Calmyrin1 binds to SCG10 protein (stathmin2) to modulate neurite outgrowth.

Calmyrin1 (CaMy1) is an EF-hand Ca(2+)-binding protein expressed in several cell types, including brain neurons. Using a yeast two-hybrid screen of a human fetal brain cDNA library, we identified SCG10 protein (stathmin2) as a CaMy1 partner. SCG10 is a microtubule-destabilizing factor involved in neuronal growth during brain development. We found increased mRNA and protein levels of CaMy1 during neuronal development, which paralleled the changes in SCG10 levels. In developing primary rat hippocampal neurons in culture, CaMy1 and SCG10 colocalized in cell soma, neurites, and growth cones. Pull-down, coimmunoprecipitation, and proximity ligation assays demonstrated that the interaction between CaMy1 and SCG10 is direct and Ca(2+)-dependent in vivo and requires the C-terminal domain of CaMy1 (residues 99-192) and the N-terminal domain of SCG10 (residues 1-35). CaMy1 did not interact with stathmin1, a protein that is homologous with SCG10 but lacks the N-terminal domain characteristic of SCG10. CaMy1 interfered with SCG10 inhibitory activity in a microtubule polymerization assay. Moreover, CaMy1 overexpression inhibited SCG10-mediated neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells. This CaMy1 activity did not occur when an N-terminally truncated SCG10 mutant unable to interact with CaMy1 was expressed. Altogether, these data suggest that CaMy1 via SCG10 couples Ca(2+) signals with the dynamics of microtubules during neuronal outgrowth in the developing brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

In vitro findings of alterations in intracellular calcium homeostasis in schizophrenia.

The pathogenesis of schizophrenia involves several complex cellular mechanisms and is not well understood. Recent research has demonstrated an association between primary disturbances characteristic of the disease, including altered dopaminergic and glutamatergic neurotransmission, and impairments in neuronal calcium (Ca(2+)) homeostasis and signaling. Emerging Ca(2+) hypothesis links and unifies various cellular processes involved in the pathogenesis of schizophrenia and suggests a central role of dysregulation of Ca(2+) homeostasis in the etiology of the disease. This review explores the in vitro data on Ca(2+) homeostasis and signaling in schizophrenia. Major limitation in this research is the lack of schizophrenia markers and validated disease models. As indicated in this review, one way to overcome these limitations may be analyses of Ca(2+) signalosomes in peripheral cells from schizophrenia patients. Validation of animal models of schizophrenia may permit the application of advanced Ca(2+) imaging techniques in living animals.

Resistance of the dopamine D4 receptor to agonist-induced internalization and degradation.

Dopamine receptors are G-protein-coupled receptors involved in the control of motivation, learning, and fine-tuning of motor movement, as well as modulation of neuroendocrine signalling. Stimulation of G-protein-coupled receptors normally results in attenuation of signalling through desensitization, followed by internalization and down-regulation of the receptor. These processes allow the cell to regain homeostasis after exposure to extracellular stimuli and offer protection against excessive signalling. Here, we have investigated the agonist-mediated attenuation properties of the dopamine D4 receptor. We found that several hallmarks of signal attenuation such as receptor phosphorylation, internalization and degradation showed a blunted response to agonist treatment. Moreover, we did not observe recruitment of beta-arrestins upon D4 receptor stimulation. We also provide evidence for the constitutive phosphorylation of two serine residues in the third intracellular loop of the D4 receptor. These data demonstrate that, when expressed in CHO, HeLa and HEK293 cells, the human D4 receptor shows resistance to agonist-mediated internalization and down-regulation. Data from neuronal cell lines, which have been reported to show low endogenous D4 receptor expression, such as the hippocampal cell line HT22 and primary rat hippocampal cells, further support these observations.

Biochemical characterization and expression analysis of a novel EF-hand Ca2+ binding protein calmyrin2 (Cib2) in brain indicates its function in NMDA receptor mediated Ca2+ signaling.

Calmyrin2 (CaMy2, Cib2) is a novel EF-hand calcium-binding protein found recently in skeletal muscles. CaMy2 mRNA was also detected in brain, but nothing is known about CaMy2 protein localization and properties in the brain. We report cloning and characterization of CaMy2 in rat brain: its expression pattern, intracellular localization and biochemical features. CaMy2 binds Ca2+ and exhibits Ca2+/conformational switch. Moreover, CaMy2 undergoes N-myristoylation without Ca2+/myristoyl switch, is membrane-associated and localizes in neurons together with Golgi apparatus and dendrite markers. CaMy2 transcript and protein are present mainly in the hippocampus and cortex. In cultured hippocampal neurons, CaMy2 is induced upon neuronal activation. Most prominent increase in CaMy2 protein (7-fold), and mRNA (2-fold) occurs upon stimulation of NMDA receptor (NMDAR). The induction is blocked by translation inhibitors, specific antagonists of NMDAR, the Ca2+-chelator BAPTA, and inhibitors of ERK1/2 and PKC, kinases transmitting NMDAR-linked Ca2+ signal. Our results show that CaMy2 level is controlled by NMDAR and Ca2+ and suggest CaMy2 role in Ca2+ signaling underlying NMDAR activation.