Functional communication between IP3R and STIM2 at subthreshold stimuli is a critical checkpoint for initiation of SOCE

Stromal interaction molecules, STIM1 and STIM2, sense decreases in the endoplasmic reticulum (ER) [Ca2+] ([Ca2+]ER) and cluster in ER–plasma membrane (ER–PM) junctions where they recruit and activate Orai1. While STIM1 responds when [Ca2+]ER is relatively low, STIM2 displays constitutive clustering in the junctions and is suggested to regulate basal Ca2+ entry. The cellular cues that determine STIM2 clustering under basal conditions is not known. By using gene editing to fluorescently tag endogenous STIM2, we report that endogenous STIM2 is constitutively localized in mobile and immobile clusters. The latter associate with ER–PM junctions and recruit Orai1 under basal conditions. Agonist stimulation increases immobile STIM2 clusters, which coordinate recruitment of Orai1 and STIM1 to the junctions. Extended synaptotagmin (E-Syt)2/3 are required for forming the ER–PM junctions, but are not sufficient for STIM2 clustering. Importantly, inositol 1,4,5-triphosphate receptor (IP3R) function and local [Ca2+]ER are the main drivers of immobile STIM2 clusters. Enhancing, or decreasing, IP3R function at ambient [IP3] causes corresponding increase, or attenuation, of immobile STIM2 clusters. We show that immobile STIM2 clusters denote decreases in local [Ca2+]ER mediated by IP3R that is sensed by the STIM2 N terminus. Finally, under basal conditions, ambient PIP2-PLC activity of the cell determines IP3R function, immobilization of STIM2, and basal Ca2+ entry while agonist stimulation augments these processes. Together, our findings reveal that immobilization of STIM2 clusters within ER–PM junctions, a first response to ER-Ca2+ store depletion, is facilitated by the juxtaposition of IP3R and marks a checkpoint for initiation of Ca2+ entry.

Orai1α, but not Orai1β, co-localizes with TRPC1 and is required for its plasma membrane location and activation in HeLa cells

The identification of two variants of the canonical pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel Orai1, Orai1α and Orai1β, in mammalian cells arises the question whether they exhibit different functional characteristics. Orai1α and Orai1β differ in the N-terminal 63 amino acids, exclusive of Orai1α, and show different sensitivities to Ca2+-dependent inactivation, as well as distinct ability to form arachidonate-regulated channels. We have evaluated the role of both Orai1 variants in the activation of TRPC1 in HeLa cells. We found that Orai1α and Orai1β are required for the maintenance of regenerative Ca2+ oscillations, while TRPC1 plays a role in agonist-induced Ca2+ influx but is not essential for Ca2+ oscillations. Using APEX2 proximity labeling, co-immunoprecipitation and the fluorescence of G-GECO1.2 fused to Orai1α our results indicate that agonist stimulation and Ca2+ store depletion enhance Orai1α–TRPC1 interaction. Orai1α is essential for TRPC1 plasma membrane location and activation. Thus, TRPC1 function in HeLa cells depends on Ca2+ influx through Orai1α exclusively.

Single-molecule imaging reveals distinct effects of ligands on CCR5 dynamics depending on its dimerization status

G protein-coupled receptors (GPCR) are present at the cell surface in different conformational and oligomeric states. However, how these states impact GPCRs biological function and therapeutic targeting remains incompletely known. Here, we investigated this issue in living cells for the CC chemokine receptor 5 (CCR5), a major receptor in inflammation and the principal entry co-receptor for Human Immunodeficiency Viruses (HIV-1). We used TIRF microscopy and an original statistical method to track and classify the motion of different receptors subpopulations. We showed a diversity of ligand-free forms of CCR5 at the cell surface constituted of various oligomeric states and exhibiting transient Brownian and restricted motions. These forms were stabilized differently by distinct ligands. In particular, agonist stimulation restricted the mobility of CCR5 and led to its clustering, a feature depending on b-arrestin, while inverse agonist stimulation exhibited the opposite effect. These results suggest a link between receptor activation and immobilization. Applied to HIV-1 envelope glycoproteins gp120, our quantitative analysis revealed agonist-like properties of gp120s. Distinct gp120s influenced CCR5 dynamics differently, suggesting that they stabilize different CCR5 conformations. Then, using a dimerization-compromized mutant, we showed that dimerization (i) impacts CCR5 precoupling to G proteins, (ii) is a pre-requisite for the immobilization and clustering of receptors upon activation, and (iii) regulates receptor endocytosis, thereby impacting the fate of activated receptors. This study demonstrates that tracking the dynamic behavior of a GPCR is an efficient way to link GPCR conformations to their functions, therefore improving the development of drugs targeting specific receptor conformations.

Regulation of Serotonin 1A Receptor SUMOylation by SENP2 and PIASxα

Serotonin 1A receptors (5-HT1ARs) are implicated in the control of mood, cognition, and memory and in various neuropsychiatric disorders such as depression and anxiety. As such, understanding the regulation of 5-HT1ARs will inform the development of better treatment approaches. We previously demonstrated 5-HT1ARs are SUMOylated by SUMO1 in the rat brain. Agonist stimulation increased SUMOylation and was further enhanced when combined with 17β-estradiol-3-benzoate (EB), which are treatments that cause the transient and prolonged desensitization of 5-HT1AR signaling, respectively. In the current study, we identified the protein inhibitor of activated STAT (PIAS)xα as the enzyme that facilitates SUMOylation, and SENP2 as the protein that catalyzes the deSUMOylation of 5-HT1ARs. We demonstrated that PIASxα significantly increased in the membrane fraction of rats co-treated with EB and an agonist, compared to either the EB-treated or vehicle-treated groups. The acute treatment with an agonist alone shifted the location of SENP2 from the membrane to the cytoplasmic fraction, but it has little effect on PIASxα. Hence, two separate mechanisms regulate SUMOylation and the activity of 5-HT1ARs by an agonist and EB. The effects of EB on 5-HT1AR SUMOylation and signaling may be related to the higher incidence of mood disorders in women during times with large fluctuations in estrogens. Targeting the SUMOylation of 5-HT1ARs could have important clinical relevance for the therapy for several neuropsychiatric disorders in which 5-HT1ARs are implicated.

The Diagnostic Utility of the Basal Luteinizing Hormone Level and Single 60-Minute Post GnRH Agonist Stimulation Test for Idiopathic Central Precocious Puberty in Girls

ObjectiveThe present study aimed to assess the diagnostic utility of the Luteinizing hormone (LH) levels and single 60-minute post gonadotropin-releasing hormone (GnRH) agonist stimulation test for idiopathic central precocious puberty (CPP) in girls.MethodsData from 1,492 girls diagnosed with precocious puberty who underwent GnRH agonist stimulation testing between January 1, 2016, and October 8, 2020, were retrospectively reviewed. LH levels and LH/follicle-stimulating hormone (FSH) ratios were measured by immuno-chemiluminescence assay before and at several timepoints after GnRH analogue stimulation testing. Mann–Whitney U test, Spearman’s correlation, χ2 test, and receiver operating characteristic (ROC) analyses were performed to determine the diagnostic utility of these hormone levels.ResultsThe 1,492 subjects were split into two groups: an idiopathic CPP group (n = 518) and a non-CPP group (n = 974). Basal LH levels and LH/FSH ratios were significantly different between the two groups at 30, 60, 90, and 120 minutes after GnRH analogue stimulation testing. Spearman’s correlation analysis showed the strongest correlation between peak LH and LH levels at 60 minutes after GnRH agonist stimulation (r = 0.986, P < 0.001). ROC curve analysis revealed that the 60-minute LH/FSH ratio yielded the highest consistency, with an area under the ROC curve (AUC) of 0.988 (95% confidence interval [CI], 0.982–0.993) and a cut-off point of 0.603 mIU/L (sensitivity 97.3%, specificity 93.0%). The cut-off points of basal LH and LH/FSH were 0.255 mIU/L (sensitivity 68.9%, specificity 86.0%) and 0.07 (sensitivity 73.2%, specificity 89.5%), respectively, with AUCs of 0.823 (95% CI, 0.799–0.847) and 0.843 (95% CI, 0.819–0.867), respectively.ConclusionsA basal LH value greater than 0.535 mIU/L can be used to diagnose CPP without a GnRH agonist stimulation test. A single 60-minute post-stimulus gonadotropin result of LH and LH/FSH can be used instead of a GnRH agonist stimulation test, or samples can be taken only at 0, 30, and 60 minutes after a GnRH agonist stimulation test. This reduces the number of blood draws required compared with the traditional stimulation test, while still achieving a high level of diagnostic accuracy.

Receptor Activity-Modifying Protein 2 (RAMP2) alters glucagon receptor trafficking in hepatocytes with functional effects on receptor signalling

Objectives: Receptor Activity-Modifying Protein 2 (RAMP2) is a chaperone protein which allosterically binds to and interacts with the glucagon receptor (GCGR). The aims of this study were to investigate the effects of RAMP2 on GCGR trafficking and signalling in the liver, where glucagon is important for carbohydrate and lipid metabolism. Methods: Subcellular localisation of GCGR in the presence and absence of RAMP2 was investigated using confocal microscopy, trafficking assays and radioligand binding assays in human embryonic kidney (HEK293T) and human hepatoma (Huh7) cells. Mouse embryonic fibroblasts (MEFs) lacking Wiskott Aldrich Syndrome protein and scar homologue (WASH) complex were used to investigate the effect of a halt in recycling of internalised proteins on GCGR signalling in the absence of RAMP2. NanoBiT complementation and cyclic AMP assays were used to study the functional effect of RAMP2 on recruitment and activation of GCGR signalling mediators. Response to hepatic RAMP2 up-regulation in lean and obese adult mice using a bespoke adeno-associated viral vector was also studied. Results: GCGR is predominantly localised at the plasma membrane in the absence of RAMP2 and exhibits remarkably slow internalisation in response to agonist stimulation. Rapid intracellular retention of glucagon-stimulated GCGR in cells lacking WASH complex indicates that activated GCGRs undergo continuous cycles of internalisation and recycling despite apparent GCGR plasma membrane localisation up to 40 minutes post-stimulation. Co-expression of RAMP2 induces GCGR internalisation both basally and in response to agonist-stimulation. The intracellular retention of GCGR in the presence of RAMP2 confers a bias away from β-arrestin-2 recruitment coupled to increased activation of Gαs proteins at endosomes. This is associated with increased short-term efficacy for glucagon-stimulated cAMP production, although long-term signalling is dampened by increased receptor lysosomal targeting for degradation. Despite these signalling effects, only minor disturbance of carbohydrate metabolism was observed in mice with up-regulated hepatic RAMP2. Conclusions: By retaining GCGR intracellularly, RAMP2 alters the spatiotemporal pattern of GCGR signalling. Further exploration of the effects of RAMP2 on GCGR in vivo is warranted.

Regulation of Interorganellar Ca2+ Transfer and NFAT Activation by the Mitochondrial Ca2+ Uniporter

Mitochondrial Ca2+ uptake is crucial for coupling receptor stimulation to cellular bioenergetics. Further, Ca2+ uptake by respiring mitochondria prevents Ca2+-dependent inactivation (CDI) of store-operated Ca2+ release-activated Ca2+ (CRAC) channels and inhibits Ca2+ extrusion to sustain cytosolic Ca2+ signaling. However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca2+ signaling is unknown. Here, we generated several cell lines with MCU-knockout (MCU-KO) as well as tissue-specific MCU-knockdown mice. We show that mitochondrial depolarization, but not MCU-KO, inhibits store-operated Ca2+ entry (SOCE). Paradoxically, despite enhancing Ca2+ extrusion and promoting CRAC channel CDI, MCU-KO increased cytosolic Ca2+ in response to store depletion. Further, physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca2+ oscillations, endoplasmic reticulum Ca2+ refilling, NFAT nuclear translocation and proliferation. However, MCU-KO did not affect inositol-1,4,5-trisphosphate receptor activity. Mathematical modeling supports that MCU-KO enhances cytosolic Ca2+, despite limiting CRAC channel activity.

Sensitivity and specificity of using pelvic ultrasonographic parameters combined with basal gonadotropin levels to diagnose central precocious puberty in Thai girls

Background The criterion standard gonadotropin-releasing hormone (GnRH) stimulation tests to diagnose central precocious puberty (CPP) are time-consuming, inconvenient, and expensive. Objectives To determine predictive cut-off values codetermined by ultrasonographic parameters and basal gonadotropin levels in girls with premature sexual development and compare them results of criterion standard tests in a study of diagnostic accuracy. Methods Retrospective review of hormonal investigations and ultrasonographic uterine and ovarian parameters in a consecutive sample of girls at a single center, tertiary care hospital in Bangkok, Thailand. Results We separated data from 68 girls (age range 2–12 years) into 2 groups based on their response to a GnRH analogue agonist stimulation test. A “prepubertal response” group included girls with premature thelarche and thelarche variants (n = 18, 6.37 ± 1.77 years) and a “pubertal response” group, including girls with CPP (n = 50, 8.46 ± 1.46 years); excluding patients with pathological causes (n = 0). The basal level of luteinizing hormone (LH) had the largest area under receiver operating characteristic curves (AUC) of 0.84; 95% confidence interval [CI] 0.74–0.93) compared with basal levels of follicle stimulating hormone (AUC 0.77; 95% CI 0.64–0.90) or estradiol (0.70; 95% CI 0.56–0.85). An optimal cut-off of 0.25 IU/L LH was related to a pubertal response to GnRH analogue agonist stimulation tests with 75.0% sensitivity, 88.9% specificity, 94.7% positive predictive value (PPV), and 57.1% negative predictive value. Uterine and ovarian cut-off volumes of 3.5 cm3 and 1.5 cm3 were related to a pubertal response with 88.6% and 76.2% PPV, respectively. A uterine width cut-off of 1.7 cm combined with a basal LH cut-off of 0.25 IU/L increased specificity and PPV to 100%. Conclusion Combining uterine and ovarian ultrasonographic parameters with basal gonadotropin levels, especially uterine width and basal LH level, appears useful for diagnosis of CPP.

Mode of induction of platelet-derived extracellular vesicles is a critical determinant of their phenotype and function

Platelet-derived extracellular vesicles (PDEVs) are the most abundant amongst all types of EVs in the circulation. However, the mechanisms leading to PDEVs release, their role in coagulation and phenotypic composition are poorly understood. PDEVs from washed platelets were generated using different stimuli and were characterised using nanoparticle tracking analysis. Procoagulant properties were evaluated by fluorescence flow cytometry and calibrated automated thrombography. EVs from plasma were isolated and concentrated using a novel protocol involving a combination of size exclusion chromatography and differential centrifugation, which produces pure and concentrated EVs. Agonist stimulation enhanced PDEV release, but did not alter the average size of EVs compared to those produced by unstimulated platelets. Agonist stimulation led to lower negatively-charged phospholipid externalization in PDEVs, which was reflected in the lower procoagulant activity compared to those generated without agonist stimulation. Circulating EVs did not have externalized negatively-charged phospholipids. None of the 4 types of EVs presented tissue factor. The mechanism by which PDEV formation is induced is a critical determinant of its phenotype and function. Importantly, we have developed methods to obtain clean, concentrated and functional EVs derived from platelet-free plasma and washed platelets, which can be used to provide novel insight into their biological functions.