The landscape of GPCR signaling in the regulation of epidermal stem cell fate and skin homeostasis.

Continuous integration of signals from the micro and macro-environment is necessary for somatic stem cells to adapt to changing conditions, maintain tissue homeostasis and activate repair mechanisms. G-protein coupled receptors (GPCRs) facilitate this integration by binding to numerous hormones, metabolites and inflammatory mediators, influencing a diverse network of pathways that regulate stem cell fate. This adaptive mechanism is particularly relevant for tissues that are exposed to environmental assault, like skin. The skin is maintained by a set of basal keratinocyte stem and progenitor cells located in the hair follicle and interfollicular epidermis, and several GPCRs and their signaling partners serve as makers and regulators of epidermal stem cell activity. GPCRs utilize heterotrimeric G protein dependent and independent pathways to translate extracellular signals into intracellular molecular cascades that dictate the activation of keratinocyte proliferative and differentiation networks, including Hedgehog GLI, Hippo YAP1 and WNT/ß-catenin, ultimately regulating stem cell identity. Dysregulation of GPCR signaling underlines numerous skin inflammatory diseases and cancer, with smoothened-driven basal cell carcinoma being a main example of a GPCR associated cancer. In this review, we discuss the impact of GPCRs and their signaling partners in skin keratinocyte biology, particularly in the regulation of the epidermal stem cell compartment.

Ric-8A, a GEF, and a Chaperone for G Protein α-Subunits: Evidence for the Two-Faced Interface.

Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a prominent non-receptor GEF and a chaperone of G protein α-subunits (Gα). Recent studies shed light on the structure of Ric-8A, providing insights into the mechanisms underlying its interaction with Gα. Ric-8A is composed of a core armadillo-like domain and a flexible C-terminal tail. Interaction of a conserved concave surface of its core domain with the Gα C-terminus appears to mediate formation of the initial Ric-8A/GαGDP intermediate, followed by the formation of a stable nucleotide-free complex. The latter event involves a large-scale dislocation of the Gα α5-helix that produces an extensive primary interface and disrupts the nucleotide-binding site of Gα. The distal portion of the C-terminal tail of Ric-8A forms a smaller secondary interface, which ostensibly binds the switch II region of Gα, facilitating binding of GTP. The two-site Gα interface of Ric-8A is distinct from that of GPCRs, and might have evolved to support the chaperone function of Ric-8A.

eIF2ß, a subunit of translation-initiation factor EIF2, is a potential therapeutic target for non-small cell lung cancer.

To identify novel therapeutic targets for non-small cell lung cancer (NSCLC), we conducted an integrative study in the following 3 stages: (i) identification of potential target gene(s) through shRNA functional screens in 2 independent NSCLC cell lines; (ii) validation of the clinical relevance of identified gene(s) using public databases; and (iii) investigation of therapeutic potential of targeting the identified gene(s) in vitro. A semi-genome-wide shRNA screen was performed in NCI-H358 cells, and was integrated with data from our previous screen in NCI-H460 cells. Among genes identified in shRNA screens, 24 were present in both NCI-H358 and NCI-H460 cells and were considered potential targets. Among the genes, we focused on eIF2ß, which is a subunit of heterotrimeric G protein EIF2 and functions as a transcription initiation factor. The eIF2ß protein is highly expressed in lung cancer cell lines compared with normal bronchial epithelial cells, and gene copy number analyses revealed that eIF2ß is amplified in a subset of NSCLC cell lines. Gene expression analysis using The Cancer Genome Atlas (TCGA) dataset revealed that eIF2ß expression is significantly upregulated in lung cancer tissues compared with corresponding normal lung tissues. Furthermore, high eIF2ß expression was correlated with poor survival in patients with lung adenocarcinoma, as shown in other cohorts using publicly available online tools. RNAi-mediated depletion of eIF2ß suppresses growth of lung cancer cells independently of p53 mutation status, in part through G1 cell cycle arrest. Our data suggest that eIF2ß is a therapeutic target for lung cancer.

Relationship between 5-hydroxytryptamine 1A receptor and G-protein β3 subunit polymorphisms and post-stroke depression / 中华神经科杂志

Objective To assess whether 5-HTR1A C( - 1019) G and GNβ3 C825T gene polymorphisms are associated with post-stroke depression (PSD) and explore the genetic mechanism of the pathogenesis of post-stroke depression. Methods All 159 patients with first stroke were divided into the PSD group and the control group according to HAMD scores. Their genotypes were determined with polymerase chain reaction and allele-specific restriction enzyme analysis. Results The frequency of 5-HTR1A (-1019) GG genotype(8/53,15. 1% ), G allele (44/106,41.5%)and GNβ3 825T allele(68/106,64. 2% ) were significantly higher in the post-stroke depression group than in the controls (5/106,4.7% ;35/212, 16. 5%; 113/212, 53.3%; ×2 = 23.204, 23. 655, 3. 392, all P ＜ 0. 05 ). Combined genotype analysis showed that individuals with both 5-HTR1A ( - 1019) G and GNβ3 825T allele ( OR =4. 980,95% CI 2. 429-10. 210,P =0. 000) had a higher risk than those with 5-HTR1 A (-1019) G allele ( OR = 3. 589,95% CI 2. 113-6. 096, P = 0. 000) or GNβ3 825T allele ( OR = 0. 638,95% CI 0.395-1. 031 ,P =0. 042) only for post-stroke depression. Conclusion The 5-HTR1A C( - 1019)G and GNβ3 C825T polymorphisms are predisposing genes of post-stroke depression. Our data also suggest a significant interaction between the 5-HTR1A ( - 1019)G allele and GNβ3 825T allele in post-stroke depression.

Negative feedback regulation of Wnt signaling by Gbetagamma-mediated reduction of Dishevelled

Wnt signaling is known to be important for diverse embryonic and post-natal cellular events and be regulated by the proteins Dishevelled and Axin. Although Dishevelled is activated by Wnt and involved in signal transduction, it is not clear how Dishevelled-mediated signaling is turned off. We report that guanine nucleotide binding protein beta 2 (Gnb2; Gbeta2) bound to Axin and Gbeta2 inhibited Wnt mediated reporter activity. The inhibition involved reduction of the level of Dishevelled, and the Gbeta2gamma2 mediated reduction of Dishevelled was countered by increased expression of Axin. Consistent with these effects in HEK293T cells, injection of Gbeta2gamma2 into Xenopus embryos inhibited the formation of secondary axes induced either by XWnt8 or Dishevelled, but not by beta-catenin. The DEP domain of Dishevelled is necessary for both interaction with Gbeta2gamma2 and subsequent degradation of Dishevelled via the lysosomal pathway. Signaling induced by Gbeta2gamma2 is required because a mutant of Gbeta2, Gbeta2 (W332A) with lower signaling activity, had reduced ability to downregulate the level of Dishevelled. Activation of Wnt signaling by either of two methods, increased Frizzled signaling or transient transfection of Wnt, also led to increased degradation of Dishevelled and the induced Dishevelled loss is dependent on Gbeta1 and Gbeta2. Other studies with agents that interfere with PLC action and calcium signaling suggested that loss of Dishevelled is mediated through the following pathway: Wnt/Frizzled-->Gbetagamma-->PLC-->Ca+2/PKC signaling. Together the evidence suggests a novel negative feedback mechanism in which Gbeta2gamma2 inhibits Wnt signaling by degradation of Dishevelled.

Inhibition of gamma ray-induced apoptosis by stimulatory heterotrimeric GTP binding protein involves Bcl-xL down-regulation in SH-SY5Y human neuroblastoma cells

Heterotrimeric GTP-binding proteins (G proteins) transduce extracellular signals into intracellular signals by activating effector molecules including adenylate cyclases that catalyze cAMP formation, and thus regulate various cellular responses such as metabolism, proliferation, and apoptosis. cAMP signaling pathways have been reported to protect cells from ionizing radiation-induced apoptosis, but however, the protective mechanism is not clear. Therefore, this study aimed to investigate the signaling molecules and the mechanism mediating the anti-apoptotic action of cAMP signaling system in radiation-induced apoptosis. Stable expression of a constitutively active mutant of G alpha s (G alpha sQL) protected gamma ray-induced apoptosis which was assessed by analysis of the cleavages of PARP, caspase-9, and caspase-3 and cytochrome C release in SH-SY5Y human neuroblastoma cells. G alpha sQL repressed the gamma ray-induced down-regulation of Bcl-xL protein, but transfection of Bcl-xL siRNA increased the gamma ray-induced apoptosis and abolished the anti-apoptotic effect of G alpha sQL. G alpha sQL decreased the degradation rate of Bcl-xL protein, and it also restrained the decrease in Bcl-xL mRNA by increasing the stability following ionizing irradiation. Furthermore, prostaglandin E2 that activates G alpha s was found to protect gamma ray-induced apoptosis, and the protective effect was abolished by treatment with prostanoid receptor antagonist specific to EP2/4R subtype. Moreover, specific agonists for adenosine A1 receptor that inhibits cAMP signaling pathway augmented gamma ray-induced apoptosis. From this study, it is concluded that Galphas-cAMP signaling system can protect SH-SY5Y cells from gamma ray-induced apoptosis partly by restraining down-regulation of Bcl-xL expression, suggesting that radiation-induced apoptosis can be modulated by GPCR ligands to improve the efficiency of radiation therapy.

THE PRELIMINARY STUDY ON THE CHANGES IN THE EXPRESSION OF G PROTEINS ? SUBUNIT mRNA IN THYROID TUMOR / 解放军医学杂志

To investigate the changes in the expression of G proteins ? subunit mRNA in non functioning thyroid adenoma (NFTA) and thyroid papillary cancer (TPC). Eleven thyroid specimens were obtained during surgery, 6 of which from patients with NFTA and 5 from patients with TPC. The expression levels of stimulating and inhibiting G protein ? subunit mRNAs were determined by reverse transcription polymerase chain reaction (RT PCR). The expression levels of Gsa mRNA in TPCs were significantly higher than those in normal thyroids and NFTAs( P