Biomedical subjects

Shuzo Sugita

Publications and source records attributed to Shuzo Sugita.

10 recordsLinked to original sources

PubMed2007

RalA and RalB function as the critical GTP sensors for GTP-dependent exocytosis.

Although it has been established that the activation of GTPases by non-hydrolyzable GTP stimulates neurotransmitter release from many different secretory cell types, the underlying mechanisms remain unclear. In the present study we aimed to elucidate the functional role(s) for endogenous Ras-like protein A (RalA) and RalB GTPases in GTP-dependent exocytosis. For this purpose stable neuroendocrine pheochromocytoma 12 (PC12) cell lines were generated in which the expressions of both RalA and RalB were strongly downregulated. In these double knock-down cells GTP-dependent exocytosis was reduced severely and was restored after the expression of RalA or RalB was reintroduced by transfection. In contrast, Ca2+-dependent exocytosis and the docking of dense core vesicles analyzed by electron microscopy remained unchanged in the double knock-down cells. Furthermore, the transfected RalA and RalB appeared to be localized primarily on the dense core vesicles in undifferentiated and nerve growth factor-differentiated PC12 cells. Our results indicate that endogenous RalA and RalB function specifically as GTP sensors for the GTP-dependent exocytosis of dense core vesicles, but they are not required for the general secretory pathways, including tethering of vesicles to the plasma membrane and Ca2+-dependent exocytosis.

Animals↗

PubMed2006

Recent advances in basic neurosciences and brain disease: from synapses to behavior.

Understanding basic neuronal mechanisms hold the hope for future treatment of brain disease. The 1st international conference on synapse, memory, drug addiction and pain was held in beautiful downtown Toronto, Canada on August 21-23, 2006. Unlike other traditional conferences, this new meeting focused on three major aims: (1) to promote new and cutting edge research in neuroscience; (2) to encourage international information exchange and scientific collaborations; and (3) to provide a platform for active scientists to discuss new findings. Up to 64 investigators presented their recent discoveries, from basic synaptic mechanisms to genes related to human brain disease. This meeting was in part sponsored by Molecular Pain, together with University of Toronto (Faculty of Medicine, Department of Physiology as well as Center for the Study of Pain). Our goal for this meeting is to promote future active scientific collaborations and improve human health through fundamental basic neuroscience researches. The second international meeting on Neurons and Brain Disease will be held in Toronto (August 29-31, 2007).

Behavior↗

PubMed2006

Crystal structure of the second LNS/LG domain from neurexin 1alpha: Ca2+ binding and the effects of alternative splicing.

Neurexins mediate protein interactions at the synapse, playing an essential role in synaptic function. Extracellular domains of neurexins, and their fragments, bind a distinct profile of different proteins regulated by alternative splicing and Ca2+. The crystal structure of n1alpha_LNS#2 (the second LNS/LG domain of bovine neurexin 1alpha) reveals large structural differences compared with n1alpha_LNS#6 (or n1beta_LNS), the only other LNS/LG domain for which a structure has been determined. The differences overlap the so-called hyper-variable surface, the putative protein interaction surface that is reshaped as a result of alternative splicing. A Ca2+-binding site is revealed at the center of the hyper-variable surface next to splice insertion sites. Isothermal titration calorimetry indicates that the Ca2+-binding site in n1alpha_LNS#2 has low affinity (Kd approximately 400 microm). Ca2+ binding ceases to be measurable when an 8- or 15-residue splice insert is present at the splice site SS#2 indicating that alternative splicing can affect Ca2+-binding sites of neurexin LNS/LG domains. Our studies initiate a framework for the putative protein interaction sites of neurexin LNS/LG domains. This framework is essential to understand how incorporation of alternative splice inserts expands the information from a limited set of neurexin genes to produce a large array of synaptic adhesion molecules with potentially very different synaptic function.

Alternative Splicing↗

PubMed2005

N-terminal insertion and C-terminal ankyrin-like repeats of alpha-latrotoxin are critical for Ca2+-dependent exocytosis.

Alpha-latrotoxin, a potent stimulator of exocytosis from neurons and neuroendocrine cells, has been studied intensively, but the mechanisms of its actions are poorly understood. Here, we developed a new method to generate active recombinant alpha-latrotoxin and conducted a structure/function analysis of the toxin in stimulating Ca2+-dependent exocytosis. alpha-Latrotoxin consists of a conserved N-terminal domain and C-terminal ankyrin-like repeats. After cleavage of an N-terminally fused purification tag of glutathione S-transferase (GST), the recombinant toxin strongly stimulated exocytosis, whereas the GST-fused toxin was much less potent. The GST-fused toxin bound to the receptors [neurexin 1alpha; CL1 (CIRL/latrophilin 1)] as efficiently as did the GST-cleaved toxin but was much less effective in inserting into the plasma membrane and inducing cation conductance. The toxin with deletion of the last two ankyrin-like repeats still bound the receptors but could neither stimulate exocytosis nor induce cation conductance efficiently. The abilities of the mutated toxins to stimulate exocytosis correlated well with their abilities to induce cation conductance, but not their binding to the receptors. Our results indicate that (1) C-terminal ankyrin-like repeats and a free (unfused) N terminus are both required for the toxin to form pores, which is essential for Ca2+-dependent exocytosis, and (2) receptor binding alone is not sufficient to stimulate Ca2+-dependent exocytosis.

Animals↗

PubMed2005

A central kinase domain of type I phosphatidylinositol phosphate kinases is sufficient to prime exocytosis: isoform specificity and its underlying mechanism.

Exocytosis, a critical process for neuronal communication and hormonal regulation, involves several distinct steps including MgATP-dependent priming (which involves the synthesis of phosphatidylinositol 4,5-bisphosphate). Type I phosphatidylinositol phosphate kinases (PIPKIs) were purified biochemically as a priming factor. PIPKI consists of three domains: the N-terminal region, the central kinase domain, and the C-terminal region. Three isoforms (alpha, beta, and gamma) of PIPKI have been identified, and each is alternatively spliced at the C-terminal region. In the present study, we conducted a structure/function analysis of PIPKIs in the priming of exocytosis, and we found that recombinant PIPKIalpha and PIPKIgamma had priming activity. However, an unexpected finding of these results was that PIPKIbeta did not prime exocytosis. The N- or C-terminal region of PIPKIalpha and PIPKIgamma was not required for priming, which indicates that the central kinase domain is sufficient for this process. Alternative splicing in each isoform did not affect the isoform specificity in priming. Priming activity by isoforms is strongly correlated with their phosphatidylinositol phosphate kinase activity because PIPKIalpha and PIPKIgamma had higher kinase activity than PIPKIbeta. These results suggest that PIPKIalpha and PIPKIgamma are the critical priming factors for exocytosis; it also suggests that the levels of phosphatidylinositol phosphate kinase activity in producing phosphatidylinositol 4,5-bisphosphate specify the function of PIPKI isoforms in priming.

Amino Acid Sequence↗

PubMed2004

RalA-exocyst interaction mediates GTP-dependent exocytosis.

Many secretory cells utilize a GTP-dependent pathway, in addition to the well characterized Ca2+-dependent pathway, to trigger exocytotic secretion. However, little is currently known about the mechanism by which this may occur. Here we show the key signaling pathway that mediates GTP-dependent exocytosis. Incubation of permeabilized PC12 cells with soluble RalA GTPase, but not RhoA or Rab3A GTPases, strongly inhibited GTP-dependent exocytosis. A Ral-binding fragment from Sec5, a component of the exocyst complex, showed a similar inhibition. Point mutations in both RalA (RalA(E38R)) and the Sec5 (Sec5(T11A)) fragment, which abolish RalA-Sec5 interaction also abolished the inhibition of GTP-dependent exocytosis. Moreover, transfection with wild-type RalA, but not RalA(E38R), enhanced GTP-dependent exocytosis. In contrast the RalA and the Sec5 fragment showed no inhibition of Ca2+-dependent exocytosis, but cleavage of a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein by Botulinum neurotoxin blocked both GTP- and Ca2+-dependent exocytosis. Our results indicate that the interaction between RalA and the exocyst complex (containing Sec5) is essential for GTP-dependent exocytosis. Furthermore, GTP- and Ca2+-dependent exocytosis use different sensors and effectors for triggering exocytosis whereas their final fusion steps are both SNARE-dependent.

Animals↗

PubMed2004

Human growth hormone co-transfection assay to study molecular mechanisms of neurosecretion in PC12 cells.

The recent introduction of transient co-transfection assays, with human growth hormone (hGH) as a marker of transfection, has allowed us to study the function of transfected proteins in neurosecretion using intact PC12 cells and adrenal chromaffin cells [J. Biol Chem. 268 (1993) 10983]. Since this assay requires several non-trivial steps (transfection of the genes, triggering of secretion, and measurement of hGH), care should be taken before attributing any observed effect to the transfected proteins. In this article, we describe a detailed procedure for the hGH transfection assay and show that this approach can be used to study the molecular events involved in neurosecretion.

Animals↗

PubMed2003

A conformational switch in the Piccolo C2A domain regulated by alternative splicing.

C2 domains are widespread Ca2+-binding modules. The active zone protein Piccolo (also known as Aczonin) contains an unusual C2A domain that exhibits a low affinity for Ca2+, a Ca2+-induced conformational change and Ca2+-dependent dimerization. We show here that removal of a nine-residue sequence by alternative splicing increases the Ca2+ affinity, abolishes the conformational change and abrogates dimerization of the Piccolo C2A domain. The NMR structure of the Ca2+-free long variant provides a structural basis for these different properties of the two splice forms, showing that the nine-residue sequence forms a beta-strand otherwise occupied by a nonspliced sequence. Consequently, Ca2+-binding to the long Piccolo C2A domain requires a marked rearrangement of secondary structure that cannot occur for the short variant. These results reveal a novel mechanism of action of C2 domains and uncover a structural principle that may underlie the alteration of protein function by short alternatively spliced sequences.

Alternative Splicing↗

PubMed2003

Sr2+ binding to the Ca2+ binding site of the synaptotagmin 1 C2B domain triggers fast exocytosis without stimulating SNARE interactions.

Sr(2+) triggers neurotransmitter release similar to Ca(2+), but less efficiently. We now show that in synaptotagmin 1 knockout mice, the fast component of both Ca(2+)- and Sr(2+)-induced release is selectively impaired, suggesting that both cations partly act by binding to synaptotagmin 1. Both the C(2)A and the C(2)B domain of synaptotagmin 1 bind Ca(2+) in phospholipid complexes, but only the C(2)B domain forms Sr(2+)/phospholipid complexes; therefore, Sr(2+) binding to the C(2)B domain is sufficient to trigger fast release, although with decreased efficacy. Ca(2+) induces binding of the synaptotagmin C(2) domains to SNARE proteins, whereas Sr(2+) even at high concentrations does not. Thus, triggering of the fast component of release by Sr(2+) as a Ca(2+) agonist involves the formation of synaptotagmin/phospholipid complexes, but does not require stimulated SNARE binding.

Animals↗

PubMed2002

Synaptotagmins form a hierarchy of exocytotic Ca(2+) sensors with distinct Ca(2+) affinities.

Synaptotagmins constitute a large family of membrane proteins implicated in Ca(2+)-triggered exocytosis. Structurally similar synaptotagmins are differentially localized either to secretory vesicles or to plasma membranes, suggesting distinct functions. Using measurements of the Ca(2+) affinities of synaptotagmin C2-domains in a complex with phospholipids, we now show that different synaptotagmins exhibit distinct Ca(2+) affinities, with plasma membrane synaptotagmins binding Ca(2+) with a 5- to 10-fold higher affinity than vesicular synaptotagmins. To test whether these differences in Ca(2+) affinities are functionally important, we examined the effects of synaptotagmin C2-domains on Ca(2+)-triggered exocytosis in permeabilized PC12 cells. A precise correlation was observed between the apparent Ca(2+) affinities of synaptotagmins in the presence of phospholipids and their action in PC12 cell exocytosis. This was extended to PC12 cell exocytosis triggered by Sr(2+), which was also selectively affected by high-affinity C2-domains of synaptotagmins. Together, our results suggest that Ca(2+) triggering of exocytosis involves tandem Ca(2+) sensors provided by distinct plasma membrane and vesicular synaptotagmins. According to this hypothesis, plasma membrane synaptotagmins represent high-affinity Ca(2+) sensors involved in slow Ca(2+)-dependent exocytosis, whereas vesicular synaptotagmins function as low-affinity Ca(2+) sensors specialized for fast Ca(2+)-dependent exocytosis.

Animals↗