Meltrin Beta Research Articles

Meltrin β/ADAM19 Interacting with EphA4 in Developing Neural Cells Participates in Formation of the Neuromuscular Junction

BackgroundDevelopment of the neuromuscular junction (NMJ) is initiated by the formation of postsynaptic specializations in the central zones of muscles, followed by the arrival of motor nerve terminals opposite the postsynaptic regions. The post- and presynaptic components are then stabilized and modified to form mature synapses. Roles of ADAM (A Disintegrin And Metalloprotease) family proteins in the formation of the NMJ have not been reported previously.Principal FindingsWe report here that Meltrin β, ADAM19, participates in the formation of the NMJ. The zone of acetylcholine receptor α mRNA distribution was broader and excess sprouting of motor nerve terminals was more prominent in meltrin β–deficient than in wild-type embryonic diaphragms. A microarray analysis revealed that the preferential distribution of ephrin-A5 mRNA in the synaptic region of muscles was aberrant in the meltrin β–deficient muscles. Excess sprouting of motor nerve terminals was also found in ephrin-A5 knockout mice, which lead us to investigate a possible link between Meltrin β and ephrin-A5-Eph signaling in the development of the NMJ. Meltrin β and EphA4 interacted with each other in developing motor neurons, and both of these proteins localized in the NMJ. Coexpression of Meltrin β and EphA4 strongly blocked vesicular internalization of ephrin-A5–EphA4 complexes without requiring the protease activity of Meltrin β, suggesting a regulatory role of Meltrin β in ephrin-A5-Eph signaling.ConclusionMeltrin β plays a regulatory role in formation of the NMJ. The endocytosis of ephrin-Eph complexes is required for efficient contact-dependent repulsion between ephrin and Eph. We propose that Meltrin β stabilizes the interaction between ephrin-A5 and EphA4 by regulating endocytosis of the ephrinA5-EphA complex negatively, which would contribute to the fine-tuning of the NMJ during development.

Meltrin β (ADAM19) mediates ectodomain shedding of Neuregulin β1 in the Golgi apparatus: fluorescence correlation spectroscopic observation of the dynamics of ectodomain shedding in living cells

Membrane-anchored Neuregulin beta1 sheds its ectodomain as soluble factors. Two proteases that belong to a disintegrin and metalloprotease (ADAM) family are known to cleave Neuregulin beta1. One is tumor necrosis factor-alpha converting enzyme (TACE/ADAM17). The other is Meltrin beta (ADAM19). Against our expectation that shedding by ADAM proteases occurs at the cell surface, here we found that Meltrin beta mediates the ectodomain shedding of Neuregulin beta1 in the Golgi apparatus. Meltrin beta was localized in and around the Golgi apparatus in developing sensory neurons. Subcellular fractionation revealed that Meltrin beta generated soluble Neuregulin beta1 in Golgi-enriched fractions while TACE-cleaved Neuregulin beta1 was recovered in lighter fractions. To examine whether Meltrin beta-mediated ectodomain shedding occurs in the Golgi apparatus in living cells, we took advantage of different diffusion properties of cleavage products from those of membrane-anchored precursor proteins. Fluorescence correlation spectroscopy (FCS) is the most sensitive method to determine milli approximately submillisecond diffusion in vivo. Protease-active Meltrin beta caused a shift in autocorrelation function in FCS of green fluorescent protein (GFP)-tagged Neuregulin beta1 in the Golgi apparatus, suggesting a conversion of Neuregulin beta1 molecules from membrane-anchored to soluble forms in that organelle. The Golgi apparatus is a site of processing Neuregulin beta1 by Meltrin beta.

Developmental and hormonal regulation of meltrin beta (ADAM19) expression in mouse testes during embryonic and postnatal life

More than half of ADAM (a disintegrin and metalloprotease) family members are expressed in mammalian male reproductive organs such as testis and epididymis. The ADAM19 gene identified in mouse is a member of the ADAM family and is highly enriched in testes of a newborn mouse. The present study was performed to determine its expression pattern in whole mouse testes in vivo as well as its in vitro action and regulation in testis cells from 2-day-old mice. Reverse transcriptase polymerase chain reaction (RT-PCR) detected ADAM19 mRNA from 15.5 days postcoitum (dpc) to 21 days postpartum (dpp), with high expression during the perinatal period. Immunohistochemistry demonstrated ADAM19 protein localization to the seminiferous cords at both embryonic and postnatal ages examined (from 15.5–19.5 dpc to 2 dpp). In particular, we obtained new evidence that a neutralizing antibody to ADAM19 had no influence on the proliferation of 2 dpp testis cells cultured in serum-free medium when compared to controls. Interestingly, it inhibited the 2 dpp testis cell proliferation elicited by stimulation with 10% FCS ( P < 0.01) or FSH ( P < 0.05). Lastly, using a model of 2 dpp testis cell cultures and RT-PCR procedures, we demonstrated that follicle stimulating-hormone (FSH) reduced the levels of ADAM19 mRNA in a time-dependent manner. Taken together, these results indicate that the expression of ADAM19 may be subject to regulation by FSH during mouse testis development. Furthermore, ADAM19 can act to regulate the proliferation of perinatal testis cells in the perinatal period.

Intracellular Activation of Human Adamalysin 19/Disintegrin and Metalloproteinase 19 by Furin Occurs via One of the Two Consecutive Recognition Sites

Adamalysin 19 (a disintegrin and metalloproteinase 19, ADAM19, or meltrin beta) is a plasma membrane metalloproteinase. Human ADAM19 zymogen contains two potential furin recognition sites (RX(K/R)R), (196)KRPR(200)R and (199)RRMK(203)R, between its pro- and catalytic domains. Protein N-terminal sequencing revealed that the cellular mature forms of hADAM19 started at (204)EDLNSMK, demonstrating that the preferred furin cleavage site was the (200)RMK(203)R downward arrow(204)EDLN. Those mature forms were catalytically active. Both Pittsburgh mutant of alpha(1)-proteinase inhibitor and dec-Arg-Val-Lys-Arg-chloromethyl ketone, two specific furin inhibitors, blocked the activation of hADAM19. Activation of hADAM19 was also blocked by brefeldin A, which inhibits protein trafficking from the endoplasmic reticulum to the Golgi, or, a calcium ionophore known to inhibit the autoactivation of furin. When (202)KR were mutated to AA, the proenzyme was also activated, suggesting that (197)RPRR is an alternative activation site. Furthermore, only pro-forms of hADAM19 were detected in the (199)RR to AA mutant, which abolished both furin recognition sites. Moreover, the zymogens were not converted into their active forms in two furin-deficient mammalian cell lines; co-expression of hADAM19 and furin in these two cell lines restored zymogen activation. Finally, co-localization between furin and hADAM19 was identified in the endoplasmic reticulum-Golgi complex and/or the trans-Golgi network. This report is the first thorough investigation of the intracellular activation of adamalysin 19, demonstrating that furin activated pro-hADAM19 in the secretory pathway via one of the two consecutive furin recognition sites.

Roles of Meltrin β/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.

Cloning and Initial Characterization of Mouse Meltrin β and Analysis of the Expression of Four MetalloproteaseDisintegrins in Bone Cells

Here we report the cloning and initial biochemical characterization of the mouse metalloprotease/disintegrin/cysteine-rich (MDC) protein meltrin beta and the analysis of the mRNA expression of four MDC genes (meltrin alpha, meltrin beta, mdc9, and mdc15) in bone cells, including osteoclasts and osteoblasts. Like most other MDC proteins, the predicted meltrin beta protein consists of a signal sequence, prodomain, metalloprotease domain with a predicted catalytic site, disintegrin domain, cysteine-rich region, epidermal growth factor repeat, transmembrane domain, and cytoplasmic domain with putative signaling motifs, such as potential SH3 ligand domains. Northern blot analysis indicates that meltrin beta is widely expressed, with the highest expression in bone, heart, and lung. RNase protection studies revealed expression of all four MDC genes analyzed here in osteoblasts, whereas only mdc9 and mdc15 mRNAs were detectable in osteoclast-like cells generated in vitro. Treatment of primary osteoblasts with 10 nM calcitriol increased meltrin beta expression more than 3-fold, and both meltrin alpha and meltrin beta expression is apparently regulated in a differentiation-associated manner in a mouse osteoblastic cell line, MC3T3E1. Collectively, these results suggest that meltrin alpha and meltrin beta may play a role in osteoblast differentiation and/or function but are not likely to be involved in osteoclast fusion.