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With technology advancing, many prediction algorithms have been developed to facilitate the modeling of inherently dynamic and flexible macromolecules such as proteins. Improvements in the prediction of protein structures have attracted a great deal of attention due to the advantages they offer, e.g., in drug design. While trusted experimental methods, such as X-ray crystallography, NMR spectroscopy, and electron microscopy, are preferred structure analysis techniques, in silico approaches are also being widely used. Two computational methods, which are on opposite ends of the spectrum with respect to their modus operandi, i.e., homology modeling and AlphaFold, have been established to provide high-quality structures. Here, a comparative study of the quality of structures either predicted by homology modeling or by AlphaFold is presented based on the characteristics determined by experimental studies using structure validation servers to fulfill the purpose. Although AlphaFold is able to predict high-quality structures, high-confidence parts are sometimes observed to be in disagreement with experimental data. On the other hand, while the structures obtained from homology modeling are successful in incorporating all aspects of the experimental structure used as a template, this method may struggle to accurately model a structure in the absence of a suitable template. In general, although both methods produce high-quality models, the criteria by which they are superior to each other are different and thus discussed in detail. [ABSTRACT FROM AUTHOR]

Gα proteins promote dynamic adjustments of cell shape directed by actin-cytoskeleton reorganization via their respective RhoGEF effectors. For example, Gα13 binding to the RGS-homology (RH) domains of several RH-RhoGEFs allosterically activates these proteins, causing them to expose their catalytic Dbl-homology (DH)/pleckstrin-homology (PH) regions, which triggers downstream signals. However, whether additional Gα proteins might directly regulate the RH-RhoGEFs was not known. To explore this question, we first examined the morphological effects of expressing shortened RH-RhoGEF DH/PH constructs of p115RhoGEF/ARHGEF1, PDZ-RhoGEF (PRG)/ARHGEF11, and LARG/ARHGEF12. As expected, the three constructs promoted cell contraction and activated RhoA, known to be downstream of Gα13 Intriguingly, PRG DH/PH also induced filopodia-like cell protrusions and activated Cdc42. This pathway was stimulated by constitutively active Gαs (GαsQ227L), which enabled endogenous PRG to gain affinity for Cdc42. A chemogenetic approach revealed that signaling by Gs-coupled receptors, but not by those coupled to Gi or Gq, enabled PRG to bind Cdc42. This receptor-dependent effect, as well as CREB phosphorylation, was blocked by a construct derived from the PRG:Gαs-binding region, PRG-linker. Active Gαs interacted with isolated PRG DH and PH domains and their linker. In addition, this construct interfered with GαsQ227Ls ability to guide PRGs interaction with Cdc42. Endogenous Gs-coupled prostaglandin receptors stimulated PRG binding to membrane fractions and activated signaling to PKA, and this canonical endogenous pathway was attenuated by PRG-linker. Altogether, our results demonstrate that active Gαs can recognize PRG as a novel effector directing its DH/PH catalytic module to gain affinity for Cdc42.

The single most frequent cancer-causing mutation across all heterotrimeric G proteins is R201C in Gαs. The current model explaining the gain-of-function activity of the R201 mutations is through the loss of GTPase activity and resulting inability to switch off to the GDP state. Here, we find that the R201C mutation can bypass the need for GTP binding by directly activating GDP-bound Gαs through stabilization of an intramolecular hydrogen bond network. Having found that a gain-of-function mutation can convert GDP into an activator, we postulated that a reciprocal mutation might disrupt the normal role of GTP. Indeed, we found R228C, a loss-of-function mutation in Gαs that causes pseudohypoparathyroidism type 1a (PHP-Ia), compromised the adenylyl cyclase-activating activity of Gαs bound to a non-hydrolyzable GTP analog. These findings show that disease-causing mutations in Gαs can subvert the canonical roles of GDP and GTP, providing new insights into the regulation mechanism of G proteins.

GS, the stimulatory heterotrimeric G protein, is an essential regulator of osteogenesis and bone turnover. To determine if increasing GaS in osteoblasts alters bone responses to hyperparathyroidism, we used a transgenic mouse line overexpressing GaS in osteoblasts (GS-Tg mice). Primary osteoblasts from GS-Tg mice showed increased basal and parathyroid hormone (PTH)-stimulated cAMP and greater responses to PTH than cells from WT mice. Skeletal responses to 2-week continuous PTH administration (cPTH) in female mice resulted in trabecular bone loss in WT mice but 74% and 34% increase in trabecular bone mass in long bones and vertebrae, respectively, in GS-Tg mice. Vertebral biomechanical strength was compromised by cPTH treatment in WT mice but not in GS-Tg. Increased peritrabecular fibrosis was greatly increased by cPTH in Gs-Tg compared to WT mice and corresponded with greater increases in Wnt pathway proteins in trabecular bone. Cortical bone responded negatively to cPTH in WT and Gs-Tg mice with large increases in porosity, decreased cortical thickness and compromised biomechanical properties. These results demonstrate that hyperparathyroidism can increase trabecular bone when GS expression and cAMP stimulation in osteoblasts are increased but this is not the case in cortical bone where increased GS expression exacerbates cortical bone loss. [ABSTRACT FROM AUTHOR]

Background: Membrane cholesterol dysregulation has been shown to alter the activity of the adenosine A2A receptor (A2AR), a G protein-coupled receptor, thereby implicating cholesterol levels in diseases such as Alzheimer's and Parkinson's. A limited number of A2AR crystal structures show the receptor interacting with cholesterol, as such molecular simulations are often used to predict cholesterol interaction sites. Methods: Here, we use experimental methods to determine whether a specific interaction between amino acid side chains in the cholesterol consensus motif (CCM) of full length, wild-type human A2AR, and cholesterol modulates activity of the receptor by testing the effects of mutational changes on functional consequences, including ligand binding, G protein coupling, and downstream activation of cyclic AMP. Results and conclusions: Our data, taken with previously published studies, support a model of receptor state-dependent binding between cholesterol and the CCM, whereby cholesterol facilitates both G protein coupling and downstream signaling of A2AR. [ABSTRACT FROM AUTHOR]

Objective: Upon activation, G protein coupled receptors (GPCRs) associate with heterotrimeric G proteins at the plasma membrane to initiate second messenger signaling. Subsequently, the activated receptor experiences desensitization, internalization, and recycling back to the plasma membrane, or it undergoes lysosomal degradation. Recent reports highlight specific cases of persistent cyclic AMP generation by internalized GPCRs, although the functional significance and mechanistic details remain to be defined. Cyclic AMP generation from internalized Glucagon-Like Peptide-1 Receptor (GLP-1R) has previously been reported from our laboratory. This study aimed at deciphering the molecular mechanism by which internalized GLP-R supports sustained cyclic AMP generation upon receptor activation in pancreatic beta cells. Methods: We studied the time course of cyclic AMP generation following GLP-1R activation with particular emphasis on defining the location where cyclic AMP is generated. Detection involved a novel GLP-1 conjugate coupled with immunofluorescence using specific endosomal markers. Finally, we employed co-immunoprecipitation as well as immunofluorescence to assess the protein–protein interactions that regulate GLP-1R mediated cyclic AMP generation at endosomes. Results: Our data reveal that prolonged association of G protein α subunit Gαs with activated GLP-1R contributed to sustained cyclic AMP generation at Rab 5 endosomal compartment. Conclusions: The findings provide the mechanism of endosomal cyclic AMP generation following GLP-1R activation. We identified the specific compartment that serves as an organizing center to generate endosomal cyclic AMP by internalized activated receptor complex.

McCune-Albright syndrome (MAS) is a rare, mosaic disorder presenting along a broad clinical spectrum. Disease arises from somatic-activating GNAS mutations, leading to constitutive Gαs activation and ligand-independent signaling of the Gαs-coupled protein receptor. The phenotype is largely determined by location and extent of tissues in which the GNAS mutation is expressed, as well as the pathophysiologic effects of Gαs activation within these tissues. Patients pre-sent clinically with a variable combination of fibrous dysplasia of bone (FD), café-au-lait skin macules, and hyperfunctioning endocrinopathies. In bone, Gαs leads to impaired differentiation of skeletal stem cells and formation of discrete, expansile FD lesions, resulting in fractures, pain, and functional impairment. A systematic approach to diagnosis and management is critically important to optimize outcomes for patients with FD/MAS. There are no medical therapies capable of altering the disease course in FD; however, screening and treatment for endocrinopathies can mitigate some skeletal morbidities. This review summarizes current understanding of MAS pathophysiology, describes the spectrum of clinical features, and includes a detailed discussion of the recommended approach to diagnosis and management. [ABSTRACT FROM AUTHOR]

The Gαs subunit is classically involved in the signal transduction of G protein-coupled receptors (GPCRs) at the plasma membrane. Recent evidence has revealed noncanonical roles for Gαs in endosomal sorting of receptors to lysosomes. However, the mechanism of action of Gαs in this sorting step is still poorly characterized. Here, we report that Gαs interacts with ubiquitin to regulate the endosomal sorting of receptors for lysosomal degradation. We reveal that the N-terminal extremity of Gαs contains a ubiquitin-interacting motif (UIM), a sorting element usually found in the endosomal sorting complex required for transport (ESCRT) machinery responsible for sorting ubiquitinated receptors into intraluminal vesicles (ILVs) ofmultivesicular bodies (MVBs). Mutation of the UIM in Gαs confirmed the importance of ubiquitin interaction for the sorting of epidermal growth factor receptor (EGFR) into ILVs for lysosomal degradation. These findings demonstrate a role for Gαs as an integral component of the ubiquitin-dependent endosomal sorting machinery and highlight the dual role of Gαs in receptor trafficking and signaling for the fine-tuning of the cellular response. [ABSTRACT FROM AUTHOR]

Objective Upon activation, G protein coupled receptors (GPCRs) associate with heterotrimeric G proteins at the plasma membrane to initiate second messenger signaling. Subsequently, the activated receptor experiences desensitization, internalization, and recycling back to the plasma membrane, or it undergoes lysosomal degradation. Recent reports highlight specific cases of persistent cyclic AMP generation by internalized GPCRs, although the functional significance and mechanistic details remain to be defined. Cyclic AMP generation from internalized Glucagon-Like Peptide-1 Receptor (GLP-1R) has previously been reported from our laboratory. This study aimed at deciphering the molecular mechanism by which internalized GLP-R supports sustained cyclic AMP generation upon receptor activation in pancreatic beta cells. Methods We studied the time course of cyclic AMP generation following GLP-1R activation with particular emphasis on defining the location where cyclic AMP is generated. Detection involved a novel GLP-1 conjugate coupled with immunofluorescence using specific endosomal markers. Finally, we employed co-immunoprecipitation as well as immunofluorescence to assess the protein–protein interactions that regulate GLP-1R mediated cyclic AMP generation at endosomes. Results Our data reveal that prolonged association of G protein α subunit Gαs with activated GLP-1R contributed to sustained cyclic AMP generation at Rab 5 endosomal compartment. Conclusions The findings provide the mechanism of endosomal cyclic AMP generation following GLP-1R activation. We identified the specific compartment that serves as an organizing center to generate endosomal cyclic AMP by internalized activated receptor complex. [ABSTRACT FROM AUTHOR]

Proteins exist as conformational ensembles, exchanging between substates to perform their function. Advances in experimental techniques yield unprecedented access to structural snapshots of their conformational landscape. However, computationally modeling how proteins use collective motions to transition between substates is challenging owing to a rugged landscape and large energy barriers. Here, we present a new, robotics-inspired motion planning procedure called dCC-RRT that navigates the rugged landscape between substates by introducing dynamic, interatomic constraints to modulate frustration. The constraints balance non-native contacts and flexibility, and instantaneously redirect the motion towards sterically favorable conformations. On a test set of eight proteins determined in two conformations separated by, on average, 7.5 Å root mean square deviation (RMSD), our pathways reduced the Cα atom RMSD to the goal conformation by 78%, outperforming peer methods. We then applied dCC-RRT to examine how collective, small-scale motions of four side-chains in the active site of cyclophilin A propagate through the protein. dCC-RRT uncovered a spatially contiguous network of residues linked by steric interactions and collective motion connecting the active site to a recently proposed, non-canonical capsid binding site 25 Å away, rationalizing NMR and multi-temperature crystallography experiments. In all, dCC-RRT can reveal detailed, all-atom molecular mechanisms for small and large amplitude motions. Source code and binaries are freely available at . [ABSTRACT FROM AUTHOR]

Mammalian Ric-8 proteins act as chaperones to regulate the cellular abundance of heterotrimeric G protein α subunits. The Ric-8A isoform chaperones Gαi/o, Gα12/13, and Gαq/11 subunits, while Ric-8B acts on Gαs/olf subunits. Here, we determined cryoelectron microscopy (cryo-EM) structures of Ric-8B in complex with Gαs and Gαolf, revealing isoform differences in the relative positioning and contacts between the C-terminal α5 helix of Gα within the concave pocket formed by Ric-8 α-helical repeat elements. Despite the overall architectural similarity with our earlier structures of Ric-8A complexed to Gαq and Gαi1, Ric-8B distinctly accommodates an extended loop found only in Gαs/olf proteins. The structures, along with results from Ric-8 protein thermal stability assays and cell-based Gαolf folding assays, support a requirement for the Gα C-terminal region for binding specificity, and highlight that multiple structural elements impart specificity for Ric-8/G protein binding. [Display omitted] • Structures of Ric-8B in complex with its folding clients Gαolf and Gαs • Ric-8B has a more permissive Gα subunit α5 helix binding pocket than Ric-8A • Ric-8B accommodates the unique i3 loop of Gαs/olf • A new RIC-8B -deletion cell line with Gαolf folding sensor was created Papasergi-Scott et al. present the cryo-EM structures of Ric-8B in complex with open, nucleotide-free Gαolf and Gαs. These structures provide molecular insight into the process of Gα subunit protein folding and define a basis for Ric-8A and Ric-8B specificity differences for Gα subunit substrates. [ABSTRACT FROM AUTHOR]

Heterotrimeric G protein signaling plays major roles during different cellular events. However, there is a limited understanding of the molecular mechanisms underlying G protein control during embryogenesis. G proteins are highly conserved and can be grouped into four subfamilies according to sequence homology and function. To further studies on G protein function during embryogenesis, the present analysis identified four Gα subunits representative of the different subfamilies and determined their spatiotemporal expression patterns during Xenopus tropicalis embryogenesis. Each of the Gα subunit transcripts was maternally and zygotically expressed, and, as development progressed, dynamic expression patterns were observed. In the early developmental stages, the Gα subunits were expressed in the animal hemisphere and dorsal marginal zone. While expression was observed at the somite boundaries, in vascular structures, in the eye, and in the otic vesicle during the later stages, expression was mainly found in neural tissues, such as the neural tube and, especially, in the cephalic vesicles, neural crest region, and neural crest-derived structures. Together, these results support the pleiotropism and complexity of G protein subfamily functions in different cellular events. The present study constitutes the most comprehensive description to date of the spatiotemporal expression patterns of Gα subunits during vertebrate development. [ABSTRACT FROM AUTHOR]

Background: The role of cAMP in regulating T cell activation and function has been controversial. cAMP is generally known as an immunosuppressant, but it is also required for generating optimal immune responses. As the effect of cAMP is likely to depend on its cellular context, the current study investigated whether the mechanism of activation of Gαs and adenylyl cyclase influences their effect on T cell receptor (TCR)-stimulated interleukin-2 (IL-2) mRNA levels. Methods: The effect of blocking Gs-coupled receptor (GsPCR)-mediated Gs activation on TCR-stimulated IL-2 mRNA levels in CD4+ T cells was compared with that of knocking down Gαs expression or inhibiting adenylyl cyclase activity. The effect of knocking down Gαs expression on TCR-stimulated cAMP accumulation was compared with that of blocking GsPCR signaling. Results: ZM-241385, an antagonist to the Gs-coupled A2A adenosine receptor (A2AR), enhanced TCR-stimulated IL-2 mRNA levels in primary human CD4+ T helper cells and in Jurkat T cells. A dominant negative Gαs construct, GαsDN3, also enhanced TCR-stimulated IL-2 mRNA levels. Similar to GsPCR antagonists, GαsDN3 blocked GsPCR-dependent activation of both Gαs and Gβγ. In contrast, Gαs siRNA and 2',5'-dideoxyadenosine (ddA), an adenylyl cyclase inhibitor, decreased TCR-stimulated IL-2 mRNA levels. Gαs siRNA, but not GαsDN3, decreased TCR-stimulated cAMP synthesis. Potentiation of IL-2 mRNA levels by ZM-241385 required at least two days of TCR stimulation, and addition of ddA after three days of TCR stimulation enhanced IL-2 mRNA levels. Conclusions: GsPCRs play an inhibitory role in the regulation of TCR-stimulated IL-2 mRNA levels whereas Gαs and cAMP can play a stimulatory one. Additionally, TCR-dependent activation of Gαs does not appear to involve GsPCRs. These results suggest that the context of Gαs/cAMP activation and the stage of T cell activation and differentiation determine the effect on TCR-stimulated IL-2 mRNA levels. [ABSTRACT FROM AUTHOR]

The G protein-coupled receptor cascade leading to production of the second messenger cAMP is replete with pharmacologically targetable proteins, with the exception of the Gα subunit, Gαs. GTPases remain largely undruggable given the difficulty of displacing high-affinity guanine nucleotides and the lack of other drug binding sites. We explored a chemical library of 1012 cyclic peptides to expand the chemical search for inhibitors of this enzyme class. We identified two macrocyclic peptides, GN13 and GD20, that antagonize the active and inactive states of Gαs, respectively. Both macrocyclic peptides fine-tune Gαs activity with high nucleotide-binding-state selectivity and G protein class-specificity. Co-crystal structures reveal that GN13 and GD20 distinguish the conformational differences within the switch II/α3 pocket. Cell-permeable analogs of GN13 and GD20 modulate Gαs/Gβγ signaling in cells through binding to crystallographically defined pockets. The discovery of cyclic peptide inhibitors targeting Gαs provides a path for further development of state-dependent GTPase inhibitors. [Display omitted] • Discovery of nucleotide-state-selective cyclic peptide binders for Gαs • Co-crystal structures reveal G protein class specificity of the cyclic peptides • Inhibition of the Gαs ON-state reduces isoproterenol-stimulated cAMP production • Sequestering the Gαs OFF-state prolongs Gβγ activation upon receptor activation Cell-permeable Gαs inhibitors regulate G protein signal transduction with high nucleotide-binding-state selectivity and class specificity. [ABSTRACT FROM AUTHOR]

Abstract: The A2A adenosine receptor (A2AR) is a G-protein-coupled receptor that contains a long cytoplasmic carboxyl terminus (A2AR-C). We report here that Gas-2 like 2 (G2L2) is a new interacting partner of A2AR-C. The interaction between A2AR and G2L2 was verified by GST pull-down, co-immunoprecipitation, immunocytochemical staining, and fluorescence resonance energy transfer. Expression of G2L2 increased the intracellular cAMP content evoked by A2AR in an A2AR-C-dependent manner. Immunoprecipitation and pull-down assays demonstrated that G2L2 selectively bound to A2AR-C and the inactive form of Gαs to facilitate the recruitment of the trimeric G protein complex to the proximal position of A2AR for efficient activation. Collectively, G2L2 is a new effector that controls the action of A2AR by modulating its ability to regulate the Gαs-mediated cAMP contents. [Copyright &y& Elsevier]

Abstract: One important function of melanocytes (MCs) is to produce and transfer melanin to neighbouring keratinocytes (KCs) to protect epithelial cells from UV radiation. The mechanisms regulating the specific migration and localisation of the MC lineage remain unknown. We have found three heterozygous mutations that cause amino acid substitutions in the SASH1 gene in individuals with a kind of dyschromatosis. In epidermal tissues from an affected individual, we observed the increased transepithelial migration of melanocytes. Functional analyses indicate that these SASH1 mutations not only cause the increased migration of A375 cells and but also induce intensive bindings with two novel cell adhesion partners IQGAP1 and Gαs. Further, SASH1 mutations induce uniform loss of E-Cadherin in human A375 cells. Our findings suggest a new scaffold protein SASH1 to regulate IQGAP1–E-Cadherin signalling and demonstrate a novel crosstalking between GPCR signalling, calmodulin signalling for the modulation of MCs invasion. [Copyright &y& Elsevier]

Extra-large stimulatory Gα (XLα s ) is a large variant of G protein α s subunit (Gα s ) that uses an alternative promoter and thus differs from Gα s at the first exon. XLα s activation by G protein-coupled receptors mediates cAMP generation, similarly to Gα s ; however, Gα s and XLα s have been shown to have distinct cellular and physiological functions. For example, previous work suggests that XLα s can stimulate inositol phosphate production in renal proximal tubules and thereby regulate serum phosphate levels. In this study, we show that XLα s directly and specifically stimulates a specific isoform of phospholipase Cβ (PLCβ), PLCβ4, both in transfected cells and with purified protein components. We demonstrate that neither the ability of XLα s to activate cAMP generation nor the canonical G protein switch II regions are required for PLCβ stimulation. Furthermore, this activation is nucleotide independent but is inhibited by Gβγ, suggesting a mechanism of activation that relies on Gβγ subunit dissociation. Surprisingly, our results indicate that enhanced membrane targeting of XLα s relative to Gα s confers the ability to activate PLCβ4. We also show that PLCβ4 is required for isoproterenol-induced inositol phosphate accumulation in osteocyte-like Ocy454 cells. Taken together, we demonstrate a novel mechanism for activation of phosphoinositide turnover downstream of G s -coupled receptors that may have a critical role in endocrine physiology., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, we show that myometrial transcriptional complexes consisting of Sp1, Sp3, histone deacetylase (HDAC)1/2, RbAp48, and mSin3A are recruited to 4 out of the 6 Sp1-4 sites within the Gαs promoter. Moreover disruption in the binding of these complexes via mithramycin administration results in a substantial decrease in expression of Gαs proteins in myometrial cell cultures. In many instances, these transcriptional regulatory complexes repress expression of genes having a high CG content within their promoter region. This repression can be attenuated by inhibition of HDAC activity by the class I/II HDAC inhibitor trichostatin A (TSA) resulting in increased gene transcription. However, although a substantial increase in Gαs protein levels was observed upon administration of TSA to primary cultures of human myometrial cells, this was not preceded by an increase in messenger RNA (mRNA) and thus an elevation in gene transcription. Importantly the increase in Gαs protein levels occurred via ubiquitination and inhibition of proteasomal activity, indicating that this pathway is also involved in regulating Gαs protein expression during pregnancy and parturition. [ABSTRACT FROM AUTHOR]

Abstract: The role of Gβγ in adenylyl cyclase (AC) signaling is complicated due to its role as a conditional activator (AC2, AC4 and AC7) and an inhibitor (AC1, AC3 and AC8). AC2 is stimulated by Gαs and if Gβγ is present the stimulation is synergistic. The precise mechanism of this synergistic activation is still not known. In order to further elucidate the role of Gβγ in AC2 activation by Gαs, peptides derived from the C1 domains of AC2 were synthesized and the ability of the various peptides to regulate AC2 function was tested. Our results identify two new Gβγ-binding sites in the AC2 C1 domain, AC2 C1a 339–360 and AC2 C1b 578–602 that are involved with stimulation of AC2 by Gβγ. These two regions are different from the previously described QEHA motif in the C2 domain of AC2. Further, the recently discovered PFAHL motif was confirmed to bind and to be involved with stimulation of AC2 by Gβγ. These functional studies indicate that multiple regions of AC2 are involved in the interaction with Gβγ. [Copyright &y& Elsevier]

GNAS is a complex gene that through use of alternative first exons encodes signaling proteins Gαs and XLαs plus neurosecretory protein NESP55. Tissue-specific expression of these proteins is regulated through reciprocal genomic imprinting in fully differentiated and developed tissue. Mutations in GNAS account for several human disorders, including McCune-Albright syndrome and Albright hereditary osteodystrophy, and further knowledge of GNAS imprinting may provide insights into variable phenotypes of these disorders. We therefore analyzed expression of Gαs, NESP55, and XLαs prior to tissue differentiation in cell cultures derived from human primordia germ cells. We found that the expression of Gαs was biallelic (maternal allele: 52.6%± 2.5%; paternal allele: 47.2%± 2.5%; p= 0.07), whereas NESP55 was expressed preferentially from the maternal allele (maternal allele: 81.9%± 10%; paternal allele: 18.1%± 10%; p= 0.002) and XLαs was preferentially expressed from the paternal allele (maternal allele: 2.7%± 0.3%; paternal allele: 97.3%± 0.3%; p= 0.007). These results demonstrate that imprinting of NESP55 occurs very early in development, although complete imprinting appears to take place later than 5–11 weeks postfertilization, and that imprinting of XLαs occurs very early postfertilization. By contrast, mprinting of Gαs most likely occurs after 11 weeks postfertilization and after tissue differentiation. [ABSTRACT FROM AUTHOR]

Objectives/Hypothesis: In previous studies, we have demonstrated that the T-allele of a specific single nucleotide polymorphism (SNP) in the Gαs gene (T393C) correlates with increased Gαs expression and hence apoptosis. The T-allele was associated with a favorable outcome in a variety of human cancers, for example, carcinoma of the urinary bladder, kidney, colorectal, oro- and hypopharynx. Study Design: The prognostic value of the T393C SNP was retrospectively evaluated in an unselected series of patients treated with curative intent for laryngeal squamous cell carcinomas including all tumor stages with different therapeutic regimens. Methods: DNA analysis was performed using DNA from paraffin-embedded tissue samples from 157 patients (142 men, 15 women) with a median follow-up of 68 (3-143) months. The various genotypes were correlated with the overall survival. Results: Survival was significantly dependent on the T393C genotype in advanced American Joint Committee on Cancer (AJCC) stages (III-IV) with an apparent gene-dose effect ( P = .0437). Five-year survival rates were 76% for TT, 49% for TC, and 43.5% for CC. In multivariate analysis including age at diagnosis, AJCC stage, grade, gender, and T393C genotypes, patients with CC genotype displayed a higher risk for death with a hazard ratio of 2.59 (95% confidence interval: 1.01-6.64, P = .047) compared with the reference group consisting of T393 homozygous individuals. Conclusions: The T393C SNP is a prognostic marker that could help to identify high risk patients suffering from head and neck cancer. [ABSTRACT FROM AUTHOR]

Abstract: Human prostacyclin receptor (hIP) stimulates STAT3 via pertussis toxin-insensitive G proteins in human erythroleukemia (HEL) cells. Since hIP can utilize Gs and Gq proteins for signal transduction and that both G proteins can induce STAT3 phosphorylation and activation via complex signaling networks, we sought to determine if one of them is predominant in mediating the hIP signal. Stimulation of STAT3 Tyr705 and Ser727 phosphorylations by the IP-specific agonist, cicaprost, was sensitive to inhibition of protein kinase A, phospholipase Cβ, protein kinase C, calmodulin-dependent protein kinase II and Janus kinase 2/3. Unlike Gα16-mediated regulation of STAT3 in the same cells, cicaprost-induced STAT3 Tyr705 phosphorylation was resistant to inhibition of Src and MEK while STAT3 Ser727 phosphorylation distinctly required phosphatidylinositol-3 kinase. This unique inhibitor-sensitivity pattern of STAT3 phosphorylation was reproduced in HEL cells by stimulating the G16-coupled C5a receptor in the presence of dibutyryl-cAMP, suggesting that the change in inhibitor-sensitivity was due to activation of the Gs pathway. This postulation was confirmed by expressing constitutively active Gα16QL and GαsQL in human embryonic kidney 293 cells and the inhibitor-sensitivity of Gα16QL-induced STAT3 phosphorylations could be converted by the mere presence of GαsQL to resemble that obtained with cicaprost in HEL cells. In addition, the restoration of the Gα16-mediated inhibitor-sensitivity upon cicaprost induction in Gαs-knocked down HEL cells again verified the pivotal role of Gs signal. Taken together, our observations illustrate that co-stimulation of Gs and Gq can result in the fine-tuning of STAT3 activation status, and this may provide the basis for cell type-specific responses following activation of hIP. [Copyright &y& Elsevier]

Abstract: Treatment of Xenopus laevis oocytes with cholesterol-depleting methyl-β-cyclodextrin (MeβCD) stimulates phosphorylation of mitogen-activated protein kinase (MAPK) and oocyte maturation, as reported previously [Sadler, S.E., Jacobs, N.D., 2004. Stimulation of Xenopus laevis oocyte maturation by methyl-β-cyclodextrin. Biol. Reprod. 70, 1685–1692.]. Here we report that treatment of oocytes with MeβCD increased levels of immunodetectable 39-kDa mos protein. The protein synthesis inhibitor, cycloheximide, blocked the appearance of Mos, blocked MeβCD-stimulated phosphorylation of MAPK, and inhibited MeβCD-induced oocyte maturation. These observations suggest that MeβCD activates the progesterone-signaling pathway. Chemical inhibition of steroid synthesis and mechanical removal of follicle cells were used to verify that MeβCD acts at the level of the oocyte and does not require production of steroid by surrounding follicle cells. Cortical Gαs is contained in low-density membrane; and treatment of oocytes with progesterone or MeβCD reduced immunodetectable levels of Gαs protein in cortices and increased internal levels of 45-kDa Gαs in cortical-free extracts. Dose-dependent increases in internal Gαs after treatment of oocytes with progesterone correlated with the steroid-induced maturation response, and the increase in internal Gαs after hormone treatment was comparable to the decrease in cortical Gαs. These results are consistent with a model in which release of Gαs from the plasma membrane is involved in activation of the progesterone-signaling pathway that leads to amphibian oocyte maturation. [Copyright &y& Elsevier]

Abstract: G protein-coupled receptors (GPCRs) are regulated by multiple families of kinases including GPCR kinases (GRKs). GRK4 is constitutively active towards GPCRs, and polymorphisms of GRK4γ are linked to hypertension. We examined, through co-immunoprecipitation, the interactions between GRK4γ and the Gα and Gβ subunits of heterotrimeric G proteins. Because GRK4 has been shown to inhibit Gαs-coupled GPCR signaling and lacks a PH domain, we hypothesized that GRK4γ would interact with active Gαs, but not Gβ. Surprisingly, GRK4γ preferentially interacts with inactive Gαs and Gβ to a greater extent than active Gαs. GRK4γ also interacts with inactive Gα13 and Gβ. Functional studies demonstrate that wild-type GRK4γ, but not kinase-dead GRK4γ, ablates isoproterenol-mediated cAMP production indicating that the kinase domain is responsible for GPCR regulation. This evidence suggests that binding to inactive Gαs and Gβ may explain the constitutive activity of GRK4γ towards Gαs-coupled receptors. [Copyright &y& Elsevier]

S-acylated peptides have many potential uses for elucidating the biophysical, structural and other properties of the numerous S-acylated proteins of mammalian cells. However, with the currently available reagents, preparation of specifically S-acylated derivatives of peptides is generally laborious or simply unfeasible. We here show that novel, easily preparable aryl and alkyl thioester derivatives of palmitic acid can mediate S-acylation of peptides corresponding to physiologically S-acylated sequences from the proteins p56lck and H-ras and the Po glycoprotein of peripheral myelin, with high selectivity for cysteine over other amino acid functional groups (including hydroxyl and both α- and ℇ-amino residues), and with much greater efficiency than is obtained using acyl-coenzyme A derivatives. Efficient and selective S-acylation can be accomplished under very mild conditions in aqueous systems containing lipid vesicles or detergent micelles, or in homogenous aqueous/acetonitrile mixtures. Using these novel thioesterifying reagents, we confirm previous suggestions that the N-terminal cysteine residue of Hedgehog proteins can exhibit rapid, uncatalyzed S-to-N acyl transfer following S-acylation to produce the N-palmitoylated amino terminus found in the mature protein. By contrast, we demonstrate that spontaneous S-to-N acyl transfer from the cysteine to the terminal glycine residue in the amino-terminal peptide of G αs is far less rapid and is likely too slow to explain the physiological N-palmitoylation of the amino terminus of this protein. [ABSTRACT FROM AUTHOR]

Abstract: The serotonin type 6 (5-HT6) receptor is a G-protein coupled receptor (GPCR) coupled to a stimulatory G-protein (GS). To identify the structural basis for the interaction of the 5-HT6 receptor with the GS protein, we have dissected the interaction between GST-fusion proteins containing the second intracellular loop (iL2), the third intracellular loop (iL3), or the C-terminal tail of the 5-HT6 receptor and the α subunit of GS (GαS). The direct interaction of iL3 and GαS was demonstrated by co-immunoprecipitation. Furthermore, the kinetic parameters of the interaction between iL3 and GαS were measured by surface plasmon resonance, and the apparent dissociation constant was determined to be 0.9×10−6M. In contrast, the second intracellular loop and C-terminal tail regions showed negligible affinity to GαS. The critical residues within the iL3 region for the interaction with GαS were identified as conserved positively charged residues near the C-terminus of iL3 by measuring the cellular levels of cAMP produced in response to 5-HT stimulation of cells transfected with 5-HT6 receptor mutants. [Copyright &y& Elsevier]

CCAAT/enhancer-binding proteins, C/EBPα and C/EBPβ, are required for fat cell differentiation and maturation. Previous studies showed that replacement of C/EBPα with C/EBPβ, generating the β/β alleles in the mouse genome, prevents lipid accumulation in white adipose tissue (WAT). In this study, β/β mice lived longer and had higher energy expenditure than their control littermates due to increased WAT energy oxidation. The WAT of β/β mice was enriched with metabolically active, thermogenic mitochondria known for energy burning. The β/β allele exerted its effect through the elevated expression of the G protein α stimulatory subunit (Gαs) in WAT. Gαs, when overexpressed in fat-laden 3T3-L1 cells, stimulated mitochondrial biogenesis similar to that seen in the WAT of β/β mice, and effectively diminished the stored lipid pool. [ABSTRACT FROM AUTHOR]

The Melanocortin-4 Receptor (MC4R) plays a pivotal role in energy homeostasis. We used human MC4R mutations associated with an increased or decreased risk of obesity to dissect mechanisms that regulate MC4R function. Most obesity-associated mutations impair trafficking to the plasma membrane (PM), whereas obesity-protecting mutations either accelerate recycling to the PM or decrease internalization, resulting in enhanced signaling. MC4R mutations that do not affect canonical Gα s protein-mediated signaling, previously considered to be non-pathogenic, nonetheless disrupt agonist-induced internalization, β-arrestin recruitment, and/or coupling to Gα s , establishing their causal role in severe obesity. Structural mapping reveals ligand-accessible sites by which MC4R couples to effectors and residues involved in the homodimerization of MC4R, which is disrupted by multiple obesity-associated mutations. Human genetic studies reveal that endocytosis, intracellular trafficking, and homodimerization regulate MC4R function to a level that is physiologically relevant, supporting the development of chaperones, agonists, and allosteric modulators of MC4R for weight loss therapy. [Display omitted] • Obesity-associated MC4R mutations that do not reduce cAMP disrupt other processes • MC4R mutations impact receptor homodimerization, endocytosis, and trafficking • Obesity-protecting mutations increase plasma membrane MC4Rs and enhance signaling • Multiple mechanisms regulate melanocortin tone to a physiologically relevant level Using mutations in the human Melanocortin-4 Receptor (MC4R), Brouwers et al. identify receptor trafficking and endocytosis, coupling to Gα s /β-arrestins, and homodimerization as mechanisms involved in the regulation of body weight that may be targeted for weight loss therapy. [ABSTRACT FROM AUTHOR]

Gα proteins promote dynamic adjustments of cell shape directed by actin-cytoskeleton reorganization via their respective RhoGEF effectors. For example, Gα 13 binding to the RGS-homology (RH) domains of several RH-RhoGEFs allosterically activates these proteins, causing them to expose their catalytic Dbl-homology (DH)/pleckstrin-homology (PH) regions, which triggers downstream signals. However, whether additional Gα proteins might directly regulate the RH-RhoGEFs was not known. To explore this question, we first examined the morphological effects of expressing shortened RH-RhoGEF DH/PH constructs of p115RhoGEF/ARHGEF1, PDZ-RhoGEF (PRG)/ARHGEF11, and LARG/ARHGEF12. As expected, the three constructs promoted cell contraction and activated RhoA, known to be downstream of Gα 13 Intriguingly, PRG DH/PH also induced filopodia-like cell protrusions and activated Cdc42. This pathway was stimulated by constitutively active Gα s (Gα s Q227L), which enabled endogenous PRG to gain affinity for Cdc42. A chemogenetic approach revealed that signaling by G s -coupled receptors, but not by those coupled to G i or G q , enabled PRG to bind Cdc42. This receptor-dependent effect, as well as CREB phosphorylation, was blocked by a construct derived from the PRG:Gα s -binding region, PRG-linker. Active Gα s interacted with isolated PRG DH and PH domains and their linker. In addition, this construct interfered with Gα s Q227L's ability to guide PRG's interaction with Cdc42. Endogenous G s -coupled prostaglandin receptors stimulated PRG binding to membrane fractions and activated signaling to PKA, and this canonical endogenous pathway was attenuated by PRG-linker. Altogether, our results demonstrate that active Gα s can recognize PRG as a novel effector directing its DH/PH catalytic module to gain affinity for Cdc42., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Castillo-Kauil et al.)

Following stimulation, G protein-coupled receptors (GPCRs) are internalized and transported to early endosomes where they are either recycled back to the plasma membrane for another round of activation or targeted to the lysosomes for degradation and long-term signal termination. This latter requires internalization of receptors into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs) for complete degradation following fusion with lysosomes. This endosomal sorting step is highly regulated and has profound functional consequences. This chapter describes how RNAi and confocal microscopy methods can be combined to evaluate whether a protein of interest (herein Gαs) is involved in GPCR sorting into ILVs of MVBs.

Dilated cardiomyopathy (DCM) is the most common form of non-ischemic cardiomyopathy. It is characterized by ventricular chamber dilation, and myocyte hypertrophy. Human tumorous imaginal disc 1 (Tid1), a chaperone protein and response to regulate number of signaling molecules in the mitochondria or cytosol. Tid1 also plays a major role in preventing DCM; however, the role of Tid1 in isoproterenol (ISO)-induced cardiac apoptosis and hypertrophy remains unclear. H9c2 cells were pretreated Tid1 before ISO-induced hypertrophy and apoptosis and then evaluated by IHC, TUNEL assay, IFC, Co-IP, and Western blot. From the IHC experiment, we found that Tid1 proteins were increased in tissues from different stages of human myocardial infarction. Using H9c2 cardiomyoblast cells we found that Tid1 was decreased by ISO treatment. However, over-expression of Tid1S suppressed NFATc3, BNP and calcineurin protein expression and inhibited NFATc3 nuclear translocation in ISO induced cardiomyoblast cells. On the other hand, Tid1S over-expression activated survival proteins p-AKT ser473 and decreased caspase-3 and cytochrome c expression. We also found that overexpression of Tid1 enhanced CHIP expression, and induced CHIP to ubiquitinate Gαs, resulting in increased Gαs degradation. Our study showed that Gαs is a novel substrate of CHIP, and we also found that the Tid1-CHIP complex plays an essential role in inhibiting ISO induced cardiomyoblast hypertrophy and apoptosis., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest.

Gα S is a heterotrimeric G protein that transduces signals from activated G protein-coupled receptors on the cell surface to stimulate adenylyl cyclase/cyclic adenosine monophosphate (AMP) signaling. Gα S plays a central role in mediating numerous growth and maintenance processes including osteogenesis and bone turnover. Decreased Gα S expression or activating mutations in Gα S both affect bone, suggesting that modulating Gα S protein levels may be important for bone health and development. To examine the effects of increased osteoblastic Gα S expression on bone development in vivo, we generated transgenic mice with Gα S overexpression in osteoblasts (HOM-Gs mice) driven by the 3.6-kilobase (kb) Col1A1 promoter. Both male and female HOM-Gs mice exhibit increased bone turnover with overactive osteoblasts and osteoclasts, resulting in a high bone mass phenotype with significantly reduced bone quality. At 9 weeks of age, HOM-Gs mice have increased trabecular number, volumetric BMD (vBMD), and bone volume; however, the bone was woven and disorganized. There was also increased cortical bone volume despite an overall reduction in size in HOM-Gs mice along with increased cortical porosity and brittleness. The skeletal phenotype of HOM-Gs mice progressed into maturity at 26 weeks of age with further accrual of trabecular bone, whereas WT mice lost trabecular bone at this age. Although cortical bone volume and geometry were similar between mature HOM-Gs and WT mice, increased porosity persisted and the bone was weaker. At the cellular level, these alterations were mediated by an increase in bone resorption by osteoclasts and an overwhelmingly higher increase in bone formation by osteoblasts. In summary, our findings demonstrate that high osteoblastic Gα S expression results in aberrant skeletal development in which bone production is favored at the cost of bone quality. © 2017 American Society for Bone and Mineral Research., (© 2017 American Society for Bone and Mineral Research.)

G protein-coupled receptors (GPCRs) are critical players in tumor growth and progression. The redundant roles of GPCRs in tumor development confound effective treatment; therefore, targeting a single common signaling component downstream of these receptors may be efficacious. GPCRs transmit signals through heterotrimeric G proteins composed of Gα and Gβγ subunits. Hyperactive Gαs signaling can mediate tumor progression in some tissues; however, recent work in medulloblastoma and basal cell carcinoma revealed that Gαs can also function as a tumor suppressor in neoplasms derived from ectoderm cells including neural and epidermal stem/progenitor cells. In these stem-cell compartments, signaling through Gαs suppresses self-renewal by inhibiting the Sonic Hedgehog (SHH) and Hippo pathways. The loss of GNAS, which encodes Gαs, leads to activation of these pathways, over-proliferation of progenitor cells, and tumor formation. Gαs activates the cAMP-dependent protein kinase A (PKA) signaling pathway and inhibits activation of SHH effectors Smoothened-Gli. In addition, Gαs-cAMP-PKA activation negatively regulates the Hippo pathway by blocking the NF2-LATS1/2-Yap signaling. In this review, we will address the novel function of the signaling network regulated by Gαs in suppression of SHH-driven tumorigenesis and the therapeutic approaches that can be envisioned to harness this pathway to inhibit tumor growth and progression.