Protein-mediated Signaling Research Articles

Is FAM19A5 an adipokine? Peripheral FAM19A5 in wild-type, FAM19A5 knockout, and LacZ knockin mice

FAM19A5 is a novel secretory protein expressed primarily in the brain. However, a recent study reported that FAM19A5 is an adipocyte-derived adipokine that regulates vascular smooth muscle function through sphingosine-1-phosphate receptor 2 (S1PR2). In our study, we investigated FAM19A5 transcript and protein levels in peripheral tissues, including adipose tissues, from wild-type, FAM19A5 knockout, and FAM19A5 LacZ knockin mice. We found that the FAM19A5 transcript levels in the central nervous system were much greater than those in any of the peripheral tissues, including adipose tissues. Furthermore, the FAM19A5 protein levels in adipose and reproductive tissues were below detectable limits for Western blot analysis and ELISA. Additionally, we found that the FAM19A5 protein did not interact with S1PR2 in terms of G protein-mediated signal transduction, β-arrestin recruitment, or ligand-mediated internalization. Taken together, our findings revealed basal levels of FAM19A5 transcripts and proteins in peripheral tissues, confirming its primary expression in the central nervous system and lack of significant interaction with S1PR2.

Opioid receptors reveal a discrete cellular mechanism of endosomal G protein activation.

Many GPCRs initiate a second phase of G protein-mediated signaling from endosomes, which inherently requires an increase in G protein activity on the endosome surface. Gs-coupled GPCRs are thought to achieve this by internalizing and allosterically activating cognate G proteins again on the endosome membrane. Here we demonstrate that the μ-opioid receptor (MOR), a Gi-coupled GPCR, increases endosomal G protein activity in a different way. Leveraging conformational biosensors, we resolve the subcellular activation dynamics of endogenously expressed MOR and Gi/o-subclass G proteins. We show that MOR activation triggers a transient increase of active-state Gi/o on the plasma membrane that is followed by a prolonged increase on endosomes. Contrary to the Gs-coupled GPCR paradigm, however, we show that the MOR-induced increase of active-state Gi/o on endosomes requires neither internalization of MOR nor activation of MOR in the endosome membrane. We propose a distinct and additional cellular mechanism for GPCR-triggered elevation of G protein activity on endosomes that is mediated by regulated trafficking of the activated G protein rather than its activating GPCR.

International Union of Basic and Clinical Pharmacology. CXVIII. Update on the Nomenclature for Atypical Chemokine Receptors including ACKR5.

Chemokines signal through classical G protein-coupled receptors (GPCRs) to induce cell migration during development, immune homeostasis and multiple diseases. Over the last decade a subfamily of atypical chemokine receptors (ACKRs) was delineated from GPCRs based on their inability to trigger conventional G protein signaling or mediate cell migration in response to chemokines. These receptors nevertheless play an important role within the chemokine system by sequestering, transporting or internalizing chemokines thereby regulating their availability and shaping their gradients. GPR182, the recently deorphanized chemokine receptor, shares about 30% of sequence similarity with its closest relative ACKR3. GPR182 is mainly expressed on endothelial cells and was proposed to act as a scavenger regulating the availability of a large set of chemokines from the CXC, CC and XC families and to act cooperatively with ACKR3 and ACKR4. Unlike other ACKRs, GPR182 was shown to have a strong constitutive interaction with β-arrestins that is required for intracellular receptor trafficking and chemokine scavenging. Chemokine ligation of GPR182 has no additional detectable impact on β-arrestin recruitment. Genetic ablation of GPR182 affects spleen size, myelopoiesis, and serum chemokine levels, indicating its role in chemokine homeostasis and immune regulation. GPR182 was also reported to regulate immune responses to bloodborne antigens and tumorigenesis. Taken together, compelling cumulative evidence indicates that GPR182 does not trigger G protein-mediated signaling but acts as a scavenger for chemokines in vitro and in vivo strongly supporting its inclusion as ACKR5 in the systematic nomenclature of chemokine receptors. Significance Statement The summarized presented findings strongly support the designation of GPR182 as ACKR5 and its formal inclusion in the family of atypical chemokine receptors.

Abstract MP05: A Preliminary 10X Genomics Xenium Profiler Spatial Transcriptomics Map of Renin-Angiotensin System Genes in the Mouse Subfornical Organ

The subfornical organ (SFO) is a brain region rich in angiotensin-II (ANG) AT1 receptors (AT1R) which regulates water and salt intake and communicates with other hypothalamic nuclei to regulate blood pressure (BP). Activation of the classical G protein-mediated AT1R signaling in the brain induces pressor and natri-dipsogenic responses. TRV027, a synthetic AT1R β-arrestin-biased agonist, decreases BP and increases saline avoidance. We examined the spatial transcriptomic makeup of the SFO to balanced or biased AT1R activation using 10X Genomics Xenium. We treated C57BL/6J mice with either aCSF, ANG (0.64 µg/h), or TRV027 (0.56 µg/h) through continuous ICV infusion for 11-days. Water intake was monitored on days 6-11. ANG caused an increase in water intake compared to aCSF or TRV027 groups (7.0±2.0, 3.0±0.2, and 1.9±0.6 mL/day, respectively, n=4, p<0.05). On day 11, brains were perfused, collected, and fixed. Subsequently, 5 µm paraffin-embedded coronal sections were mounted on Xenium slides and tested with the 10X mouse brain set of 247 genes plus 100 custom add-on genes including all RAS genes. A third of the cells expressed AT1R, and the pattern of AT1R-expressing cells Xenium-detected was qualitatively similar to NZ44 mice, which express GFP under the control of the AT1R locus. Expression of the prorenin receptor ( Atp6ap2 ) was abundant throughout the SFO. A few cells in the SFO and more prominently in the adjacent blood vessels were angiotensinogen positive. Single cells were identified that expressed the ANG type 2 receptors (AT2R). Expression of Mas receptor was undetectable. Most of the AT1R-expressing cells were excitatory glutamatergic neurons (expressing Slc17a6 , VGLUT2) but a few were inhibitory GABAergic neurons co-expressing Slc32a1 (VGAT). Each section had cells classified as astrocytes, neurons, ependymal cells, and immune cells (e.g., microglia or macrophages). Increased numbers of immune cells were detected in the SFO of ANG-treated mice. Interestingly, although there was no evidence for expression of renin in the SFO, renin-expressing cells were identified in the choroid plexus which is consistent with highly sensitive in situ RNA hybridization data. Additional qualitative and quantitative analyses are ongoing to compare gene expression patterns in AT1R-positive cell types. Future studies will identify gene expression patterns in SFO neurons with connectivity to other brain regions controlling water/salt intake and BP.

Gβγ dimers mediate low K+ stress-inhibited root growth via modulating auxin redistribution in Arabidopsis.

In the investigation of heterotrimeric G protein-mediated signal transduction in planta, their roles in the transmittance of low K+ stimuli remain to be elucidated. Here, we found that the primary root growth of wild-type Arabidopsis was gradually inhibited with the decrease of external K+ concentrations, while the primary root of the mutants for G protein β subunit AGB1 and γ subunits AGG1, AGG2 and AGG3 could still grow under low K+ conditions (LK). Exogenous NAA application attenuated primary root elongation in agb1 and agg1/2/3 but promoted the growth in wild-type seedlings under LK stress. Using ProDR5:GFP, ProPIN1:PIN1-GFP and ProPIN2:PIN2-GFP reporter lines, a diminishment in auxin concentration at the radicle apex and a reduction in PIN1and PIN2 efflux carrier abundance were observed in wild-type roots under LK, a phenomenon not recorded in the agb1 and agg1/2/3. Further proteolytic and transcriptional assessments revealed an enhanced degradation of PIN1 and a suppressed expression of PIN2 in the wild-type background under LK, contrasting with the stability observed in the agb1 and agg1/2/3 mutants. Our results indicate that the G protein β and γ subunits play pivotal roles in suppressing of Arabidopsis root growth under LK by modulating auxin redistribution via alterations in PIN1 degradation and PIN2 biosynthesis.

Identifying key genes and functionally enriched pathways in acute myeloid leukemia by weighted gene co-expression network analysis.

Acute myeloid leukemia (AML) is characterized by the uncontrolled proliferation of myeloid leukemia cells in the bone marrow and other hematopoietic tissues and is highly heterogeneous. While with the progress of sequencing technology, understanding of the AML-related biomarkers is still incomplete. The purpose of this study is to identify potential biomarkers for prognosis of AML. Based on WGCNA analysis of gene mutation expression, methylation level distribution, mRNA expression, and AML-related genes in public databases were employed for investigating potential biomarkers for the prognosis of AML. This study screened a total of 6153 genes by analyzing various changes in 103 acute myeloid leukemia (AML) samples, including gene mutation expression, methylation level distribution, mRNA expression, and AML-related genes in public databases. Moreover, seven AML-related co-expression modules were mined by WGCNA analysis, and twelve biomarkers associated with the AML prognosis were identified from each top 10 genes of the seven co-expression modules. The AML samples were then classified into two subgroups, the prognosis of which is significantly different, based on the expression of these twelve genes. The differentially expressed 7 genes of two subgroups (HOXB-AS3, HOXB3, SLC9C2, CPNE8, MEG8, S1PR5, MIR196B) are mainly involved in glucose metabolism, glutathione biosynthesis, small G protein-mediated signal transduction, and the Rap1 signaling pathway. With the utilization of WGCNA mining, seven gene co-expression modules were identified from the TCGA database, and there are unreported genes that may be potential driver genes of AML and may be the direction to identify the possible molecular signatures to predict survival of AML patients and help guide experiments for potential clinical drug targets.

Left Ventricular Dilation Resulting from the Absence of Either Rgs2 or Rgs5 Is Associated with Increased Immune Cell Infiltration in the Myocardium

Sustained sympathoexcitation is a hallmark of heart failure, with equally sustained activation of downstream signaling pathways involving Gs- and Gi/o-coupled β-adrenergic receptors (βAR) stimulating cAMP production and PKA activation. A critical mechanism controlling G protein-mediated βAR signaling is the activity of regulator of G protein signaling (RGS) proteins, including members of the B/R4 subfamily, which accelerate the termination of Gq/11 and Gi/o protein signaling. Previous studies by us and others reported that moderation of Gi/o activity separately by RGS2 and RGS5 of the R4 subfamily is key to maintaining normal ventricular rhythm. The loss of one or both R4 RGS proteins also results in left ventricular (LV) dilation and reduced ejection fraction. Here, we tested the hypothesis that the LV structural remodeling is due to the disruption of the coordinated activity of RGS2 and 5 resulting in sensitization of βAR signaling. To test this hypothesis, we challenged wild type (WT), Rgs2 KO ,Rgs5 KO, and Rgs2/5 double knockout (dbKO) adult male mice with sub-chronic infusion of isoproterenol (ISO, 30 mg/kg/day, 3 days) via a peritoneal osmotic minipump implantation. Rgs2 KO and Rgs5 KO mice showed increased LV chamber size at baseline, which was unchanged by ISO administration. In saline-treated animals, the loss of Rgs2 increased Rgs5 mRNA expression, while Rgs5 absence increased Rgs4 mRNA expression. ISO infusion induced concentric cardiac hypertrophy in WT mice, as evidenced by increased heart weight/tibia length (HW/TL) ratio and cardiomyocyte cross sectional area. While ISO infusion increased interstitial fibrosis in midventricular tissue and HW/TL ratio in dbKO mice, it did not affect cardiomyocyte cross sectional area or exacerbate LV dilation, despite increased expression of the cardiac fetal gene Nppa as well as the sarcomeric protein α-actinin and Ca2+/calmodulin-dependent kinase II (CaMKII). Cardiact tissue from saline-treated Rgs2 KO and Rgs5 KO mice had markedly increased CD45+ cells vs WT, while tissue from Rgs5 KO mice showed increased CD68+ cells, as revealed by immunohistochemistry. Taken together, these results indicate that ventricular remodeling seen in Rgs2 KO, Rgs5 KO, and Rgs2/5 dbKO mice is insensitive to sustained βAR stimulation but may be due to increased cardiac resident immune cell population. This work was supported by NIH R56 DK132859-01A1 and R01 HL139754, and Craig H. Neilsen Foundation Grant 382566 to Patrick Osei-Owusu. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Comparison of the function of two novel human dopamine D2 receptor variants identifies a likely mechanism for their pathogenicity

Two recently discovered DRD2 mutations, c.634A > T, p.Ile212Phe and c.1121T > G, p.Met374Arg, cause hyperkinetic movement disorders that have overlapping features but apparently differ in severity. The two known carriers of the Met374Arg variant had early childhood disease onset and more severe motor, cognitive, and neuropsychiatric deficits than any known carriers of the Ile212Phe variant, whose symptoms were first apparent in adolescence. Here, we evaluated if differences in the function of the two variants in cultured cells could explain differing pathogenicity. Both variants were expressed less abundantly than the wild type receptor and exhibited loss of agonist-induced arrestin binding, but differences in expression and arrestin binding between the variants were minor. Basal and agonist-induced activation of heterotrimeric Gi/o/z proteins, however, showed clear differences; agonists were generally more potent at Met374Arg than at the Ile212Phe or wild type variants. Furthermore, all Gα subtypes tested were constitutively activated more by Met374Arg than by Ile212Phe. Met374Arg produced greater constitutive inhibition of cyclic AMP accumulation than Ile212Phe or the wild type D2 receptor. Met374Arg and Ile212Phe were more sensitive to thermal inactivation than the wild type D2 receptor, as reported for other constitutively active receptors, but Ile212Phe was affected more than Met374Arg. Additional pharmacological characterization suggested that the mutations differentially affect the shape of the agonist binding pocket and the potency of dopamine, norepinephrine, and tyramine. Molecular dynamics simulations provided a structural rationale for enhanced constitutive activation and agonist potency. Enhanced constitutive and agonist-induced G protein-mediated signaling likely contributes to the pathogenicity of these novel variants.

Structural determinants for activation of the Tau kinase CDK5 by the serotonin receptor 5-HT7R

BackgroundMultiple neurodegenerative diseases are induced by the formation and deposition of protein aggregates. In particular, the microtubule-associated protein Tau leads to the development of so-called tauopathies characterized by the aggregation of hyperphosphorylated Tau within neurons. We recently showed that the constitutive activity of the serotonin receptor 7 (5-HT7R) is required for Tau hyperphosphorylation and aggregation through activation of the cyclin-dependent kinase 5 (CDK5). We also demonstrated physical interaction between 5-HT7R and CDK5 at the plasma membrane suggesting that the 5-HT7R/CDK5 complex is an integral part of the signaling network involved in Tau-mediated pathology.MethodsUsing biochemical, microscopic, molecular biological, computational and AI-based approaches, we investigated structural requirements for the formation of 5-HT7R/CDK5 complex.ResultsWe demonstrated that 5-HT7R domains responsible for coupling to Gs proteins are not involved in receptor interaction with CDK5. We also created a structural model of the 5-HT7R/CDK5 complex and refined the interaction interface. The model predicted two conserved phenylalanine residues, F278 and F281, within the third intracellular loop of 5-HT7R to be potentially important for complex formation. While site-directed mutagenesis of these residues did not influence Gs protein-mediated receptor signaling, replacement of both phenylalanines by alanine residues significantly reduced 5-HT7R/CDK5 interaction and receptor-mediated CDK5 activation, leading to reduced Tau hyperphosphorylation and aggregation. Molecular dynamics simulations of 5-HT7R/CDK5 complex for wild-type and receptor mutants confirmed binding interface stability of the initial model.ConclusionsOur results provide a structural basis for the development of novel drugs targeting the 5-HT7R/CDK5 interaction interface for the selective treatment of Tau-related disorders, including frontotemporal dementia and Alzheimer’s disease.

Gasdermins: a dual role in pyroptosis and tumor immunity.

The gasdermin (GSDM) protein family plays a pivotal role in pyroptosis, a process critical to the body's immune response, particularly in combatting bacterial infections, impeding tumor invasion, and contributing to the pathogenesis of various inflammatory diseases. These proteins are adept at activating inflammasome signaling pathways, recruiting immune effector cells, creating an inflammatory immune microenvironment, and initiating pyroptosis. This article serves as an introduction to the GSDM protein-mediated pyroptosis signaling pathways, providing an overview of GSDMs' involvement in tumor immunity. Additionally, we explore the potential applications of GSDMs in both innovative and established antitumor strategies.

Optimized design and in vivo application of optogenetically functionalized Drosophila dopamine receptors

Neuromodulatory signaling via G protein-coupled receptors (GPCRs) plays a pivotal role in regulating neural network function and animal behavior. The recent development of optogenetic tools to induce G protein-mediated signaling provides the promise of acute and cell type-specific manipulation of neuromodulatory signals. However, designing and deploying optogenetically functionalized GPCRs (optoXRs) with accurate specificity and activity to mimic endogenous signaling in vivo remains challenging. Here we optimize the design of optoXRs by considering evolutionary conserved GPCR-G protein interactions and demonstrate the feasibility of this approach using two Drosophila Dopamine receptors (optoDopRs). These optoDopRs exhibit high signaling specificity and light sensitivity in vitro. In vivo, we show receptor and cell type-specific effects of dopaminergic signaling in various behaviors, including the ability of optoDopRs to rescue the loss of the endogenous receptors. This work demonstrates that optoXRs can enable optical control of neuromodulatory receptor-specific signaling in functional and behavioral studies.

DNA Extraction- and Amplification-Free Nucleic Acid Biosensor for the Detection of Foodborne Pathogens Based on CRISPR/Cas12a and Argonaute Protein-Mediated Cascade Signal Amplification.

A novel method for detecting low levels of viable foodborne pathogens, specifically Salmonella typhimurium (S. typhimurium), has been developed. Traditional nucleic acid assay, such as polymerase chain reaction (PCR), often requires complex DNA extraction and amplification, making it challenging to differentiate between viable and nonviable pathogens. This assay employed a phage as the recognition element to precisely identify and lyse viable S. typhimurium that can undergo DNA extraction. It combined the efficient trans-cleavage activities of CRISPR/Cas12a with the specific cleavage advantages of Argonaute proteins, enabling ultrasensitive detection. This double-enzyme-mediated nucleic acid test can accurately distinguish viable and nonviable S. typhimurium with a detection limit of 23 CFU/mL without DNA amplification. The method was successfully applied to common food samples, producing results consistent with quantitative PCR tests. This work provides a promising platform for easily detecting viable foodborne pathogens with high sensitivity without the need for DNA extraction and amplification.

Dapagliflozin downregulates G protein-coupled receptor Kinase-2 and upregulates regulator of g protein signaling-4 in adrenals to exert sympatholysis in heart failure

Abstract Dapagliflozin is a sodium-glucose co-transporter (SGLT)-2 inhibitor with beneficial cardiovascular effects independent of its anti-diabetic actions. Among these is sympatholysis, i.e., norepinephrine (NE) lowering which is beneficial in chronic human heart failure (HF). Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and sympathetic nervous system (SNS) hyperactivity in HF due to dysregulation of adrenal sympatho-inhibitory α2-adrenergic receptors (ARs) [1]. On the other hand, we recently reported that signaling by neuronal free fatty acid receptor (FFAR)-3, which stimulates NE release from sympathetic neurons, is blocked by Regulator of G protein Signaling (RGS)-4 [2,3]. We hypothesized herein that dapagliflozin may lower SNS hyperactivity in the adrenal gland by improving α2AR, and antagonizing FFAR3 signaling in adrenal chromaffin cells. We used the rat pheochromocytoma PC12 cell line expressing human α2A-AR, as well as freshly isolated adrenal glands from rats treated with dapagliflozin in vivo. Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217+67 pg/ml, n=6), compared to control, vehicle-treated rats (363+77 pg/ml, n=6, p<.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and tyrosine hydroxylase (TH) mRNA and protein levels in dapagliflozin-treated rat adrenals vs. vehicle-treated animal-derived glands. In contrast, RGS4 mRNA levels were increased in dapagliflozin-treated adrenals. Adrenal α2AR density was higher in dapagliflozin- vs. vehicle-treated rats (51.3+7.3 vs. 26.1+8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p<.05). These results (i.e., GRK2 & TH downregulation with RGS4 upregulation) were completely recapitulated in PC12 cells in culture, treated with 5 microM dapagliflozin (or vehicle) for 24 hours. Importantly, α2AR-dependent G protein-mediated signaling towards inhibition of CA secretion was markedly enhanced, whereas FFAR3-dependent phospholipase C/calcium signaling was reduced, at 24 hrs post-dapagliflozin treatment of PC12 cells. GRK2 overexpression partially reversed dapagliflozin`s effect on cholinergic-induced CA secretion. In conclusion, dapagliflozin exerts a sympatholytic action in the adrenal medulla via a) downregulation of TH, which reduces CA biosynthesis, and GRK2, which reduces α2AR desensitization; and b) upregulation of RGS4 to block FFAR3-stimulated CA secretion (Figure).Figure

Gαs is dispensable for β-arrestin coupling but dictates GRK selectivity and is predominant for gene expression regulation by β2-adrenergic receptor

β-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether β-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., β-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate β-arrestins, thereby limiting the ability to distinguish G protein from β-arrestin-mediated signaling events. We used β2-adrenergic receptor (β2AR) and its β2AR-C tail mutant expressed in human embryonic kidney 293cells wildtype or CRISPR-Cas9 gene edited for Gαs, β-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and β-arrestins in controlling gene expression. We found that Gαs is not required for β2AR and β-arrestin conformational changes, β-arrestin recruitment, and receptor internalization, but that Gαs dictates the GPCR kinase isoforms involved in β-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of β2AR in wildtype and β-arrestin1/2-KO cells but absent in Gαs-KO cells. These results were validated by re-expressing Gαs in the corresponding KO cells and silencing β-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of β2AR. Overall, our results support that Gs is essential for β2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas β-arrestins initiate signaling events modulating Gαs-driven nuclear transcriptional activity.

Adiponectin receptor 1-mediated stimulation of Cav3.2 channels in trigeminal ganglion neurons induces nociceptive behaviors in mice

BackgroundAdipokines, including adiponectin, are implicated in nociceptive pain; however, the underlying cellular and molecular mechanisms remain unknown.MethodsUsing electrophysiological recording, immunostaining, molecular biological approaches and animal behaviour tests, we elucidated a pivotal role of adiponectin in regulating membrane excitability and pain sensitivity by manipulating Cav3.2 channels in trigeminal ganglion (TG) neurons.ResultsAdiponectin enhanced T-type Ca2+ channel currents (IT) in TG neurons through the activation of adiponectin receptor 1 (adipoR1) but independently of heterotrimeric G protein-mediated signaling. Coimmunoprecipitation revealed a physical association between AdipoR1 and casein kinase II alpha-subunits (CK2α) in the TG, and inhibiting CK2 activity by chemical inhibitor or siRNA targeting CK2α prevented the adiponectin-induced IT response. Adiponectin significantly activated protein kinase C (PKC), and this effect was abrogated by CK2α knockdown. Adiponectin increased the membrane abundance of PKC beta1 (PKCβ1). Blocking PKCβ1 pharmacologically or genetically abrogated the adiponectin-induced IT increase. In heterologous expression systems, activation of adipoR1 induced a selective enhancement of Cav3.2 channel currents, dependent on PKCβ1 signaling. Functionally, adiponectin increased TG neuronal excitability and induced mechanical pain hypersensitivity, both attenuated by T-type channel blockade. In a trigeminal neuralgia model induced by chronic constriction injury of infraorbital nerve, blockade of adipoR1 signaling suppressed mechanical allodynia, which was prevented by silencing Cav3.2.ConclusionOur study elucidates a novel signaling cascade wherein adiponectin stimulates TG Cav3.2 channels via adipoR1 coupled to a novel CK2α-dependent PKCβ1. This process induces neuronal hyperexcitability and pain hypersensitivity. Insight into adipoR-Cav3.2 signaling in sensory neurons provides attractive targets for pain treatment.

Heterotrimeric G Protein-Mediated Signaling Is Involved in Stress-Mediated Growth Inhibition in Arabidopsis thaliana.

Heterotrimeric G protein-mediated signaling plays a vital role in physiological and developmental processes in eukaryotes. On the other hand, because of the absence of a G protein-coupled receptor and self-activating mechanism of the Gα subunit, plants appear to have different regulatory mechanisms, which remain to be elucidated, compared to canonical G protein signaling established in animals. Here we report that Arabidopsis heterotrimeric G protein subunits, such as Gα (GPA1) and Gβ (AGB1), regulate plant growth under stress conditions through the analysis of heterotrimeric G protein mutants. Flg22-mediated growth inhibition in wild-type roots was found to be caused by a defect in the elongation zone, which was partially blocked in agb1-2 but not gpa1-4. These results suggest that AGB1 may negatively regulate plant growth under biotic stress conditions. In addition, GPA1 and AGB1 exhibited genetically opposite effects on FCA-mediated growth inhibition under heat stress conditions. Therefore, these results suggest that plant G protein signaling is probably related to stress-mediated growth regulation for developmental plasticity in response to biotic and abiotic stress conditions.

Mechanisms of Arrestin-Mediated Signaling.

Arrestins were first discovered as proteins that selectively bind active phosphorylated GPCRs and suppress (arrest) their G protein-mediated signaling. Nonvisual arrestins are also recognized as signaling proteins regulating a variety of cellular pathways. Arrestins are highly flexible; they can assume many different conformations. In their receptor-bound conformation, arrestins have higher affinity for a subset of binding partners. This explains how receptor activation regulates certain branches of arrestin-dependent signaling via arrestin recruitment to GPCRs. However, free arrestins are also active molecular entities that regulate other signaling pathways and localize signaling proteins to particular subcellular compartments. Recent findings suggest that the two visuals, arrestin-1 and arrestin-4, which are expressed in photoreceptor cells, not only regulate signaling via binding to photopigments but also interact with several nonreceptor partners, critically affecting the health and survival of photoreceptor cells. Detailed in this overview are GPCR-dependent and independent modes of arrestin-mediated regulation of cellular signaling. © 2023 Wiley Periodicals LLC.

Loss of cholinergic receptor muscarinic 1 impairs cortical mitochondrial structure and function: implications in Alzheimer's disease.

Introduction: Cholinergic Receptor Muscarinic 1 (CHRM1) is a G protein-coupled acetylcholine (ACh) receptor predominantly expressed in the cerebral cortex. In a retrospective postmortem brain tissues-based study, we demonstrated that severely (≥50% decrease) reduced CHRM1 proteins in the temporal cortex of Alzheimer's patients significantly correlated with poor patient outcomes. The G protein-mediated CHRM1 signal transduction cannot sufficiently explain the mechanistic link between cortical CHRM1 loss and the appearance of hallmark Alzheimer's pathophysiologies, particularly mitochondrial structural and functional abnormalities. Therefore, the objective of this study was to analyze the molecular, ultrastructural, and functional properties of cortical mitochondria using CHRM1 knockout (Chrm1-/-) and wild-type mice to identify mitochondrial abnormalities. Methods: Isolated and enriched cortical mitochondrial fractions derived from wild-type and Chrm1-/- mice were assessed for respiratory deficits (oxygen consumption) following the addition of different substrates. The supramolecular assembly of mitochondrial oxidative phosphorylation (OXPHOS)-associated protein complexes (complex I-V) and cortical mitochondrial ultrastructure were investigated by blue native polyacrylamide gel electrophoresis and transmission electron microscopy (TEM), respectively. A cocktail of antibodies, specific to Ndufb8, Sdhb, Uqcrc2, Mtco1, and Atp5a proteins representing different subunits of complexes I-V, respectively was used to characterize different OXPHOS-associated protein complexes. Results: Loss of Chrm1 led to a significant reduction in cortical mitochondrial respiration (oxygen consumption) concomitantly associated with reduced oligomerization of ATP synthase (complex V) and supramolecular assembly of complexes I-IV (Respirasome). Overexpression of Chrm1 in transformed cells (lacking native Chrm1) significantly increased complex V oligomerization and respirasome assembly leading to enhanced respiration. TEM analysis revealed that Chrm1 loss led to mitochondrial ultrastructural defects and alteration in the tinctorial properties of cortical neurons causing a significant increase in the abundance of dark cortical neurons (Chrm1-/- 85% versus wild-type 2%). Discussion: Our findings indicate a hitherto unknown effect of Chrm1 deletion in cortical neurons affecting mitochondrial function by altering multiple interdependent factors including ATP synthase oligomerization, respirasome assembly, and mitochondrial ultrastructure. The appearance of dark neurons in Chrm1-/- cortices implies potentially enhanced glutamatergic signaling in pyramidal neurons under Chrm1 loss condition. The findings provide novel mechanistic insights into Chrm1 loss with the appearance of mitochondrial pathophysiological deficits in Alzheimer's disease.

B-Arrestin2 Deficiency in the Brain Alters Blood Pressure Regulation

Low-renin hypertension is a common subset of hypertensive patients, which is associated with salt sensitivity. Deoxycorticosterone (DOCA)-salt hypertension, a prototypical model of low-renin hypertension, requires activation of the angiotensin type-1 receptor (AT 1 R) in specific brain areas. G protein-mediated signaling of the AT 1 R within the brain is known to induce dipsogenic and pressor responses to Ang II stimulation. Non-canonical or b-arrestin-mediated signaling is thought to counterbalance the maladaptive G-protein (Gαq) signaling during disease. Recently, we found that global deletion of β-arrestin2 (ARRB2) exhibited an exacerbated increase in blood pressure (BP) in response to DOCA-salt compared to WT suggesting a protective role for ARRB2. However, the role of the non-canonical AT1R/β-arrestin pathway within the brain is understudied. Consequently, we hypothesized that b-Arrestin activation within the brain contributes to BP regulation. Global male and female β-arrestin1 ( Arrb1)- and β-arrestin2 ( Arrb2)-KO mice were employed to evaluate acute BP response upon treatment with intracerebroventricular (ICV) infusion of AngII (1ug). Age- and sex-matched C57BL/6 mice served as controls. Mice were simultaneously instrumented with radiotelemeters and ICV-cannula, BP was continuously recorded before and after AngII infusion in awake animals. At baseline, Arrb2-KO mice showed a slight increase in BP WT (WT=134.0±10.7 vs Arrb2-KO=143.7±17.2 mmHg; n=14 and 19, respectively). Further, Arrb2-KO mice exhibited significantly higher BP in response to AngII (WT=143.7±17.2 vs Arrb2-KO=176.9±24.2 mmHg; n=14 and 19, respectively p<0.05). These findings suggest that loss of ARRB2 exacerbates the pressor response to AngII within the brain. Additionally, we evaluated BP upon activation β-arrestin using TRV120027 (TRV27), a β-arrestin biased agonist specific for the AT 1 R. We used the BPH2/J genetic mouse model of low renin hypertension. Mice were subjected to continuous infusion of TRV27 (6ug/h), and telemetric BP was continuously recorded. BPN/2J mice were used as the normotensive controls. At baseline, BPH2/J exhibited significantly higher BP when compared to normotensive controls (BPN=123.9±5.3 vs. BPH=153.4±3.4 mmHg; n=4 p<0.05). Further, chronic delivery of ICV-TRV showed a higher magnitude of BP reduction in the BPH2/J group compared to controls (BPN=-3.8±3.0 vs. BPH=-8.8±1.3 mmHg; n=4 p<0.05), suggesting that the effects of b-arrestin activation are predominant under hypertensive states. Overall, endogenous ARRB2 counterbalances the deleterious effects of AT 1 R overactivation in the brain. The stimulation of the AT 1 R β-arrestin axis in the brain using biased AT 1 R agonist that penetrate the brain-blood barrier might be a potential strategy to treat low renin hypertension. HL084207 and HL144807 to CDS, and 22PRE898004 to NMM This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The roles of RGS proteins in cardiometabolic disease.

G protein-coupled receptors (GPCRs) are the most prominent receptors on the surface of the cell and play a central role in the regulation of cardiac and metabolic functions. GPCRs transmit extracellular stimuli to the interior of the cells by activating one or more heterotrimeric G proteins. The duration and intensity of G protein-mediated signalling are tightly controlled by a large array of intracellular mediators, including the regulator of G protein signalling (RGS) proteins. RGS proteins selectively promote the GTPase activity of a subset of Gα subunits, thus serving as negative regulators in a pathway-dependent manner. In the current review, we summarise the involvement of RGS proteins in cardiometabolic function with a focus on their tissue distribution, mechanisms of action and dysregulation under various disease conditions. We also discuss the potential therapeutic applications for targeting RGS proteins in treating cardiometabolic conditions and current progress in developing RGS modulators. LINKED ARTICLES: This article is part of a themed issue Therapeutic Targeting of G Protein-Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc.