Roles of Meltrin beta /ADAM19 in the processing of neuregulin

Meltrin beta/ADAM19 is a member of ADAMs (a disintegrin and metalloproteases), which are a family of membrane-anchored glycoproteins that play important roles in fertilization, myoblast fusion, neurogenesis, and proteolytic processing of several membrane-anchored proteins. The expression pattern of meltrin beta during mouse development coincided well with that of neuregulin-1 (NRG), a member of the epidermal growth factor family. Then we examined whether meltrin beta participates in the proteolytic processing of membrane-anchored NRGs. When NRG-beta1 was expressed in mouse L929 cells, its extracellular domain was constitutively processed and released into the culture medium. This basal processing activity was remarkably potentiated by overexpression of wild-type meltrin beta, which lead to the significant decrease in the cell surface exposure of extracellular domains of NRG-beta1. Furthermore, expression of protease-deficient mutants of meltrin beta exerted dominant negative effects on the basal processing of NRG-beta1. These results indicate that meltrin beta participates in the processing of NRG-beta1. Since meltrin beta affected the processing of NRG-beta4 but not that of NRG-alpha2, meltrin beta was considered to have a preference for beta-type NRGs as substrate. Furthermore, the effects of the secretory pathway inhibitors suggested that meltrin beta participates in the intracellular processing of NRGs rather than the cleavage on the cell surface.     

Intracellular amyloid-beta 1-42, but not extracellular soluble amyloid-beta peptides, induces neuronal apoptosis

Alzheimer disease (AD), the most frequent cause of dementia, is characterized by an important neuronal loss. A typical histological hallmark of AD is the extracellular deposition of beta-amyloid peptide (A beta), which is produced by the cleavage of the amyloid precursor protein (APP). Most of the gene mutations that segregate with the inherited forms of AD result in increasing the ratio of A beta 42/A beta 40 production. A beta 42 also accumulates in neurons of AD patients. Altogether, these data strongly suggest that the neuronal production of A beta 42 is a critical event in AD, but the intraneuronal A beta 42 toxicity has never been demonstrated. Here, we report that the long term expression of human APP in rat cortical neurons induces apoptosis. Although APP processing leads to production of extracellular A beta 1-40 and soluble APP, these extracellular derivatives do not induce neuronal death. On the contrary, neurons undergo apoptosis as soon as they accumulate intracellular A beta 1-42 following the expression of full-length APP or a C-terminal deleted APP isoform. The inhibition of intraneuronal A beta 1-42 production by a functional gamma-secretase inhibitor increases neuronal survival. Therefore, the accumulation of intraneuronal A beta 1-42 is the key event in the neurodegenerative process that we observed.     

KIF2beta, a new kinesin superfamily protein in non-neuronal cells, is associated with lysosomes and may be implicated in their centrifugal translocation.

Lysosomes concentrate juxtanuclearly in the region around the microtubule-organizing center by interaction with microtubules. Different experimental and physiological conditions can induce these organelles to move to the cell periphery by a mechanism implying a plus-end-directed microtubule-motor protein (a kinesin-like motor). The responsible kinesin-superfamily protein, however, is unknown. We have identified a new mouse isoform of the kinesin superfamily, KIF2beta, an alternatively spliced isoform of the known, neuronal kinesin, KIF2. Developmental expression pattern and cell-type analysis in vivo and in vitro reveal that KIF2beta is abundant at early developmental stages of the hippocampus but is then downregulated in differentiated neuronal cells, and it is mainly or uniquely expressed in non-neuronal cells while KIF2 remains exclusively neuronal. Electron microscopy of mouse fibroblasts and immunofluorescence of KIF2beta-transiently-transfected fibroblasts show KIF2 and KIF2beta primarily associated with lysosomes, and this association can be disrupted by detergent treatment. In KIF2beta-overexpressing cells, lysosomes (labeled with anti-lysosome-associated membrane protein-1) become abnormally large and peripherally located at some distance from their usual perinuclear positions. Overexpression of KIF2 or KIF2beta does not change the size or distribution of early, late and recycling endosomes nor does overexpression of different kinesin superfamily proteins result in changes in lysosome size or positioning. These results implicate KIF2beta as a motor responsible for the peripheral translocation of lysosomes.     

Endogenous meltrin alpha is ubiquitously expressed and associated with the plasma membrane but exogenous meltrin alpha is retained in the endoplasmic reticulum

Meltrin alpha (ADAM12) is a member of the ADAM (MDC) protein family characterized by the presence of metalloprotease and disintegrin domains. ADAM proteins contain single transmembrane domains, and the processed mature proteins are postulated to span the plasma membrane. It has been reported that transfection of a truncated meltrin alpha cDNA lacking the prodomain and metalloprotease domain promotes skeletal muscle cell fusion. We show here that meltrin alpha was constitutively expressed in both undifferentiated and differentiated C2 skeletal muscle cells and also in fibroblasts. Both its precursor and processed mature forms were present in these cells. Thus, meltrin alpha may play general roles in addition to its roles in myogenesis. Since endogenous meltrin alpha cannot be detected by immunofluorescence microscopy, we examined the location of the exogenously expressed protein by transfection. Unexpectedly, the exogenously expressed meltrin alpha was located to a network structure of the endoplasmic reticulum (ER) but not to the plasma membrane. Cell fractionation revealed that the intrinsic mature protein was associated with the plasma membrane. However, the exogenously expressed protein remained unprocessed. These results seem to imply that the exogenously expressed meltrin alpha is not translocated from the ER to the trans-Golgi network, where a processing enzyme resides, and that it is consequently not converted to the mature form. Thus, the transfected meltrin alpha is unlikely to exert its physiological functions. Conversely, the ER may serve as a reservoir of the latent form of intrinsic meltrin alpha.     

Guanylyl cyclase/PSD-95 interaction: targeting of the nitric oxide-sensitive alpha2beta1 guanylyl cyclase to synaptic membranes

The signaling molecule nitric oxide (NO) exerts most of its effects by the stimulation of the NO-sensitive guanylyl cyclase. Two isoforms of the NO receptor molecule exist: the ubiquitously occurring alpha(1)beta(1) and the alpha(2)beta(1) with a more limited distribution. As the isoforms are functionally indistinguishable, the physiological relevance of these isoforms remained unclear. The neuronal NO synthase has been reported to be associated with PSD-95. Here, we demonstrate the interaction of the so far unnoticed alpha(2)beta(1) isoform with PSD-95 in rat brain as shown by coprecipitation. The interaction is mediated by the alpha(2) C-terminal peptide and the third PDZ domain of PSD-95. As a consequence of the PSD-95 interaction, the so far considered "soluble" alpha(2)beta(1) isoform is recruited to the membrane fraction of synaptosomes, whereas the alpha(1)beta(1) isoform is found in the cytosol. Our results establish the alpha(1)beta(1) as the cytosolic and the alpha(2)beta(1) as the membrane-associated NO-sensitive guanylyl cyclase and suggest the alpha(2)beta(1) isoform as the sensor for the NO formed by the PSD-95-associated neuronal NO synthase.     

Guanylyl cyclase: NO hits its target

The NO receptor, NO-sensitive guanylyl cyclase, plays a key role in the NO/cGMP signal-transduction cascade. Two isoforms of the enzyme are currently known, the widely distributed vascular alpha1beta1 isoform and the neuronal alpha2beta1 isoform predominantly expressed in brain. Interaction with the PSD-95 (postsynaptic density protein-95) family of scaffolding proteins targets the neuronal alpha2beta1 isoform to synaptic membranes. The NO sensor of the guanylyl cyclase is formed by the prosthetic haem group, where NO binding takes place and induces the up to 200-fold activation of the enzyme. The haem group allows tight regulation of enzymic activity by NO and represents the most striking feature of the enzyme, as it differs in many aspects from the well-characterized haem groups of other haemoproteins. The new NO sensitizers such as YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole] affect activation by NO and CO by mechanisms that are currently subject to intense research.     

Identification and characterization of a novel splice variant of beta3 subunit of GABA(A) receptor in rat testis and spermatozoa

Gamma-aminobutyric acid type A (GABA(A)) receptors are the major sites of inhibitory action of fast synaptic neurotransmission in the brain. Their receptors are also widely distributed in peripheral and endocrine tissues. A full-length cDNA encoding a novel splice variant of beta3 subunit of GABA(A) receptor, designated as beta3t, was identified in rat testis. This isoform contains a segment, having identical amino acid sequence as the beta3 subunit of neuronal GABA(A) receptors except for a section composed of 25 different amino acid sequence in the N-terminus. Northern blot shows that this isoform is found in rat testis. The beta3t isoform mRNA was detected in germ cells in the late step of spermatogenesis by in situ hybridization assay. Results of immunohistochemical and immunocytochemical assays indicate that the beta3t isoform is expressed in rat testis and spermatozoa. To determine a possible function of the N-terminal 25 amino acid segment, a recombinant plasmid of beta3t-EGFPC was constructed by fusing green fluorescent protein to the C-terminus of the beta3t isoform. The chimera product failed to be translocated unto the cell surface when expressed in HEK 293 cells; whereas, the beta3 subunit of rat brain is incorporated into the plasma membrane. In conclusion, the present results show that one variant of beta3 subunit of GABA(A) receptor, designated as beta3t, is found in germ cells of rat testis and sperm. The inability of the beta3t variant to target into the plasma membrane maybe a consequence of the unique 25 amino acid segment in the N-terminus.     

Dystrophin Dp71f associates with the beta1-integrin adhesion complex to modulate PC12 cell adhesion

Dystrophin Dp71 is the main product of the Duchenne muscular dystrophy gene in the brain; however, its function is unknown. To study the role of Dp71 in neuronal cells, we previously generated by antisense treatment PC12 neuronal cell clones with decreased Dp71 expression (antisense-Dp71 cells). PC12 cells express two different splicing isoforms of Dp71, a cytoplasmic variant called Dp71f and a nuclear isoform called Dp71d. We previously reported that antisense-Dp71 cells display deficient adhesion to substrate and reduced immunostaining of beta1-integrin in the cell area contacting the substrate. In this study, we isolated additional antisense-Dp71 clones to analyze in detail the potential involvement of Dp71f isoform with the beta1-integrin adhesion system of PC12 cells. Immunofluorescence analyses as well as immunoprecipitation assays demonstrated that the PC12 cell beta1-integrin adhesion complex is composed of beta1-integrin, talin, paxillin, alpha-actinin, FAK and actin. In addition, our results showed that Dp71f associates with most of the beta1-integrin complex components (beta1-integrin, FAK, alpha-actinin, talin and actin). In the antisense-Dp71 cells, the deficiency of Dp71 provokes a significant reduction of the beta1-integrin adhesion complex and, consequently, the deficient adhesion of these cells to laminin. In vitro binding experiments confirmed the interaction of Dp71f with FAK and beta1-integrin. Our data indicate that Dp71f is a structural component of the beta1-integrin adhesion complex of PC12 cells that modulates PC12 cell adhesion by conferring proper complex assembly and/or maintenance.     

Functional significance of isoform diversification in the protocadherin gamma gene cluster

The mammalian Protocadherin (Pcdh) alpha, beta, and gamma gene clusters encode a large family of cadherin-like transmembrane proteins that are differentially expressed in individual neurons. The 22 isoforms of the Pcdhg gene cluster are diversified into A-, B-, and C-types, and the C-type isoforms differ from all other clustered Pcdhs in sequence and expression. Here, we show that mice lacking the three C-type isoforms are phenotypically indistinguishable from the Pcdhg null mutants, displaying virtually identical cellular and synaptic alterations resulting from neuronal apoptosis. By contrast, mice lacking three A-type isoforms exhibit no detectable phenotypes. Remarkably, however, genetically blocking apoptosis rescues the neonatal lethality of the C-type isoform knockouts, but not that of the Pcdhg null mutants. We conclude that the role of the Pcdhg gene cluster in neuronal survival is primarily, if not specifically, mediated by its C-type isoforms, whereas a separate role essential for postnatal development, likely in neuronal wiring, requires isoform diversity.     

Versican V1 isoform induces neuronal differentiation and promotes neurite outgrowth

The chondroitin sulfate proteoglycan versican is one of the major extracellular components in the developing and adult brain. Here, we show that isoforms of versican play different roles in neuronal differentiation and neurite outgrowth. Expression of versican V1 isoform in PC12 cells induced complete differentiation, whereas expression of V2 induced an aborted differentiation accompanied by apoptosis. V1 promoted neurite outgrowth of hippocampal neurons, but V2 failed to do so. V1 transfection enhanced expression of epidermal growth factor receptor and integrins, and facilitated sustained extracellular signal-regulated kinase/MAPK phosphorylation. Blockade of the epidermal growth factor receptor, beta1 integrin, or Src significantly inhibited neuronal differentiation. Finally, we demonstrated that versican V1 isoform also promoted differentiation of neural stem cells into neurons. Our results have implications for understanding how versican regulates neuronal development, function, and repair.     

Major occurrence of the new alpha2beta1 isoform of NO-sensitive guanylyl cyclase in brain

NO-sensitive guanylyl cyclase (GC) acts as the effector molecule for NO and therefore plays a key role in the NO/cGMP signalling cascade. Besides the long known GC isoform (alpha(1)beta(1)), another heterodimer (alpha(2)beta(1)) has recently been identified to be associated with PSD-95 in brain.Here, we report on the tissue distribution of all known guanylyl cyclase subunits to elucidate the isoform content in different tissues of the mouse. The guanylyl cyclase subunit levels were assessed with quantitative real-time PCR, and the most important results were verified in Western blots. We demonstrate the major occurrence of the alpha(2)beta(1) heterodimer in brain, find a significant amount in lung and lower amounts in all other tissues tested. In brain, the levels of the alpha(2)beta(1) and alpha(1)beta(1) isoforms were comparable; in all other tissues, the alpha(1)beta(1) heterodimer was the predominating isoform. The highest guanylyl cyclase content was found in lung; here the GC amounted to approximately twice as much as in brain.In sum, the major occurrence of the alpha(2)beta(1) heterodimer suggests a special role in synaptic transmission; whether this isoform outside the brain also occurs in neuronal networks has to be addressed in future studies.     

Characterization of a novel synGAP isoform, synGAP-beta

We cloned a cDNA encoding a novel synGAP, synGAP-d (GenBank(TM) accession number ), from a rat brain cDNA library. The clone consisted of 4801 nucleotides with a coding sequence of 3501 nucleotides, encoded a protein consisting of 1166 amino acids with >99% homology with 1092 amino acid overlaps to synGAP, and contained a 13-nucleotide insertion to the previously reported synGAP mRNAs, which suggested that the clone was a splice variant of synGAP. We also found that there are at least seven variants in the 3' portion of the synGAP mRNA and that they encoded five different protein isoforms. The coding sequence of these C-terminal variants were classified into alpha1, alpha2, beta1, beta2, beta3, beta4, and gamma, and synGAP-d was classified as the beta1 form. The previously reported synGAPs (synGAP-a, -b, and -c and p135synGAP) can be classified as the alpha1 isoform. All isoforms were expressed specifically in the brain. Unexpectedly, the beta isoform, which lacks a C-terminal PSD-95-binding motif ((S/T)XV), was more restricted to the postsynaptic density fraction than the motif-containing alpha1 isoform. The beta isoform did not interact with PSD-95 but specifically interacted with a nonphosphorylated alpha subunit of Ca(2+)/calmodulin-dependent protein kinase II through its unique C-terminal tail.     

Regulation and function of glycogen synthase kinase-3 isoforms in neuronal survival

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase consisting of two isoforms, alpha and beta. The activities of GSK-3 are regulated negatively by serine phosphorylation but positively by tyrosine phosphorylation. GSK-3 inactivation has been proposed as a mechanism to promote neuronal survival. We used GSK-3 isoform-specific small interfering RNAs, dominant-negative mutants, or pharmacological inhibitors to search for functions of the two GSK-3 isoforms in regulating neuronal survival in cultured cortical neurons in response to glutamate insult or during neuronal maturation/aging. Surprisingly, RNA interference-induced depletion of either isoform was sufficient to block glutamate-induced excitotoxicity, and the resulting neuroprotection was associated with enhanced N-terminal serine phosphorylation in both GSK-3 isoforms. However, GSK-3beta depletion was more effective than GSK-3alpha depletion in suppressing spontaneous neuronal death in extended culture. This phenomenon is likely due to selective and robust inhibition of GSK-3beta activation resulting from GSK-3beta Ser9 dephosphorylation during the course of spontaneous neuronal death. GSK-3alpha silencing resulted in reduced tyrosine phosphorylation of GSK-3beta, suggesting that tyrosine phosphorylation is also a critical autoregulatory event. Interestingly, GSK-3 inhibitors caused a rapid and long-lasting increase in GSK-3alpha Ser21 phosphorylation levels, followed by a delayed increase in GSK-3beta Ser9 phosphorylation and a decrease in GSK-3alpha Tyr279 and GSK-3beta Tyr216 phosphorylation, thus implying additional levels of GSK-3 autoregulation. Taken together, our results underscore important similarities and dissimilarities of GSK-3alpha and GSK-3beta in the roles of cell survival as well as their distinct modes of regulation. The development of GSK-3 isoform-specific inhibitors seems to be warranted for treating GSK-3-mediated pathology.     

The receptor tyrosine phosphatase-like protein ICA512 binds the PDZ domains of beta2-syntrophin and nNOS in pancreatic beta-cells

Islet cell autoantigen (ICA) 512 of type I diabetes is a receptor tyrosine phosphatase-like protein associated with the secretory granules of neurons and endocrine cells including insulin-secreting beta-cells of the pancreas. Here we show that in a yeast two-hybrid assay its cytoplasmic domain binds beta2-syntrophin, a modular adapter which in muscle cells interacts with members of the dystrophin family including utrophin, as well as the signaling molecule neuronal nitric oxide synthase (nNOS). The cDNA isolated by two-hybrid screening corresponded to a novel beta2-syntrophin isoform with a predicted molecular mass of 28 kDa. This isoform included the PDZ domain, but not the C-terminal region, which in full-length beta2-syntrophin is responsible for binding dystrophin-related proteins. In vitro binding of the beta2-syntrophin PDZ domain to ICA512 required both ICA512's C-terminal region and an internal polypeptide preceding its tyrosine phosphatase-like domain. Immunomicroscopy and co-immunoprecipitations from insulinoma INS-1 cells confirmed the occurrence of ICA512-beta2-syntrophin complexes in vivo. ICA512 also interacted in vitro with the PDZ domain of nNOS and ICA512-nNOS complexes were co-immunoprecipitated from INS-1 cells. Finally, we show that INS-1 cells, like muscle cells, contain beta2-syntrophin-utrophin oligomers. Thus, we propose that ICA512, through beta2-syntrophin and nNOS, links secretory granules with the actin cytoskeleton and signaling pathways involving nitric oxide.     

Differential localization and sequence analysis of capping protein beta- subunit isoforms of vertebrates

Capping protein nucleates the assembly of actin filaments and stabilizes actin filaments by binding to their barbed ends. We describe here a novel isoform of the beta subunit of chicken capping protein, the beta 2 isoform, which arises by alternative splicing. The chicken beta 1 isoform and the beta 2 isoform are identical in their amino acid sequence except for a short region at the COOH terminus; this region of the beta subunit has been implicated in binding actin. Human and mouse cDNAs of the beta 1 and beta 2 isoforms also were isolated and among these vertebrates, the COOH-terminal region of each isoform is highly conserved. In contrast, comparison of the sequences of the vertebrate beta subunit COOH-termini to those of lower eukaryotes shows no similarities. The beta 2 isoform is the predominant isoform of nonmuscle tissues and the beta 1 isoform, which was first characterized in studies of capping protein from chicken muscle, is the predominant isoform of muscle tissues, as shown by immunoblots probed with isoform- specific antibodies and by RNAse protection analysis of mRNAs. The beta 2 isoform also is a component of dynactin complex from brain, which contains the actin-related protein Arp1. Both beta-subunit isoforms are expressed in cardiac muscle but they have non-overlapping subcellular distributions. The beta 1 isoform is at Z-discs of myofibrils, and the beta 2 isoform is enriched at intercalated discs; in cardiac myocytes grown in culture, the beta 2 isoform also is a component of cell-cell junctions and at sites where myofibrils contact the sarcolemma. The biochemical basis for the differential distribution of capping protein isoforms is likely due to interaction with specific proteins at Z-discs and cell-cell junctions, or to preferential association with different actin isoforms. Thus, vertebrates have developed isoforms of capping protein that associate with distinct actin-filament arrays.     

The Na,K-ATPase alpha 2 isoform is expressed in neurons,and its absence disrupts neuronal activity in newborn mice

Na,K-ATPase is an ion transporter that impacts neural and glial physiology by direct electrogenic activity and the modulation of ion gradients. Its three isoforms in brain have cell-type and development-specific expression patterns. Interestingly, our studies demonstrate that in late gestation, the alpha2 isoform is widely expressed in neurons, unlike in the adult brain, in which alpha2 has been shown to be expressed primarily in astrocytes. This unexpected distribution of alpha2 isoform expression in neurons is interesting in light of our examination of mice lacking the alpha2 isoform which fail to survive after birth. These animals showed no movement; however, defects in gross brain development, muscle contractility, neuromuscular transmission, and lung development were ruled out. Akinesia suggests a primary neuronal defect and electrophysiological recordings in the pre-B?tzinger complex, the brainstem breathing center, showed reduction of respiratory rhythm activity, with less regular and smaller population bursts. These data demonstrate that the Na,K-ATPase alpha2 isoform could be important in the modulation of neuronal activity in the neonate.