Retrograde transport by the microtubule-associated protein MAP 1C

Microtubules are involved in several forms of intracellular motility, including mitosis and organelle movement. Fast axonal transport is a highly ordered form of organelle motility that operates in both the anterograde (outwards from the cell body) and retrograde (from the periphery towards the cell body) direction. Similar microtubule-associated movement is observed in non-neuronal cells, and might be involved in secretion, endocytosis and the positioning of organelles within the cell. Kinesin is a mechanochemical protein that produces force along microtubules in an anterograde direction. We recently found that the brain microtubule-associated protein MAP 1C (ref. 7) is a microtubule-activated ATPase and, like kinesin, can translocate microtubules in an in vitro assay for microtubule-associated motility. MAP 1C seemed to be related to the ciliary and flagellar ATPase, dynein, which is thought to produce force in a direction opposite to that observed for kinesin. Here we report that MAP 1C, in fact, acts in a direction opposite to kinesin, and has the properties of a retrograde translocator.     

Potassium conductances in hippocampal neurons blocked by excitatory amino-acid transmitters

Excitatory amino acids mediate fast synaptic transmission in the central nervous system through the activation of at least three distinct ionotropic receptors: N-methyl-D-aspartate (NMDA), the alpha-amino-3-hydroxy-5-methyl-isoxasole-4-propionate (AMPA)/quisqualate (QUIS) and the kainate subtypes (for reviews, see refs 1, 2). They also activate the additional QUIS 'metabotropic' receptor (sensitive to trans-1-amino-cyclopentyl-1,3-dicarboxylate, ACPD) linked to inositol phospholipid metabolism. We have used hippocampal slice cultures to study the electrophysiological consequences of the metabotropic response. We find that activation of an ACPD-sensitive QUIS receptor produces a 'slow' excitation of CA3 pyramidal cells, resulting from depression of a Ca2(+)-dependent K+ current and a voltage-gated K+ current. Combined voltage-clamp and microfluorometric recordings show that, although these receptors can trigger an increase in intracellular Ca2+ concentration, suppression of K+ currents is independent of changes in intracellular Ca2+. These effects closely resemble those induced by activating muscarinic acetylcholine receptors in the same neurons and suggest that excitatory amino acids not only act as fast ionotropic transmitters but also as slow neuromodulatory transmitters.     

Alternative splicing directs the expression of two D2 dopamine receptor isoforms

Dopamine receptors are classified into D1 and D2 subtypes on the basis of their pharmacological properties and the intracellular responses they mediate. The cerebral D2 dopamine receptor is the target of drugs used to alleviate the main symptoms of schizophrenia. Although it is considered to be a single molecular entity, there is evidence that multiple D2-receptor subtypes exist. A complementary DNA encoding a D2 receptor has recently been cloned and the deduced 415-amino-acid sequence indicates that it belongs to the large superfamily of receptors coupled to G proteins, and that its topology consists of seven transmembrane domains. In this family, the genes are frequently without introns and each is believed to encode a unique polypeptide product. Here we show that the gene for the D2 receptor produces two receptor isoforms by alternative messenger RNA splicing, providing a route to receptor diversity in this family. One isoform corresponds to the D2(415) receptor, but the second contains an additional sequence encoding a 29-amino-acid fragment, defining a novel D2(444) receptor isoform. Expression of the two isoforms is tissue-specific, and both are regulated by guanyl nucleotides. As the extra sequence is located within a putative cytoplasmic loop that binds to G proteins, the two isoforms might interact with different G proteins and thereby initiate distinct intracellular signals.     

T-cell receptor delta-chain can substitute for alpha to form a beta delta heterodimer

Specific monoclonal antibodies have made possible the identification of two T-cell antigen receptor (TCR) heterodimers, alpha beta TCR and gamma delta TCR. Formation of these receptors is largely separated by the preferential pairing of alpha-TCR with beta and gamma-TCR with delta, the sequential rearrangement and expression of the TCR loci during thymic development and the deletion of the delta-loci either prior to or concomitant with alpha-rearrangement in alpha beta TCR cells. Here we show that delta-TCR can substitute for alpha in pairing with beta to form a beta delta heterodimer. This receptor is expressed on the cell surface of the T-leukaemia cell line DND41 as analysed with beta- and delta-specific monoclonal antibodies. We suggest that a variety of factors including, for example, the deletion of the delta-TCR loci, can now be understood as exclusion mechanisms operating to prevent not only the formation of gamma delta receptors, but also of beta delta T-cell receptors, thereby promoting the numerically dominant alpha beta TCR lineage. Nevertheless, some developing T-cells that do not rearrange the alpha-loci may express the beta delta TCR as described here.     

Coexistence of A- and B-form DNA in a single crystal lattice

It is well known that DNA can exist in a variety of conformations which can be interconverted by relatively mild changes in conditions. The in vivo conformation of DNA is usually thought to be the B form, but there is recent evidence that other conformations may be important in DNA-protein recognition. Different fragments of DNA crystallized under virtually identical conditions can form A, B or Z helices. A fragment that adopted an A conformation in a crystal was found in the B conformation in solution, whereas NMR spectroscopy of A-DNA films revealed the presence of a substantial amount of disordered B-DNA. Until now, however, a DNA fragment of a given sequence has not been crystallized in more than one global conformation. We report here an X-ray diffraction study of crystals of the DNA octamer dGGBrUABrUACC. In addition to a 'framework' of A-DNA, which gives discrete X-ray reflections, there are partially disordered B-DNA helices, recognized by their diffuse scattering features.     

Dynamin is a GTPase stimulated to high levels of activity by microtubules.

Dynamin was initially identified in calf brain tissue as a protein of relative molecular mass 100,000 which induced nucleotide-sensitive bundling of microtubules. Purified dynamin showed only trace ATPase activity. But in combination with an activating factor removed during the purification, it exhibited microtubule-activated ATPase activity and dynamin-induced bundles showed evidence of ATP-dependent force production. Dynamin is the product of the Drosophila gene shibire, which has been implicated in synaptic vesicle recycling and, more generally, in the budding of endocytic vesicles from the plasma membrane. Dynamin also shows extensive homology with proteins that participate in vacuolar protein sorting and spindle pole-body separation in yeast, and in interferon-induced viral resistance in mammals. All members of this family contain consensus sequence elements consistent with GTP binding near their amino termini, although none has been shown to have GTPase activity. We report here that dynamin is a specific GTPase which can be stimulated to very high levels of activity by microtubules.     

Potentiation of NMDA receptor currents by arachidonic acid.

Arachidonic acid is released by phospholipase A2 when activation of N-methyl-D-aspartate (NMDA) receptors by neurotransmitter glutamate raises the calcium concentration in neurons, for example during the initiation of long-term potentiation and during brain anoxia. Here we investigate the effect of arachidonic acid on glutamate-gated ion channels by whole-cell clamping isolated cerebellar granule cells. Arachidonic acid potentiates, and makes more transient, the current through NMDA receptor channels, and slightly reduces the current through non-NMDA receptor channels. Potentiation of the NMDA receptor current results from an increase in channel open probability, with no change in open channel current. We observe potentiation even with saturating levels of agonist at the glutamate- and glycine-binding sites on these channels; it does not result from conversion of arachidonic acid to lipoxygenase or cyclooxygenase derivatives, or from activation of protein kinase C. Arachidonic acid may act by binding to a site on the NMDA receptor, or by modifying the receptor's lipid environment. Our results suggest that arachidonic acid released by activation of NMDA (or other) receptors will potentiate NMDA receptor currents, and thus amplify increases in intracellular calcium concentration caused by glutamate. This may explain why inhibition of phospholipase A2 blocks the induction of long-term potentiation.     

Isolation and characterization of a cDNA clone encoding the murine homologue of the human 20K T3/T-cell receptor glycoprotein

The antigen receptor on the surface of human T lymphocytes, which consists of a heterodimer of relative molecular mass (Mr) 90,000 (90K) (alpha- and beta-chains), is associated with the T3 antigen (gamma = 25K, delta = 20K and epsilon = 20K). A working model for the mode of action of the T3/T-cell receptor complex is that the clonotypic alpha- and beta-chains are involved in the recognition and binding of antigen in the context of polymorphic major histocompatibility complex (MHC) gene products on the surface of target cells. Antigen binding by the clonotypic receptor probably results in conformational changes in this structure which are recognized by and subsequently trigger the associated T3 complex to transmit signals into the cell, resulting in a proliferative response. The similarity in structure between murine and human clonotypic antigen receptors suggests that such a mechanism of recognition and activation also exists in mouse T lymphocytes, but so far there has been no evidence for the existence of a murine T3 complex. Here we demonstrate the existence of a T3 delta-chain mRNA in murine T lymphocytes. Our sequence data strongly suggest that this mouse mRNA codes for a complete T3 delta polypeptide chain and reveal some interesting properties of the protein.