Gabriele Grenningloh

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.

Alpha subunit variants of the human glycine receptor: primary structures, functional expression and chromosomal localization of the corresponding genes.

Two cDNAs encoding variants (alpha 1 and alpha 2) of the strychnine binding subunit of the inhibitory glycine receptor (GlyR) were isolated from a human fetal brain cDNA library. The predicted amino acid sequences exhibit approximately 99% and approximately 76% identity to the previously characterized rat 48 kd polypeptide. Heterologous expression of the human alpha 1 and alpha 2 subunits in Xenopus oocytes resulted in the formation of glycine‐gated strychnine‐sensitive chloride channels, indicating that both polypeptides can form functional GlyRs. Using a panel of rodent‐human hybrid cell lines, the gene encoding alpha 2 was mapped to the short arm (Xp21.2‐p22.1) of the human X chromosome. In contrast, the alpha 1 subunit gene is autosomally located. These data indicate molecular heterogeneity of the human GlyR at the level of alpha subunit genes.

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.

Functional expression in Xenopus oocytes of the strychnine binding 48 kd subunit of the glycine receptor

The inhibitory postsynaptic glycine receptor (GlyR) of rat spinal cord is an oligomeric transmembrane protein which forms an agonist‐gated anion channel. Expression in Xenopus oocytes of its mol. wt 48,000 subunit generated glycine‐gated chloride channels which were analysed by voltage clamp. The agonist and antagonist response properties as well as the desensitization characteristics of these 48 kd subunit receptors resembled GlyRs expressed from spinal cord poly(A)+ RNA. These data indicate that the 48 kd subunit is capable of assembling into a functional receptor homo‐oligomer which displays the pharmacology characteristic of the spinal cord GlyR.

Functional chloride channels by mammalian cell expression of rat glycine receptor subunit

Cultured human cells were transfected with cloned rat glycine receptor (GlyR) 48 kd subunit cDNA. In these cells glycine elicited large chloride currents (up to 1.5 nA), which were blocked by nanomolar concentrations of strychnine. However, no corresponding high-affinity binding of [3H]strychnine was detected in membrane preparations of the transfected cells. Analysis by monoclonal antibodies specific for the 48 kd subunit revealed high expression levels of this membrane protein. After solubilization, the 48 kd subunit behaved as a macromolecular complex when analyzed by sucrose density centrifugation. Approximately 50% of the solubilized complex bound specifically to a 2-aminostrychnine affinity column, indicating the existence of low-affinity antagonist binding sites on most of the expressed GlyR protein. Thus, the 48 kd strychnine binding subunit efficiently assembles into high molecular weight complexes, resembling the native spinal cord GlyR. However, formation of functional receptor channels of high affinity for strychnine occurs with low efficiency.

Targeting of SCG10 to the Area of the Golgi Complex Is Mediated by Its NH2-terminal Region

SCG10 is a neuronal growth-associated protein that is concentrated in the growth cones of developing neurons. SCG10 shows a high degree of sequence homology to the ubiquitous phosphoprotein stathmin, which has been recently identified as a factor that destabilizes microtubules by increasing their catastrophe rate. Whereas stathmin is a soluble cytosolic protein, SCG10 is membrane-associated, indicating that the protein acts in a distinct subcellular compartment. Identifying the precise intracellular distribution of SCG10 as well as the mechanisms responsible for its specific targeting will contribute to elucidating its function. The main structural feature distinguishing the two proteins is that SCG10 contains an NH2-terminal extension of 34 amino acids. In this study, we have examined the intracellular distribution of SCG10 in PC12 cells and in transfected COS-7 cells and the role of the NH2-terminal domain in membrane-binding and intracellular targeting. SCG10 was found to be localized to the Golgi complex region. We show that the NH2-terminal region (residues 1-34) was necessary for membrane targeting and Golgi localization. Fusion proteins consisting of the NH2-terminal 34 amino acids of SCG10 and the related protein stathmin or the unrelated protein, β-galactosidase, accumulated in the Golgi, demonstrating that this sequence was sufficient for Golgi localization. Biosynthetic labeling of transfected COS-7 cells with [3H]palmitic acid revealed that two cysteine residues contained within the NH2-terminal domain were sites of palmitoylation.

Nerve-terminal proteins: to fuse to learn

Transmitter release and membrane expansion involves the fusion of specialized vesicles to their target membranes. The mechanisms that regulate these fusion events might contribute to short- and long-term changes of synaptic efficiency that are associated with learning. A series of recently described protein-protein interactions has shed new light on vesicle binding to the cytoskeleton, vesicle docking to the target membranes and, finally, vesicle fusion and membrane retrieval. Specific steps in this pathway might be key sites for modulating the strength of synaptic connections that underlie the molecular basis of learning.

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.

MOLECULAR CLONING OF THE ANTAGONIST-BINDING SUBUNIT OF THE GLYCINE RECEPTOR

The postsynaptic receptor for the inhibitory neurotransmitter glycine is a heterooligomeric membrane protein which, after affinity purification on an antagonist column, contains three polypeptides of 48K, 58K and 93K. Sequencing of cDNA clones of the antagonist-binding 48K subunit revealed a structural organization similar to and significant amino acid homology with nicotinic acetylcholine receptor proteins. Our data suggest the existence of a set of related genes encoding transmembrane channel-forming neurotransmitter receptor polypeptides of excitable membranes.

Antisense Blockade of Expression

With the advent of modern molecular genetics and molecular biology, we will face more and more situations where novel gene products with unknown functions are identified. Genetic linkage analysis will allow the association of novel or known genes to Important diseases (1). Similarly, sensitlve differential cloning procedures will identify rare genes expressed in specific physiological or pathological situations (1, 3). In both cases, establishing the precise function of the identified gene is an essential step for the understanding of the cellular mechanisms that either lead to the disease or are pivotal in important physiological processes.