Abstract
Innervation of skeletal muscles results in expression of adult-type nicotinic acetylcholine receptors (alpha 2 beta epsilon delta) beneath the neuromuscular junction. This local expression is largely a result of selective induction of adult-type nicotinic acetylcholine receptor (nAChR) genes in endplate-associated myonuclei. The molecular mechanism by which the nerve induces gene expression in these nuclei is not known. We have shown previously that ionophore-induced calcium influx across the plasma membrane preferentially decreases expression from the adult-type specific nAChR epsilon-subunit gene (Walke, W., Staple, J., Adams, L., Gnegy, M., Chahine, K., and Goldman, D. (1994) J. Biol. Chem. 269, 19447-19456). Here we provide evidence that the genes encoding adult-type nAChRs are specifically regulated by protein-tyrosine phosphatase activity. Orthovanadate, a specific protein-tyrosine phosphatase inhibitor, caused increased expression of the epsilon-subunit gene in rat primary myotubes and was able to completely block the suppressive effects of increased calcium influx on epsilon-subunit RNA expression. Overexpression of protein-tyrosine phosphatases selectively decreased expression from the adult-type nAChR genes with no effect on the embryonic-type specific gamma-subunit gene. These results demonstrate that protein-tyrosine phosphatases regulate mammalian adult-type nAChR gene expression and suggest a mechanism by which muscle innervation selectively regulates gene expression in endplate-associated myonuclei.
Highlights
Musclaectivity-dependenstuppression of extrasynaptic nicotinic acetylcholine receptor (nAChR) gene expression has been linked to a calcium-dependent protein kinase C pathway in chick [8,9,10] and a CAMP
We have shown previously that this phosphorylation mediates ARIA-induced increasesin thaitonophore-inducedcalciuminfluaxcrostshe nAChR subunit mRNAs
Orthovanadate, a specifsieclectively regulated by protein-tyrosine phosphatase activity protein-tyrosinpehosphatasienhibitorc,auseidnand have linked this activity to mediating the effects of increased expressionof the esubunit gene in rat primary creased calcium influx on €-gene expression
Summary
This 850-bp fragment was subcloned into the luciferase expression vector, pXPl[20]. A second y-subunit promoter construct (pXPly 388) contains a 388-bp fragment of y-subunit gene spanning. The e-subunit promoterfluciferase expression construct, as described previously [14], contains about 5kilobase pairs. The minimal enkephalin promoter/chloramphenicol acetyltransferase expression vector (ENK72CAT) used to control for variability in transfection efficiency has been described previously [27]. 7l-ansfections-Myotube cultures were transfected by calcium phosphate precipitation [28, 29] with pg/60-mm plate of esubunit promoterfluciferase expression constructand 20 pg/60-mm plate of ENK72CAT. Transfections for PTP overexpression studies were carried out using 5 pg/60-mm plate of test plasmid (a-,y-, ti-, or c-subunit promoterfluciferase expression vector) along with 30 pg/60-mm plate of PTP lD, PTP IDM, PTP lB, or PTP CLlOO and 20 pg/60-mm plate of ENK72CAT. To perform luciferase and CAT assays [30,31], Vanadate (@M)
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