PKCα binds G3BP2 and regulates stress granule formation following cellular stress.

Protein kinase C (PKC) isoforms regulate a number of processes crucial for the fate of a cell. In this study we identify previously unrecognized interaction partners of PKCα and a novel role for PKCα in the regulation of stress granule formation during cellular stress. Three RNA-binding proteins, cytoplasmic poly(A)(+) binding protein (PABPC1), IGF-II mRNA binding protein 3 (IGF2BP3), and RasGAP binding protein 2 (G3BP2) all co-precipitate with PKCα. RNase treatment abolished the association with IGF2BP3 and PABPC1 whereas the PKCα-G3BP2 interaction was largely resistant to this. Furthermore, interactions between recombinant PKCα and G3BP2 indicated that the interaction is direct and PKCα can phosphorylate G3BP2 in vitro. The binding is mediated via the regulatory domain of PKCα and the C-terminal RNA-binding domain of G3BP2. Both proteins relocate to and co-localize in stress granules, but not to P-bodies, when cells are subjected to stress. Heat shock-induced stress granule assembly and phosphorylation of eIF2α are suppressed following downregulation of PKCα by siRNA. In conclusion this study identifies novel interaction partners of PKCα and a novel role for PKCα in regulation of stress granules.

The gonadotropin receptors FSH-R and LH-R of Atlantic halibut (Hippoglossus hippoglossus), 1: isolation of multiple transcripts encoding full-length and truncated variants of FSH-R.

As a first step towards understanding the regulatory mechanisms underlying the asynchronous oogenesis in repetitive spawning fish, full-length cDNAs encoding the receptors for follicle stimulating hormone (FSH-R) and luteinizing hormone (LH-R) were isolated from the gonads of the flatfish Atlantic halibut (Hippoglossus hippoglossus). The predicted halibut FSH-R and LH-R of 664 and 698 amino acids, respectively, both contain the characteristic features of a large extracellular (EC) domain, a hepta-helical transmembrane (TM) domain, and a short cytoplasmic C-terminal tail. Halibut FSH-R and LH-R share only 42% overall sequence identity mostly due to low homology in the ligand-binding EC domain. Both receptors show high sequence identity to their orthologs of Nile tilapia, but seem to be more remotely related to the receptors in catfish, zebrafish and salmonids. In contrast to the intron-less TM domain of almost all vertebrate gonadotropin receptors, three introns were identified in this domain of halibut FSH-R, thus resembling the gene structure of Drosophila glycoprotein hormone receptor type I. The FSH-R pre-mRNA was shown to be processed in alternative ways by isolating two different transcripts encoding the complete receptor and four alternative spliced transcripts encoding different truncated receptor variants. Based on the DNA sequence variation and chromosomal organization of the gonadotropin receptors in several teleosts, we propose that the encoding genes have been duplicated in the fish lineage.

The gonadotropin receptors FSH-R and LH-R of Atlantic halibut (Hippoglossus hippoglossus)--2. Differential follicle expression and asynchronous oogenesis.

The biological activity and spatio-temporal expression patterns of the gonadotropin receptors FSH-R and LH-R were examined in the repetitive spawner Atlantic halibut to elucidate the gonadotropic regulation of the asynchronous follicle development. The cloned receptors were expressed in mammalian COS-7 cells, and stimulation with sea bass FSH and LH increased the cAMP production. The halibut FSH-R and LH-R genes were shown to be highly expressed in the gonads of sexually mature fish, but the transcripts were also found in extra-gonadal tissues such as pituitary and brain. Different expression patterns of FSH-R and LH-R in the developing follicles were documented by semi-quantitative RT-PCR. Abundant FSH-R mRNA was found in the small follicles during primary growth and vitellogenesis, and the signals were localized to the granulosa cells by in situ hybridization. In contrast, follicular LH-R mRNA was hardly detectable during the early stages. Conversely, in follicles during final maturation FSH-R mRNA levels tended to decrease, while the expression of LH-R was highly upregulated. Whereas the pituitary FSH and LH are asynchronously expressed in annual spawners, both gonadotropins were expressed in the female halibut pituitary throughout the reproductive cycle, except in the prespawning females. Hence, the sequential gonadotropic activation of ovarian follicle growth and maturation in repetitive spawners is probably regulated by modulating the temporal expression of FSH-R and LH-R in the follicle membrane.

Molecular characterization and expression of FSHbeta, LHbeta, and common alpha-subunit in male Atlantic halibut (Hippoglossus hippoglossus).

To elucidate the role of the gonadotropins in the multiple spawner Atlantic halibut (Hippoglossus hippoglossus) full length cDNAs encoding FSHbeta, LHbeta, and the common alpha-subunit were cloned from pituitary glands by RACE-PCR. The three cDNAs consisted of 614, 595, and 666 nucleotides encoding peptides of 131, 146, and 124 amino acids, respectively. Halibut FSHbeta and LHbeta showed unique structural features among the vertebrate glycoprotein hormones. First, in contrast to all known FSHbeta, which contain either one or two conserved N-glycosylation sites, no potential binding site was found in Atlantic halibut FSHbeta. Second, the conserved glycosylation site in the N-terminus of all vertebrate LHbeta has been substituted with a unique C-terminal binding site in Atlantic halibut LHbeta. Furthermore, a specific cysteine residue of importance for the folding and heterodimerization of mammalian FSH is lacking in the FSHbeta from Atlantic halibut as well as many other teleosts. However, teleost FSHbeta is characterized by an additional N-terminal cysteine, which has likely replaced the missing residue, implicating a modified folding pattern of this subunit. In situ hybridization of mature male pituitaries revealed that FSHbeta and LHbeta mRNA were expressed in distinct cell types throughout the proximal pars distalis of the adenohypophysis, while alpha-subunit mRNA was identified in all parts of the proximal pars distalis, and also along the periphery of pars intermedia. Consistently, Northern blot analysis of pituitary RNA from mature males showed that FSHbeta, LHbeta, and alpha-subunit mRNAs were highly expressed. In juvenile male pituitaries very few cells containing FSHbeta, LHbeta, and alpha-subunit mRNA were identified by in situ hybridization. Low mRNA levels encoding LHbeta and the alpha-subunit were also demonstrated by Northern blot analysis of the juvenile pituitaries, while no FSHbeta mRNA was detected using this less sensitive technique. The results suggest that both FSH and LH play a role during both the very early and the final reproductive stages in Atlantic halibut males.