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.     

Molecular evidence for a glycine-gated chloride channel in macrophages and leukocytes

Recent studies have demonstrated that glycine blunts the response of Kupffer cells to endotoxin. Based on pharmacological evidence, it was hypothesized that Kupffer cells and other macrophages contain a glycine-gated chloride channel similar to the glycine receptor expressed in neuronal tissues. Moreover, glycine stimulates influx of radiolabeled chloride in Kupffer cells in a dose-dependent manner. RT-PCR was used to identify mRNA of both alpha- and beta-subunits of the glycine receptor in rat Kupffer cells, peritoneal neutrophils, and splenic and alveolar macrophages, similar to the sequence generated from rat spinal cord. Importantly, the sequence of the cloned Kupffer cell glycine receptor fragment for the beta-subunit was >95% homologous with the receptor from the spinal cord. Membranes of these cells also contain a protein that is immunoreactive with antibodies against the glycine-gated chloride channel. These data demonstrate that Kupffer cells, as well as other macrophages and leukocytes, express mRNA and protein for a glycine-gated chloride channel with both molecular and pharmacological properties similar to the channel expressed in the central nervous system.     

Purification and characterization of the glycine receptor of pig spinal cord

A large-scale purification procedure was developed to isolate the glycine receptor of pig spinal cord by affinity chromatography on aminostrychnine agarose. After an overall purification of about 10 000-fold, the glycine receptor preparations contained three major polypeptides of Mr 48 000, 58 000, and 93 000. Photoaffinity labeling with [3H]strychnine showed that the [3H]strychnine binding site is associated with the Mr 48 000 and, to a much lesser extent, the Mr 58 000 polypeptides. [3H]Strychnine binding to the purified receptor exhibited a dissociation constant KD of 13.8 nM and was inhibited by the agonists glycine, taurine, and beta-alanine. Gel filtration and sucrose gradient centrifugation gave a Stokes radius of 7.1 nm and an apparent sedimentation coefficient of 9.6 S. Peptide mapping of the [3H]strychnine-labeled Mr 48 000 polypeptides of purified pig and rat glycine receptor preparations showed that the strychnine binding region of this receptor subunit is highly conserved between these species. Also, three out of six monoclonal antibodies against the glycine receptor of rat spinal cord significantly cross-reacted with their corresponding polypeptides of the pig glycine receptor. These results show that the glycine receptor of pig spinal cord is very similar to the well-characterized rat receptor protein and can be purified in quantities sufficient for protein chemical analysis.     

Agonist binding to purified glycine receptor reconstituted into giant liposomes elicits two types of chloride currents.

Using 'inside-out' membrane patches obtained from reconstituted giant liposomes containing purified glycine receptor from rat spinal cord, we have detected chloride currents elicited in response to the presence of the agonists glycine or beta-alanine. Regardless of the agonist employed, two different patterns of single channel currents could be detected, which differ in their main conductance, complexity of substates and opening frequency. In agreement with the expectations of glycine receptor heterogeneity suggested recently at the mRNA and cDNA level, our results indicate the existence of functionally different glycine receptors in the adult rat spinal cord.     

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.     

Identification of two distinct microtubule binding domains on recombinant rat MAP 1B.

A set of partially overlapping cDNA clones covering 9 kb of continuous sequence encoding the high molecular weight microtubule-associated protein (MAP) 1B, was isolated from a rat brain library in lambda gt11. The protein encoded was immunoreactive with monoclonal antibodies raised against calf MAP 1B, rat MAP 1X, and rat MAP 5, as shown by immunoblotting. Using Northern blot analysis, it was shown that the level of MAP 1B mRNA increased dramatically upon nerve growth factor-induced PC12 cell differentiation. The expression of polypeptides encoded by cDNA constructs, in conjunction with microtubule binding assays, revealed two separate microtubule binding domains, corresponding to sequences at the 5' and 3' end of the mRNA. As shown by DNA sequencing, the binding domain encoded by 5' terminal sequences consisted of the basic repeat motif KKEE(I/V), previously identified in mouse MAP 1B (Noble, M., S. A. Lewis, N. J. Cowan, J. Cell Biol. 109, 3367-3376 (1989)). The second binding domain, too, was found to be basic, but without any apparent repeat structure. It is concluded that single proteolytically unprocessed MAP 1B molecules would have the potential to function as microtubule cross-linkers.     

Sensitive Immunoassay Shows Selective Association of Peripheral and Integral Membrane Proteins of the Inhibitory Glycine Receptor Complex

The inhibitory glycine receptor of mammalian spinal cord is a ligand-gated chloride channel that, on affinity purification, contains two subunits of 48-kilodalton (kD) and 58-kD molecular mass in addition to an associated 93-kD protein. Ligand-binding 48-kD subunit and 93-kD protein were quantified in the CNS of the adult rat using a newly developed dot receptor assay (detection limit less than or equal to 1 fmol/assay) which employs monoclonal antibodies specific for glycine receptor polypeptides. The 93-kD protein was found to codistribute at a fixed stoichiometry with the 48-kD subunit throughout the CNS of the rat. Moreover, the 93-kD protein cofractionated with the ligand-binding subunit on solubilization and affinity chromatography or immunoprecipitation. However, both proteins were separated on sucrose gradient centrifugation of detergent extracts of spinal cord membranes in accord with earlier observations on purified receptor. These data prove that the 93-kD polypeptide is selectively associated with the membrane core of the strychnine-sensitive glycine receptor. The regional distribution of glycine receptor polypeptides was also determined in the CNS of the spastic rat mutant. In contrast to hereditary spasticity in mouse and cattle, no reduction of glycine receptors was found in the spastic rat.     

cDNA sequence of the rat U snRNP-associated protein N: description of a potential Sm epitope.

Anti-Sm antibodies from a patient with systemic lupus erythematosus (SLE) were used to isolate cDNA clones encoding the snRNP-associated protein N from a rat brain derived cDNA library. The predicted primary structure of the 240 amino acid protein has a proline rich carboxyl terminus and shares a region of sequence similarity with other snRNP polypeptides, A and B/B'. Anti-Sm sera recognize a beta-galactosidase fusion protein containing only the carboxyl-terminal 80 amino acids of N; antibodies eluted from this fusion protein also react with A, B/B' and N on immunoblots, suggesting that these proteins share an Sm epitope located within this segment. Polyclonal antibodies raised against a 23 amino acid synthetic peptide derived from this conserved region of N recognize A, N and B/B' on immunoblots and can immunoprecipitate the Sm class of U snRNAs. These results confirm that this sequence defines a potential Sm epitope. RNA blotting analyses demonstrate that a 1.6 kb mRNA expressed predominantly in brain encodes the N polypeptide in both rats and humans. At low stringency rat N cDNA also hybridizes to a 1.3 kb mRNA species which encodes B/B', suggesting that N is structurally related to, but distinct from B/B'. Although B/B' proteins are thought to be expressed in all human cells, only N and B, but not B', are observed on immunoblots of human brain proteins probed with anti-Sm sera. The apparent difference in the complement of proteins associated with snRNP particles in human brain versus elsewhere suggests a possible mechanism for the regulation of brain-specific mRNA splicing.     

Isolation, localization, and cloning of a kainic acid binding protein from frog brain

Excitatory amino acids (EAA) are major neurotransmitters in the vertebrate central nervous system. EAA receptors have been divided into three major subtypes on the basis of electrophysiological and ligand binding studies: N-methyl-D-aspartate, kainate, and quisqualate receptors. To understand their molecular properties, we undertook a project aimed at isolation and cloning of these receptor subtypes. We purified a kainate binding protein (KBP) from frog brain, in which kainate binding sites are about fortyfold more abundant than in rat brain, using domoic acid affinity chromatography, and made monoclonal and polyclonal antibodies to the purified protein. These antibodies immunoprecipitate the frog KBP but not KBPs from other species. Immunocytochemical analyses show that KBP has a synaptic and extrasynaptic localization in frog optic tectum, with most labeling being extrasynaptic. The cDNA encoding frog brain KBP was isolated by screening a frog brain cDNA library with oligonucleotide probes that were based on the amino acid sequence of the purified protein. The deduced amino acid sequence of the KBP has a hydrophobic profile similar to those of other ligand-gated ion channel subunits, such as the nicotinic acetylcholine receptor, the GABAA receptor, and the glycine receptor. Frog brain KBP is very similar (36% amino acid identity to the carboxyl half) to rat brain kainate receptor, suggesting that these two proteins evolved from a common ancestor. The function of KBP in frog brain remains a major question. Preliminary results showed that Xenopus laevis oocytes injected with KBP RNA did not produce a detectable electrophysiological response when perfused with kainate. These results suggest that additional subunits may be required to form a functional receptor or that KBP is not functionally related to a neurotransmitter receptor.     

Molecular cloning and analysis of an eIF-4A-related rat liver nuclear protein.

In this paper, we report the cloning and analysis of a cDNA encoding a protein of M(r) congruent to 47,000 (p47), which is localized to the nucleus of rat hepatocytes. The cDNA showed 37% overall sequence identity with a mouse translation initiation factor, eIF-4A, which belongs to a family of putative ATP-dependent RNA helicases. We raised polyclonal antibodies against the fusion protein and by indirect immunofluorescence on primary cultures of hepatocytes have demonstrated that p47 is located in the nucleus. Although only approximately 27% of hepatocytes showed this nuclear staining, most of the nuclei in proliferating transformed cell lines such as 3T3, PtK-1, and Hela were fluorescently labeled. Studies on serum-starved cells in culture indicated that p47 was expressed in a cell cycle-dependent manner. Northern analyses demonstrated that the levels of p47 mRNA were high in fetal liver and dropped significantly after birth to low levels in adult liver. Our data suggest that p47 is developmentally regulated in rat liver at the mRNA level.     

Microtubule-associated protein 2 within axons of spinal motor neurons: associations with microtubules and neurofilaments in normal and beta,beta'-iminodipropionitrile-treated axons

We have examined the distribution of microtubule-associated protein 2 (MAP2) in the lumbar segment of spinal cord, ventral and dorsal roots, and dorsal root ganglia of control and beta,beta'-iminodipropionitrile- treated rats. The peroxidase-antiperoxidase technique was used for light and electron microscopic immunohistochemical studies with two monoclonal antibodies directed against different epitopes of Chinese hamster brain MAP2, designated AP9 and AP13. MAP2 immunoreactivity was present in axons of spinal motor neurons, but was not detected in axons of white matter tracts of spinal cord and in the majority of axons of the dorsal root. A gradient of staining intensity among dendrites, cell bodies, and axons of spinal motor neurons was present, with dendrites staining most intensely and axons the least. While dendrites and cell bodies of all neurons in the spinal cord were intensely positive, neurons of the dorsal root ganglia were variably stained. The axons of labeled dorsal root ganglion cells were intensely labeled up to their bifurcation; beyond this point, while only occasional central processes in dorsal roots were weakly stained, the majority of peripheral processes in spinal nerves were positive. beta,beta'- Iminodipropionitrile produced segregation of microtubules and membranous organelles from neurofilaments in the peripheral nervous system portion and accumulation of neurofilaments in the central nervous system portion of spinal motor axons. While both anti-MAP2 hybridoma antibodies co-localized with microtubules in the central nervous system portion, only one co-localized with microtubules in the peripheral nervous system portion of spinal motor axons, while the other antibody co-localized with neurofilaments and did not stain the central region of the axon which contained microtubules. These findings suggest that (a) MAP2 is present in axons of spinal motor neurons, albeit in a lower concentration or in a different form than is present in dendrites, and (b) the MAP2 in axons interacts with both microtubules and neurofilaments.     

Expression of 5α-reductase in bacteria as a trp E fusion protein and its use in the production of antibodies for immunocytochemical localization of 5α-reductase

A cDNA encoding a full-length rat 5α-reductase was isolated using female rat liver mRNA and the polymerase chain reaction, and fused to the Escherichia coli trp E gene in a pATH expression vector. The trp E-5α-reductase fusion protein expressed in bacteria and a synthetic oligopeptide corresponding to the C-terminus of rat 5α-reductase were used as antigens to produce rabbit polyclonal antibodies to 5α-reductase. Antibodies to the 5α-reductase portion of the fusion protein and to the peptide were purified by affinity chromatography. Antibodies against the 5α-reductase fusion protein reacted with a single component of rat liver microsomes with M_r 26,000 on Western blots, consistent with the size of 5α-reductase predicted from its cDNA, and with a M_r 23,000 component on Western blots of detergent extracts of rat ventral prostate nuclei; other rat ventral prostate cellular fractions (mitochondrial, microsomal, cytosol) bound little or no antibody. Antibody against the synthetic peptide reacted with a M_r 26,000 component of rat liver microsomes as well as with several components in various cellular fractions of rat ventral prostate. With anti-5α-reductase fusion protein antibodies, specific immunocytochemical staining was observed in the epithelial cell nuclei of the rat ventral prostate, seminal vesicle, epididymis and other accessory sex glands. This nuclear staining was specific, since antibodies from non-immunized rabbits did not give nuclear staining and preincubation of the anti-5α-reductase fusion protein antibodies with the trp E-5α-reductase fusion protein eliminated nuclear staining. Incubation of antibodies with trp E (without the 5α-reductase fusion) had no effect on nuclear staining. Specific staining was not detected in the cytoplasm of these epithelial cells. Little or no specific staining was observed in stromal cells in these rat tissuess. Human prostate was also immunocytochemically stained with this antibody. Specific staining was found in both epithelial and stromal cell nuclei.     

A green fluorescent protein kinase substrate allowing detection and localization of intracellular ERK/MAP kinase activity

We describe a versatile intracellular reporter of ERK/MAP kinase activity: a cDNA construct, pGFP.MBP, encoding amino acids 85-144 of the human myelin basic protein fused to the C-terminus of an enhanced green fluorescent protein (GFP). The fused fragment of myelin basic protein contains a single consensus ERK/MAP kinase phosphorylation motif (PRTP, where the threonine is phosphorylated). Phosphorylation of the specific motif can be detected via immunoblotting or immunofluorescence with a commercially available phospho-specific monoclonal antibody. When expressed in mammalian cells by either transient or stable transfection, the fusion protein acts as a bona fide kinase substrate, as demonstrated by rapid serum-induced phosphorylation that is blocked by a specific MEK inhibitor. Moreover, the localization of the total substrate pool is easily visualized by GFP autofluorescence and the extent of its phosphorylation simultaneously detected within intact fixed cells by immunofluorescence using the commercially available phospho-specific antibody. The approach described should be generally applicable to the intracellular analysis of many specific protein kinase substrates for which phospho-specific antibodies have been produced.     

Monoclonal antiidiotypic antibodies against delta opioid receptors as an electron microscopy probe.

Four different rat monoclonal antibodies were produced against delta opioid receptor using an antiidiotypic approach in which antibodies directed against the opioid agonist DADLE were used as immunogen. In the first step, seven hybridomas were selected on the basis of their ability to inhibit the DADLE-anti-DADLE antibody interaction. After purification from ascitic fluids, these monoclonal antibodies were characterized. Four antiidiotypic antibodies, named 5, 11, 16, and 51, directed toward different epitopes, recognized the delta opioid receptor: (i) they bound directly to the NG108-15 cells, (ii) they inhibited the [3H]DADLE binding on the NG108-15 cells, (iii) they immunoprecipitated a 52,500 dalton protein present on the surface of the NG108-15 cells. The four monoclonal antiidiotypic anti-opioid receptor antibodies were used to immunocytologically detect the opioid receptors under light and electron microscopy in the rat spinal cord. The regional distribution of the immunoreactivity corresponded to layers known to be rich delta opioid receptor subtype. Moreover, at the ultrastructural level, the labeling was located mainly on plasma membranes, especially on non-synaptic zones. Our results show that monoclonal antiidiotypic antibodies constitute a valuable tool for visualizing cell surface receptors.     

Cloning of a glycine receptor subtype expressed in rat brain and spinal cord during a specific period of neuronal development.

Complementary (c) DNAs encoding a glycine receptor (GlyR) isomer were cloned from libraries constructed in lambda ZAPII with poly (A)+ RNA of neonatal rat spinal cord. Northern blot analysis revealed that RNA hybridized to the cloned cDNA is detectable only for a period of late embryonic/early postnatal stage of the spinal cord. Moreover, other central nervous tissues, such as hippocampus and cerebral cortex, in the infant rats are also rich in this message. The 'neonatal (N) GlyR' has 71% homology to that of another GlyR isoform in which adult rad cord is rich (AGlyR). Injection of a single RNA transcribed from the NGlyr-cDNA into Xenopus oocyte induced functional formation of glycine-gated Cl- channels, however, its pharmacological property differed from that of AGlyR.