Although many G protein-coupled receptors (GPCRs) can form dimers, a possible role of this phenomenon in their activation remains elusive. A recent and exciting proposal is that a dynamic intersubunit interplay may contribute to GPCR activation. Here, we examined this possibility using dimeric metabotropic glutamate receptors (mGluRs). We first developed a system to perfectly control their subunit composition and show that mGluR dimers do not form larger oligomers. We then examined an mGluR dimer containing one subunit in which the extracellular agonist-binding domain was uncoupled from the G protein-activating transmembrane domain. Despite this uncoupling in one protomer, agonist stimulation resulted in symmetric activation of either transmembrane domain in the dimer with the same efficiency. This, plus other data, can only be explained by an intersubunit rearrangement as the activation mechanism. Although well established for other types of receptors such as tyrosine kinase and guanylate cyclase receptors, this is the first clear demonstration that such a mechanism may also apply to GPCRs.
Our study provides a new mechanism of TNFRSF1A regulation whereby three polymorphisms in the promoter, exon 1 and intron 4 have a functional and combined effect on exon 2 splicing, via a coupling mechanism between transcription and splicing. These polymorphisms may affect the phenotype of TRAPS and TRAPS-like patients.
Mutations in the MEditerranean FeVer (MEFV) gene are responsible for familial Mediterranean fever (FMF), a recessively inherited auto-inflammatory disease. Cases of dominant inheritance and phenotype-genotype heterogeneity have been reported; however, the underlying molecular mechanism is not currently understood. The FMF protein named pyrin or marenostrin (P/M) is thought to be involved in regulating innate immunity but its function remains subject to controversy. Recent studies postulate that a defect in MEFV expression regulation may play a role in FMF physiopathology. Our group, along with others, has identified several alternatively spliced MEFV transcripts in leukocytes. Since alternative splicing and nonsense-mediated decay (NMD) pathways are usually coupled in the post-transcriptional regulation of gene expression, we hypothesized that NMD could contribute to the regulation of the MEFV gene. To address this issue, we examined the effect of indirect and direct inhibition of NMD on expression of the MEFV transcripts in THP1, monocyte and neutrophil cells. We showed that MEFV is the first auto-inflammatory gene regulated by NMD in both a cell- and transcript-specific manner. These results and preliminary western-blot analyses suggest the possible translation of alternatively spliced MEFV transcripts into several P/M variants according to cell type and inflammatory state. Our results introduce the novel hypothesis that variation of NMD efficiency could play an important role in FMF physiopathology as a potent phenotypic modifier.
Familial mediterranean fever (FMF) is a hereditary autoinflammatory autosomal recessive disease caused by mutations in the MEFV gene. Despite the identification of many disease associated MEFV mutations, often the clinical diagnosis cannot be genetically confirmed. The currently used diagnostic sequencing techniques only allow the detection of point mutations, small deletions or duplications. The question as to whether larger genetic alterations are also involved in the pathophysiology of FMF remains to be answered. To address this question, we used multiplex ligation-dependent probe amplification (MLPA) on a total of 216 patients with FMF symptoms. This careful analysis revealed that not a single deletion/ duplication could be detected in this large cohort of patients. This result suggests that single or multiexon MEFV gene copy number changes do not contribute substantially, if at all, to the MEFV mutation spectrum.
The recent identification of genes implicated in hereditary recurrent fevers has allowed their specific diagnosis. So far however, only punctual mutations have been identified and a significant number of patients remain with no genetic confirmation of their disease after routine molecular approaches such as sequencing. The possible involvement of sequence rearrangements in these patients has only been examined in familial Mediterranean fever and was found to be unlikely. To assess the existence of larger genetic alterations in 3 other concerned genes, MVK (Mevalonate kinase), NLRP3 (Nod like receptor family, pyrin domain containing 3) and TNFRSF1A (TNF receptor superfamily 1A), we adapted the qPCR-HRM method to study possible intragenic deletions and duplications. This single-tube approach, combining both qualitative (mutations) and quantitative (rearrangement) screening, has proven effective in Lynch syndrome diagnosis. Using this approach, we studied 113 unselected (prospective group) and 88 selected (retrospective group) patients and identified no intragenic rearrangements in the 3 genes. Only qualitative alterations were found with a sensitivity similar to that obtained using classical molecular techniques for screening punctual mutations. Our results support that deleterious copy number alterations in MVK, NLRP3 and TNFRSF1A are rare or absent from the mutational spectrum of hereditary recurrent fevers, and demonstrate that a routine combined method such as qPCR-HRM provides no further help in genetic diagnosis. However, quantitative approaches such as qPCR or SQF-PCR did prove to be quick and effective and could still be useful after non contributory punctual mutation screening in the presence of clinically evocative signs.
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