagonist, has been exploited to affinity-purifiy the GlyR from detergent extracts of mammalian spinal cord membranes.