The developmentally regulated myelin basic proteins (MBPs), which arise from the golli (gene of oligodendrocyte lineage) complex, are highly positively charged, intrinsically disordered, multifunctional proteins having several alternatively spliced isoforms and posttranslational modifications, and they play key roles in myelin compaction. The classic 18.5-kDa MBP isoform has a proline-rich region comprising amino acids 92-99 (murine sequence -T(92)PRTPPPS(99)-) that contains a minimal SH3 ligand domain. We have previously shown that 18.5-kDa MBP binds to several SH3 domains, including that of Fyn, a member of the Src family of tyrosine kinases involved in a number of signaling pathways during CNS development. To determine the physiological role of this binding as well as the role of phosphorylation of Thr92 and Thr95, in the current study we have produced several MBP variants specifically targeting phosphorylation sites and key structural regions of MBP's SH3 ligand domain. Using isothermal titration calorimetry, we have demonstrated that, compared with the wild-type protein, these variants have lower affinity for the SH3 domain of Fyn. Moreover, overexpression of N-terminal-tagged GFP versions in immortalized oligodendroglial N19 and N20.1 cell cultures results in aberrant elongation of membrane processes and increased branching complexity and inhibits the ability of MBP to decrease Ca(2+) influx. Phosphorylation of Thr92 can also cause MBP to traffic to the nucleus, where it may participate in additional protein-protein interactions. Coexpression of MBP with a constitutively active form of Fyn kinase resulted in membrane process elaboration, a phenomenon that was abolished by point amino acid substitutions in MBP's SH3 ligand domain. These results suggest that MBP's SH3 ligand domain plays a key role in intracellular protein interactions in vivo and may be required for proper membrane elaboration of developing oligodendrocytes and, further, that phosphorylation of Thr92 and Thr95 can regulate this function.

The atomic parameters of the quartz form of germanium dioxide have been determined at room temperature from diffractometrically measured intensities with Me Ka radiation. Initial values of the parameters were obtained by Fourier techniques, and the anisotropic refinement was carried out by least-squares analysis to the unusually low R value of 1.9 %. The two independent Ge-O bond lengths are 1.737 ± 0.003 and 1.741 ± 0.002 _~; the independent O-Ge-O bond angles are 106.3 _ 0-1 ; 107.7 ±0-2, 110-4___0.1 and 113.1 ±0.1 °. The irregularity in the Gee 4 tetrahedron is thus clearly attributable to distortions in the bond angles. The Ge-O-Ge bond anglo of 130-1 _ 0-1 o is significantly smaller than the corresponding angle in SiO~. Analysis of the thermal ellipsoid data indicates that the vibration of germanium is very nearly isotropic; that of oxygen definitely anisotropic. The maximum r.m.s, displacement of oxygen is, as in SiO~, perpendicular to the plane defined by the given oxygen and the two germanium atoms which it links.

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The myelin basic protein (MBP) family arises from different transcription start sites of the golli (gene of oligodendrocyte lineage) complex, with further variety generated by differential splicing. The “classical” MBP isoforms are peripheral membrane proteins that facilitate compaction of the mature myelin sheath but also have multiple protein interactions. The early developmental golli isoforms have previously been shown to promote process extension and enhance Ca2+ influx into primary and immortalized oligodendrocyte cell lines. Here, we have performed similar studies with the classical 18.5- and 21.5-kDa isoforms of MBP. In contrast to golli proteins, overexpression of classical MBP isoforms significantly reduces Ca2+ influx in the oligodendrocyte cell line N19 as well as in primary cultures of oligodendroglial progenitor cells. Pharmacological experiments demonstrate that this effect is mediated by voltage-operated Ca2+ channels (VOCCs) and not by ligand-gated Ca2+ channels or Ca2+ release from intracellular stores. The pseudo-deiminated 18.5-kDa and the full-length 21.5-kDa isoforms do not reduce Ca2+ influx as much as the unmodified 18.5-kDa isoform. However, more efficient membrane localization (of overexpressed, pseudo-deiminated 18.5-kDa and 21.5-kDa isoforms of classical MBP containing the 21-nt 3′-untranslated region transit signal) further reduces the Ca2+ response after plasma membrane depolarization, suggesting that binding of classical MBP isoforms to the plasma membrane is important for modulation of Ca2+ homeostasis. Furthermore, we have found that the mature 18.5-kDa isoform expressed in oligodendrocytes colocalizes with VOCCs, particularly at the leading edge of extending membrane processes. In summary, our findings suggest a key role for classical MBP proteins in regulating voltage-gated Ca2+ channels at the plasma membrane of oligodendroglial cells and thus also in regulation of multiple developmental stages in this cell lineage.