G protein–coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. β-Arrestins (βArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete βArr1/2 and G proteins have cast doubt on the role of β-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of βArr1/2 and reconstitution with βArr1/2 in three different parental and CRISPR-derived βArr1/2 knockout HEK293 cell pairs to assess the effect of βArr1/2 deletion on ERK1/2 activation by four Gs-coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for β-Arr2 or β-Arr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For β2-adrenergic receptors (β2ARs) and β1ARs, βArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V2 and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with βArr1/2. Loss of desensitization and receptor internalization in CRISPR βArr1/2 knockout cells caused β2AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing βArr1/2. These data suggest that βArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from βArr1/2- or G protein–deleted cells to GPCR behavior in native systems.

Classically, the FSH receptor (FSH-R) mediates its effects through coupling to guanine nucleotide-binding protein alpha S subunit (Galpha(s)) and activation of the cAMP/protein kinase A (PKA) signaling pathway. beta-Arrestins are rapidly recruited to the FSH-activated receptor and play key roles in its desensitization and internalization. Here, we show that the FSH-R expressed in HEK 293 cells activated ERK by two temporally distinct pathways dependent, respectively, on Galpha(s)/PKA and beta-arrestins. Galpha(s)/PKA-dependent ERK activation was rapid, transient, and blocked by H89 (a PKA inhibitor), but it was insensitive to small interfering RNA-mediated depletion of beta-arrestins. beta-Arrestin-dependent ERK activation was slower but more sustained and was insensitive to H89. We identified five Ser/Thr residues in the C terminus of the receptor (638-644) as a major phosphorylation site. Mutation of these residues into Ala (5A FSH-R) significantly reduced the stability of FSH-induced beta-arrestin 1 and 2 interaction when compared with the wild-type receptor. As expected, the 5A FSH-R-mediated cAMP accumulation was enhanced, and its internalization was reduced. In striking contrast, the ability of the 5A FSH-R to activate ERK via the beta-arrestin-dependent pathway was increased. G protein-coupled receptor kinase 5 (GRK5) and GRK6 were required for beta-arrestin-dependent ERK activation by both the wild-type and 5A FSH-R. By contrast, GRK2 depletion enhanced ERK activation by the wild-type FSH-R but not by the 5A FSH-R. In conclusion, we demonstrate the existence of a beta-arrestin-dependent, GRK-regulated mechanism for ERK activation by the FSH-R. A phosphorylation cluster in the C terminus of the FSH-R, identified as a site of beta-arrestin recruitment, positively regulated both desensitization and internalization but negatively regulated beta-arrestin-dependent ERK activation.

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The A189 V inactivating mutation of the human FSH receptor (FSHR) leads to subfertility in men and primary ovarian failure in women. This mutation has previously been associated with intracellular retention of the FSHR and impaired cAMP production. Here, we show that the A189 V FSHR stably expressed in HEK293N cells provoked ERK MAP kinases phosphorylation through β-arrestins, independently of the canonical cAMP/PKA pathway. Interesting, both the A189 V and wild-type (Wt) FSHRs selectively activated cAMP-independent ERK phosphorylation when expressed at low plasma membrane densities. These data indicate that the selective intracellular signalling triggered by the A189 V FSHR resulted from reduced membrane expression rather than by switching receptor coupling. Hence, receptor density at the plasma membrane might control the balance between distinct signal transduction mechanisms. Furthermore, our results help to clarify why mutations of FSHβ are more deleterious to human fertility than the FSHR A189 V mutation which preserves parts of receptor signalling repertoire.

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The molecular mechanisms and hidden dynamics governing ERK activation by the angiotensin II type 1A receptor are studied and deciphered, revealing a signal balancing mechanism that is found to be relevant to a range of other seven transmembrane receptors.

Abstract. -At Echassières (Allier, France), arsenic speciation was determined in a soil developed over a micaschist where Hercynian hydrothermal mineralization, including arsenopyrite FeAsS and löllingite FeAs 2 , has lead to a regional As anomaly. The overlying soils which have developed from long term weathering exhibit As levels as high as 900 ppm in the richest area, where the saprolite contains up to 5200 ppm As. Analysis by powder XRD, µ-X-ray diffraction on the 20µm scale, SEM-EDS and electron micro-probe analyses revealed that As, released from arsenopyrite and/or löllingite alteration, is concentrated in a secondary iron arsenate, pharmacosiderite, (Ba x ,K 2-2x ) (Fe,Al) 4 (AsO 4 ) 3 (OH) 5 • 6H 2 O. Quantitative mineralogical analysis by Rietveld refinement indicates that the proportion of As hosted by this mineral decreases systematically from the saprolite to the topsoil (from 70 % to 30 % of the total bulk As content, respectively). EXAFS spectroscopy indicates that the main form of the As occurring with pharmacosiderite, consists of As(V) ions sorbed on iron oxides. Sorption processes, which dominate As speciation in the topsoil horizons, appear as a key mechanism able to delay As dissemination from soils to plant and surface waters, provided that pH and Eh conditions remain sufficiently acidic and oxidizing, respectively. Résumé. -La spéciation de l'arsenic a été déterminée dans un sol développé sur un micaschiste affecté par des minéra-lisations à arsénopyrite FeAsS et löllingite FeAs 2 d'âge hercynien qui ont conduit à la mise en place d'une anomalie géochimique régionale en As, à Échassières, Allier, France. Les sols qui se sont développés au cours d'une longue pé-riode d'altération présentent des concentrations atteignant 900 ppm dans les horizons de surface des zones les plus riches, où la saprolite contient jusqu'à 5200 ppm d'As. Les analyses par diffraction des rayons X sur poudre, par micro-diffraction à l'échelle de 20µm ainsi que les analyses par MEB-EDS et microsonde électronique révèlent que l'As, libéré par l'altération de l'arsénopyrite et/ou de la löllingite, est piégé dans un arséniate de fer secondaire, la pharmacosidérite, (Ba x ,K 2-2x ) (Fe,Al) 4 (AsO 4 ) 3 (OH) 5 • 6H 2 O. L'analyse minéralogique quantitative par affinement Rietveld indique que la fraction de l'As total piégé dans cette phase diminue de 70 % dans la saprolite à moins de 30 % dans les horizons de surface. La spectroscopie EXAFS indique que les ions As(V) adsorbés à la surface des oxydes de fer représentent la forme majoritaire de l'arsenic accompagnant la pharmacosidérite. Les processus d'adsorption, qui dominent la spéciation de l'arsenic dans les horizons de surface apparaissent comme un mécanisme important capable de retarder la dissémination de l'arsenic du sol vers les eaux et les plantes, à condition que les conditions de pH et de Eh du sol restent suffisamment acides et oxydantes.

FSH is a major hormonal input that drives Sertoli cells to their fully differentiated function in male reproduction. It is a physiologically important issue to define how FSH mediates its effects at the cellular level to regulate gene expression. FSH biological activities are transduced via a seven-spanned transmembrane receptor, the FSH-R, primarily leading to cAMP-dependent protein kinase A (PKA) activation and cAMP response element binding protein-mediated transcriptional responses. Nevertheless, the intracellular mechanisms interacting with PKA to control Sertoli cell differentiation by FSH are still incompletely defined. Here, we report that, in primary cultures of Sertoli cells isolated from prepubertal rats, FSH enhanced p70S6K enzymatic activity, in a PKA-dependent manner. p70S6K was constitutively phosphorylated on Thr 389, in a manner sensitive to inhibitors of phosphatidyl-inositide-3 kinase and mammalian target of rapamycin. But FSH could not enhance p70S6K phosphorylation on Thr 389. Rather, the hormone induced the dephosphorylation of Thr 421/Ser 424, located in the autoinhibitory domain of p70S6K, in a PKA-dependent manner. Consistently, FSH-induced phosphorylation of the S6 ribosomal protein, a cellular substrate of p70S6K, required PKA activity. In conclusion, these results show that FSH triggers unexpected regulations of p70S6K by dephosphorylation of Thr 421/Ser 424 mediated by PKA, and stimulates S6 phosphorylation, in Sertoli cells.