Characterization of a transneuronal cytokine family Cbln − regulation of secretion by heteromeric assembly

Abstract

Cbln1, a member of the C1q and tumor necrosis factor superfamily, plays crucial roles as a cerebellar granule cell-derived transneuronal regulator of synapse integrity and plasticity in Purkinje cells. Although other Cbln family members, Cbln2-Cbln4, have distinct spatial and temporal patterns of expression throughout the CNS, their biochemical and biological properties have remained largely uncharacterized. Here, we demonstrated that in mammalian heterologous cells, Cbln2 and Cbln4 were secreted as N-linked glycoproteins, like Cbln1. In contrast, despite the presence of a functional signal sequence, Cbln3 was not secreted when expressed alone but was retained in the endoplasmic reticulum (ER) or cis-Golgi because of its N-terminal domain. All members of the Cbln family formed not only homomeric but also heteromeric complexes with each other in vitro. Accordingly, when Cbln1 and Cbln3 were co-expressed in heterologous cells, a proportion of the Cbln1 proteins was retained in the ER or cis-Golgi; conversely, some Cbln3 proteins were secreted together with Cbln1. Similarly, in wild-type granule cells expressing Cbln1 and Cbln3, Cbln3 proteins were partially secreted and reached postsynaptic sites on Purkinje cell dendrites, while Cbln3 was almost completely degraded in cbln1-null granule cells. These results indicate that like Cbln1, Cbln2 and Cbln4 may also serve as transneuronal regulators of synaptic functions in various brain regions. Furthermore, heteromer formation between Cbln1 and Cbln3 in cerebellar granule cells may modulate each other's trafficking and signaling pathways; similarly, heteromerization of other Cbln family proteins may also have biological significance in other neurons.

Activity-Dependent Repression of Cbln1 Expression: Mechanism for Developmental and Homeostatic Regulation of Synapses in the Cerebellum

“…1-4). Although the precise role of Cbln3 in vivo remains unclear, one of its putative functions is the negative regulation of the release of Cbln1 through the formation of Cbln1-Cbln3 heteromers, which are mostly retained in the soma (Iijima et al, 2007). Interestingly, the high-K ϩ -induced repression of cbln3 mRNA was slightly but significantly delayed by that of cbln1 mRNA (Fig.…”

“…Cultured cerebellar granule cells were lysed with buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing 0.1% SDS and a protease inhibitor mixture mix (Roche Applied Science), and the soluble fractions were analyzed by immunoblotting with anti-Cbln1 or anti-Cbln3 antibody (rabbit; 1:250), which were kindly provided by Dr. Masahiko Watanabe (Hokkaido University, Sapporo, Japan) (Iijima et al, 2007;Miura et al, 2009). Horseradish peroxidase-conjugated secondary antibody and the enhanced chemiluminescence kit (both from GE Healthcare) were used for these analyses.…”

Cerebellin and des-cerebellin exert ACTH-like effects on corticosterone secretion and the intracellular signaling pathway gene expression in cultured rat adrenocortical cells - DNA microarray and QPCR studies

“…Cbln3 binds specifically to Cbln1 (6) and recently Cbln4 was identified (7) (NCBI accession number NM_175631). All members of the Cbln family form homomeric and also heteromeric complexes with each other in vitro and it was suggested that such complexes play a crucial role in normal development of the cerebellum (2,8). However, not only entire Cbln molecules are able to act as a transneuronal cytokines.…”

Precerebellin-related genes and precerebellin 1 peptide in the adrenal gland of the rat: Expression pattern, localization, developmental regulation and effects on corticosteroidogenesis

“…At present, Cbln are recognized as a new class of transneuronal regulators of synapse development and synaptic plasticity in various brain regions (6,8). With great probability this action depends on direct paracrine effects of Cbln complexes on adjacent synapses or nervous cells.…”

“…Precerebellins belong to the C1q/TNF superfamily of secreted proteins and, like other members of the family, they may form homomeric and also heteromeric complexes with each other in vitro (6)(7)(8)(9)(10)(11)(12). At present, Cbln are recognized as a new class of transneuronal regulators of synapse development and synaptic plasticity in various brain regions (6,8).…”