Tyrosine kinase inhibitors alter composition of nicotinic receptors on neurons.

Protein tyrosine kinase (PTK) inhibitors were used to examine the roles of tyrosine phosphorylation in synaptic function. We show here that two different PTK inhibitors, herbimycin A and lavendustin A, both selectively downregulate a subpopulation of nicotinic acetylcholine receptors (AChRs) on chick ciliary ganglion neurons in culture. The downregulation requires a number of hours to occur and involves only those receptors containing the alpha 3, alpha 5, and beta 4 gene products. Not affected are AchRs that additionally contain the beta 2 gene product or AchRs that are made up of the alpha 7 gene product. The downregulation preferentially targets receptors destined for the cell surface and has little effect on the large pool of intracellular receptors. The receptor loss is not additive with that seen in the presence of either cycloheximide or tunicamycin, two compounds that the block appearance of new receptors. The downregulation induced by herbimycin A in surface receptors is accompanied by a specific decrement in the amount of alpha 3 protein in the cells. The results indicate that PTKs, either by phosphorylating AChR gene products directly or by acting through intermediary proteins, regulate the size and composition of the AChR pool maintained on the cell surface. Receptor regulation by PTKs may provide a mechanism for long-term control of synaptic signaling between neurons.

Minimum intron requirements for tRNA splicing and nuclear transport in Xenopus oocytes.

The presence or absence of an intron defines two classes of eukaryotic nuclear tRNA genes whose transcripts differ in a requirement for splicing. Using quantitative nuclear microinjection, we have previously found that nucleocytoplasmic transport of these two classes of tRNAs involves pathways which differ in one or more limiting components. To examine substrate features which distinguish these two pathways, a series of variants of a Xenopus tRNA(Tyr) gene were constructed in which the intron size was altered. The splicing and transport properties of the resulting transcripts were examined in oocyte microinjection and in vitro processing assays. The addition of one or two nucleotides at the splice site equivalent in an intronless gene produced transcripts which could be transported without splicing. However, transport was reduced relative to the mature-sequence tRNA, suggesting the anticodon loop (interrupted in pre-tRNAs) may be recognized by the intronless tRNA transport apparatus. Transcripts with four- or six-nucleotide intervening sequences were incompletely spliced with cleavage at only the 3' splice site. Neither unspliced precursor nor partially processed intermediates were efficiently transported. The results of coinjection experiments using tRNA and pre-tRNA competitors suggest that simple retention by the splicing apparatus may not account for failure to export these RNAs. Finally, a requirement for splicing is not unique to transport of pre-tRNA(Tyr) since a pre-tRNA(3Leu) variant which was not spliced was also not exported.