Kinesin heavy chain and kinesin-related polypeptides (KRPs) comprise a family of motor proteins with diverse intracellular transport functions. Using pan-kinesin peptide antibodies that react with these proteins, we have previously purified from sea urchin eggs a trimeric microtubule-binding and bundling protein, KRP (85/95) (ref. 8) comprising subunits of M(r) 115,000 (115K), 95K and 85K. We report here that kinesin-related genes encode the 85K and 95K subunits, and that the protein can be immunoprecipitated from cytosol as a trimeric complex using an 85K monoclonal antibody. We also find that purified KRP(85/95) directs movements towards the 'plus' ends of microtubules. To our knowledge, this protein is the first kinesin-related motor to be purified from its natural host cell in a native multimeric state.

Plant cells assemble the bipolar spindle and phragmoplast microtubule (MT) arrays in the absence of the centrosome structure. Our recent findings in Arabidopsis thaliana indicated that AUGMIN subunit3 (AUG3), a homolog of animal dim gtubulin 3, plays a critical role in g-tubulin-dependent MT nucleation and amplification during mitosis. Here, we report the isolation of the entire plant augmin complex that contains eight subunits. Among them, AUG1 to AUG6 share low sequence similarity with their animal counterparts, but AUG7 and AUG8 share homology only with proteins of plant origin. Genetic analyses indicate that the AUG1, AUG2, AUG4, and AUG5 genes are essential, as stable mutations in these genes could only be transmitted to heterozygous plants. The sterile aug7-1 homozygous mutant in which AUG7 expression is significantly reduced exhibited pleiotropic phenotypes of seriously retarded vegetative and reproductive growth. The aug7-1 mutation caused delocalization of g-tubulin in the mitotic spindle and phragmoplast. Consequently, spindles were abnormally elongated, and their poles failed to converge, as MTs were splayed to discrete positions rendering deformed arrays. In addition, the mutant phragmoplasts often had disorganized MT bundles with uneven edges. We conclude that assembly of MT arrays during plant mitosis depends on the augmin complex, which includes two plant-specific subunits.

Cyclic GMP has been implicated as a messenger molecule involved in visual transduction. Photoexcited rhodopsin (R*) binds to a multisubunit membrane protein called transducin (T) and stimulates the exchange of a bound GDP molecule for GTP. This leads to the release of the alpha-subunit of T with bound GTP (T alpha-GTP), which activates a cyclic GMP phosphodiesterase. The question arises as to whether the hydrolysis of cyclic GMP that results from activation of the phosphodiesterase is sufficiently rapid to be involved in visual excitation, which occurs on a time scale of approximately 2 s in the single-photon limit. Previous studies have suggested that the cyclic GMP phosphodiesterase is activated in less than 100 ms at moderate light levels. We report here light scattering studies of magnetically orientated frog rod outer segments which show that a molecule of R* catalyses the activation of a molecule of T in about 1 ms. Thus, hundreds of molecules can be activated within the response time of vision in the single-photon limit, and the formation of T alpha-GTP is fast enough for it to be a key step in visual transduction.

Background The clinical efficacy of migraine therapeutic agents directed towards the calcitonin-gene related peptide (CGRP) pathway has confirmed the key role of this axis in migraine pathogenesis. Three antibodies against CGRP – fremanezumab, galcanezumab and eptinezumab – and one antibody against the CGRP receptor, erenumab, are clinically approved therapeutics for the prevention of migraine. In addition, two small molecule CGRP receptor antagonists, ubrogepant and rimegepant, are approved for acute migraine treatment. Targeting either the CGRP ligand or receptor is efficacious for migraine treatment; however, a comparison of the mechanism of action of these therapeutic agents is lacking in the literature. Methods To gain insights into the potential differences between these CGRP pathway therapeutics, we compared the effect of a CGRP ligand antibody (fremanezumab), a CGRP receptor antibody (erenumab) and a CGRP receptor small molecule antagonist (telcagepant) using a combination of binding, functional and imaging assays. Results Erenumab and telcagepant antagonized CGRP, adrenomedullin and intermedin cAMP signaling at the canonical human CGRP receptor. In contrast, fremanezumab only antagonized CGRP-induced cAMP signaling at the human CGRP receptor. In addition, erenumab, but not fremanezumab, bound and internalized at the canonical human CGRP receptor. Interestingly, erenumab also bound and internalized at the human AMY1 receptor, a CGRP receptor family member. Both erenumab and telcagepant antagonized amylin-induced cAMP signaling at the AMY1 receptor while fremanezumab did not affect amylin responses. Conclusion The therapeutic effect of agents targeting the CGRP ligand versus receptor for migraine prevention (antibodies) or acute treatment (gepants) may involve distinct mechanisms of action. These findings suggest that differing mechanisms could affect efficacy, safety, and/or tolerability in migraine patients.

In the retinal cyclic GMP phosphodiesterase (PDE), catalysis by the alpha beta-heterodimer is inhibited in the dark by two identical gamma-subunits and stimulated in the light by the GTP-bearing alpha-subunit of the heterotrimeric G-protein transducin (T beta gamma-T alpha GDP). Two T alpha GTP molecules, dissociated from T beta gamma, bind to and displace the PDE gamma subunits from their inhibitory sites on PDE alpha beta. With GTP gamma S in lieu of GTP, this association becomes persistent. Under physiological conditions, the PDE alpha beta (gamma T alpha)2 active complex stays on the membrane. But in low-salt buffers, it becomes soluble and dissociates into a partially active PDE alpha beta catalytic moiety and two PDE gamma-T alpha GTP gamma S complexes. This indicates that T alpha binds preferentially to PDE gamma. We have studied the interaction of recombinant bovine PDE gamma with purified T alpha in solution or with retinal rod outer segments (ROS) containing both T beta gamma-T alpha GDP and PDE alpha beta gamma 2. When added to dark ROS, recombinant PDE gamma did not bind to inactive PDE alpha beta gamma 2 but extracted T alpha GDP from membrane-bound holo-transducin to form a soluble PDE gamma-T alpha GDP complex. PDE gamma also bound to purified T alpha GDP in solution. The kinetics and affinity of the interaction between PDE gamma and T alpha GDP or T alpha GTP gamma S were determined by monitoring changes in the proteins' tryptophan fluorescence. The Kd's for the binding of recombinant PDE gamma to soluble T alpha GTP gamma S and T alpha GDP are < or = 0.1 and 3 nM, respectively. PDE gamma-T alpha GDP falls apart in 3 s. This slow dissociation means that, in situ, T alpha-PDE gamma cannot physically leave the active PDE alpha beta, since after GTP hydrolysis, an isolated T alpha-PDE gamma complex would dissociate too slowly to allow a fast PDE reinhibition by the liberated PDE gamma. When recombinant PDE gamma was added to PDE that had been persistently activated by T alpha GTP gamma S, reinhibition occurred and T alpha GTP gamma S, complexed to the native PDE gamma, was released, indicating that both had hitherto stayed bound to PDE alpha beta. The mutation W70F does not prevent recombinant PDE gamma from inhibiting PDE alpha beta but diminishes its affinity for T alpha GTP and T alpha GDP 100-fold.(ABSTRACT TRUNCATED AT 400 WORDS)