Coronin 3 and its role in murine brain morphogenesis.

Coronins belong to the fundamental WD40-repeat proteins. They are mainly found at the submembraneous area, they bind F-actin in vitro, and most of the seven mammalian coronins have unclear roles. Coronin 3 is abundantly expressed in the adult CNS. All murine brain areas express coronin 3 during embryogenesis and the first postnatal stages. Expression in grey matter decreases postnatally, except for hippocampal pyramidal and dentate gyrus neurons, and cerebellar Purkinje cells, while levels in white matter increase in the course of myelination. Consistently, coronin 3 is abundant in differentiating neuro-2a and PC-12 cells and in primary oligodendrocytes. Treatment with PKC activator PMA reduced coronin 3 protein levels. To address its functions, neuro-2a and PC-12 cells were transfected with GFP-tagged coronin 3 versions. Full-length coronin 3 among other areas localized to outgrowing neurites, whereas truncated proteins efficiently suppressed neurite formation. Our results favour a role for coronin 3 in neuron morphogenesis and possibly migration.

Coronin 7, the mammalian POD-1 homologue, localizes to the Golgi apparatus.

Coronins constitute an evolutionary conserved family of WD-repeat actin-binding proteins. Their primary function is thought to be regulating the actin cytoskeleton. Apart from that, several coronins were indirectly shown to participate in vesicular transport, establishment of cell polarity and cytokinesis. Here, we report a novel mammalian protein, coronin 7 (crn7), which is significantly different from other mammalian coronins in its domain architecture. Crn7 possesses two stretches of WD repeats in contrast to the other coronins only having one. The protein is expressed throughout the mouse embryogenesis and is strongly upregulated in brain and developing structures of the immune system in the course of development. In adult animals, both crn7 mRNA and protein are abundantly present in most organs, with significantly higher amounts in brain, kidney, thymus and spleen and lower amounts in muscle. At the subcellular level, the bulk of the protein appears to be present in the cytosol and in large cytosolic complexes. However, a significant portion of the protein is detected on vesicle-like cytoplasmic structures as well as on the cis-Golgi. In the Golgi region, crn7 staining appears broader than that of the cis-Golgi markers Erd2p and beta-COP, still, the trans-Golgi network appears predominantly crn7-negative. Importantly, the membrane-associated form of crn7 protein is phosphorylated on tyrosine residues, whereas the cytosolic form is not. Crn7 is the first coronin protein proven to localize to the Golgi membrane. We conclude that it plays a role in the organization of intracellular membrane compartments and vesicular trafficking rather than in remodeling the cytoskeleton.

Long-term-desensitization of prostacyclin receptors is independent of the C-terminal tail.

Persistent stimulation of the G(s) protein-coupled prostacyclin receptor (IP-R) causes its slow desensitization in a variety of cell types, a significant desensitization requiring several hours. To evaluate the role of the human IP-R C-terminus in desensitization and agonist-induced internalization, a C-terminally truncated hIP-receptor was generated. The C-terminal 68 amino acid residues were deleted by introduction of a stop codon for exchange of the original S319 codon (termed D318 mutant). Wild-type (WT) and truncated receptor were expressed in COS1 cells. Pretreatment of cells with the stable prostacyclin mimetic cicaprost (200 nM) desensitized cAMP production via WT and D318 receptors to similar extents. The cAMP response of WT and D318, respectively, was reduced by approximately 50% of maximal cAMP formation after 8 hr of continuous agonist stimulation, indicating significant long-term desensitization. Moreover, agonist-promoted sequestration of WT and D318 C-terminally tagged with green fluorescent protein was demonstrated, indicating that receptor internalization was not prevented by truncation of the C-terminus. These results demonstrated that long-term desensitization and sequestration of hIP-R did not depend on structures located in the hIP-R C-terminus.

Oligomerization, F-actin interaction, and membrane association of the ubiquitous mammalian coronin 3 are mediated by its carboxyl terminus.

Coronin 3 is a ubiquitously expressed member of the coronin protein family in mammals. In fibroblasts and HEK 293 cells, it is localized both in the cytosol and in the submembranous cytoskeleton, especially at lamellipodia and membrane ruffles. The carboxyl terminus of all coronins contains a coiled coil suggested to mediate dimerization. We show here that in contrast to other coronin homologues, the recombinant human coronin 3 carboxyl terminus forms oligomers rather than dimers, and that this part is sufficient to bind to and cross-link F-actin in vitro. The carboxyl terminus alone also conferred membrane association in vivo, and removal of the coiled coil abolished membrane localization but not in vitro F-actin binding. Coronin 3 is exclusively extracted as an oligomer from both the cytosol and the membrane fraction. Because oligomerization was not reported for other coronins, it might be a key feature governing coronin 3-specific functions. Cytosolic coronin 3 showed a high degree of phosphorylation, which is likely to regulate the subcellular localization of the protein.