AKAP12 Upregulation Associates With PDE8A to Accelerate Cardiac Dysfunction.

BACKGROUND: In heart failure, signaling downstream the ß2-adrenergic receptor is critical. Sympathetic stimulation of ß2-adrenergic receptor alters cAMP (cyclic adenosine 3',5'-monophosphate) and triggers PKA (protein kinase A)-dependent phosphorylation of proteins that regulate cardiac function. cAMP levels are regulated in part by PDEs (phosphodiesterases). Several AKAPs (A kinase anchoring proteins) regulate cardiac function and are proposed as targets for precise pharmacology. AKAP12 is expressed in the heart and has been reported to directly bind ß2-adrenergic receptor, PKA, and PDE4D. However, its roles in cardiac function are unclear. METHODS: cAMP accumulation in real time downstream of the ß2-adrenergic receptor was detected for 60 minutes in live cells using the luciferase-based biosensor (GloSensor) in AC16 human-derived cardiomyocyte cell lines overexpressing AKAP12 versus controls. Cardiomyocyte intracellular calcium and contractility were studied in adult primary cardiomyocytes from male and female mice overexpressing cardiac AKAP12 (AKAP12OX) and wild-type littermates post acute treatment with 100-nM isoproterenol (ISO). Systolic cardiac function was assessed in mice after 14 days of subcutaneous ISO administration (60 mg/kg per day). AKAP12 gene and protein expression levels were evaluated in left ventricular samples from patients with end-stage heart failure. RESULTS: AKAP12 upregulation significantly reduced total intracellular cAMP levels in AC16 cells through PDE8. Adult primary cardiomyocytes from AKAP12OX mice had significantly reduced contractility and impaired calcium handling in response to ISO, which was reversed in the presence of the selective PDE8 inhibitor (PF-04957325). AKAP12OX mice had deteriorated systolic cardiac function and enlarged left ventricles. Patients with end-stage heart failure had upregulated gene and protein levels of AKAP12. CONCLUSIONS: AKAP12 upregulation in cardiac tissue is associated with accelerated cardiac dysfunction through the AKAP12-PDE8 axis.

A Small Molecule That In Vitro Neutralizes Infection of SARS-CoV-2 and Its Most Infectious Variants, Delta, and Omicron.

The COVID-19 pandemic has underscored the urgent need to develop highly potent and safe medications that are complementary to the role of vaccines. Specifically, it has exhibited the need for orally bioavailable broad-spectrum antivirals that are able to be quickly deployed against newly emerging viral pathogens. The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and its variants Delta and Omicron are still a major threat to patients of all ages. In this brief report, we describe that the small molecule CD04872SC was able to neutralize SARS-CoV2 infection with a half-maximal effective concentration (EC50) = 248 µM. Serendipitously, we also were able to observe that CD04872SC inhibited the infection of the SARS-CoV-2 variants; Delta (EC50 = 152 µM) and Omicron (EC50 = 308 µM). These properties may define CD04872SC as a potential broad-spectrum candidate lead for the development of treatments for COVID-19.

Fusion of the ß2-adrenergic receptor with either Gαs or ßarrestin-2 produces constitutive signaling by each pathway and induces gain-of-function in BEAS-2B cells.

The ß2AR is a prototypical G protein-coupled receptor (GPCR) known to orchestrate different cellular responses by the stimulation of specific signaling pathways. The best-established signaling pathways for the ß2AR are the canonical Gs pathway and the alternative ß arrestin 2 (ßarr2) pathway. Exploring each pathway separately remains a challenging task due to the dynamic nature of the receptor. Here, we fused the ß2AR with its cognate transducers, Gαs and ßarr2, using short linkers as a novel approach for restricting the conformation of the receptor and preferentially activating one of its two signaling pathways. We characterized the behavior of our fusion proteins ß2AR-Gαs and ß2AR-ßarr2 in HEK293 cells by measuring their constitutive activity, transducer recruitment, and pharmacological modulation. Our fusion proteins show (a) steric hindrance from the reciprocal endogenous transducers, (b) constitutive activity of the ß2AR for the signaling pathway activated by the tethered transducer, and (c) pharmacologic modulation by ß2AR ligands. Based on these characteristics, we further explored the possibility of a gain-of-function mechanism in the human lung non-tumorigenic epithelial cell line, BEAS-2B cells. This immortalized human bronchial epithelial cell line has immunomodulatory properties through cytokine release mediated by ß2AR stimulation. Our findings suggest that each signaling pathway of the ß2AR is biased toward either the Th1 or Th2 inflammatory response suggesting a role in regulating the immune phenotype of respiratory diseases. Our data imply that our fusion proteins can be used as tools to isolate the function elicited by a single signaling pathway in physiologically relevant cell types.

Direct reprogramming of cardiomyocytes into cardiac Purkinje-like cells.

Currently, there are no treatments that ameliorate cardiac cell death, the underlying basis of cardiovascular disease. An unexplored cell type in cardiac regeneration is cardiac Purkinje cells; specialized cells from the cardiac conduction system (CCS) responsible for propagating electrical signals. Purkinje cells have tremendous potential as a regenerative treatment because they may intrinsically integrate with the CCS of a recipient myocardium, resulting in more efficient electrical conduction in diseased hearts. This study is the first to demonstrate an effective protocol for the direct reprogramming of human cardiomyocytes into cardiac Purkinje-like cells using small molecules. The cells generated were genetically and functionally similar to native cardiac Purkinje cells, where expression of key cardiac Purkinje genes such as CNTN2, ETV1, PCP4, IRX3, SCN5a, HCN2 and the conduction of electrical signals with increased velocity was observed. This study may help to advance the quest to finding an optimized cell therapy for heart regeneration.

A NanoBiT assay to monitor membrane proteins trafficking for drug discovery and drug development.

Internalization of membrane proteins plays a key role in many physiological functions; however, highly sensitive and versatile technologies are lacking to study such processes in real-time living systems. Here we describe an assay based on bioluminescence able to quantify membrane receptor trafficking for a wide variety of internalization mechanisms such as GPCR internalization/recycling, antibody-mediated internalization, and SARS-CoV2 viral infection. This study represents an alternative drug discovery tool to accelerate the drug development for a wide range of physiological processes, such as cancer, neurological, cardiopulmonary, metabolic, and infectious diseases including COVID-19.

CXCR7: a ß-arrestin-biased receptor that potentiates cell migration and recruits ß-arrestin2 exclusively through Gßγ subunits and GRK2.

BACKGROUND: Some chemokine receptors referred to as atypical chemokine receptors (ACKRs) are thought to non-signaling decoys because of their inability to activate typical G-protein signaling pathways. CXCR7, also known as ACKR3, binds to only two chemokines, SDF-1α and I-TAC, and recruits ß-arrestins. SDF-1α also binds to its own conventional receptor, CXCR4, involving in homeostatic modulation such as development and immune surveillance as well as pathological conditions such as inflammation, ischemia, and cancers. Recently, CXCR7 is suggested as a key therapeutic target together with CXCR4 in such conditions. However, the molecular mechanisms underlying cellular responses and functional relation with CXCR7 and CXCR4 have not been elucidated, despite massive studies. Therefore, we aimed to reveal the molecular networks of CXCR7 and CXCR4 and compare their effects on cell migration. METHODS: Base on structural complementation assay using NanoBiT technology, we characterized the distinct mechanisms underlying ß-arrestin2 recruitment by both CXCR4 and CXCR7. Crosslinking and immunoprecipitation were conducted to analyze complex formation of the receptors. Gene deletion using CRISPR and reconstitution of the receptors were applied to analysis of ligand-dependent ERK phosphorylation and cell migration. All experiments were performed in triplicate and repeated more than three times. Unpaired Student's t-tests or ANOVA using PRISM5 software were employed for statistical analyses. RESULTS: Ligand binding to CXCR7 does not result in activation of typical signaling pathways via Gα subunits but activation of GRK2 via ßγ subunits and receptor phosphorylation with subsequent ß-arrestin2 recruitment. In contrast, CXCR4 induced Gαi activation and recruited ß-arrestin2 through C-terminal phosphorylation by both GRK2 and GRK5. SDF-1α-stimulated ERK phosphorylation was facilitated by CXCR4, but not CXCR7. Heterodimerization of CXCR4 and CXCR7 was not confirmed in this study, while homodimerization of them was verified by crosslinking experiment and NanoBiT assay. Regarding chemotaxis, SDF-1α-stimulated cell migration was mediated by both CXCR4 and CXCR7. CONCLUSION: This study demonstrates that SDF-1α-stimulated CXCR7 mediates ß-arrestin2 recruitment via different molecular networking from that of CXCR4. CXCR7 may be neither a simple scavenger nor auxiliary receptor but plays an essential role in cell migration through cooperation with CXCR4.

Establishment of a NanoBiT-Based Cytosolic Ca2+ Sensor by Optimizing Calmodulin-Binding Motif and Protein Expression Levels.

Cytosolic Ca2+ levels ([Ca2+]c) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca2+]c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca2+]c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca2+]c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca2+-loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca2+]c. Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca2+]c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca2+]c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca2+]c in single cells and animal models.

Exploring the molecular structures that confer ligand selectivity for galanin type II and III receptors.

Galanin receptors (GALRs) belong to the superfamily of G-protein coupled receptors. The three GALR subtypes (GALR1, GALR2, and GALR3) are activated by their endogenous ligands: spexin (SPX) and galanin (GAL). The synthetic SPX-based GALR2-specific agonist, SG2A, plays a dual role in the regulation of appetite and depression-like behaviors. Little is known, however, about the molecular interaction between GALR2 and SG2A. Using site-directed mutagenesis and domain swapping between GALR2 and GALR3, we identified residues in GALR2 that promote interaction with SG2A and residues in GALR3 that inhibit interaction with SG2A. In particular, Phe103, Phe106, and His110 in the transmembrane helix 3 (TM3) domain; Val193, Phe194, and Ser195 in the TM5 domain; and Leu273 in the extracellular loop 3 (ECL3) domain of GALR2 provide favorable interactions with the Asn5, Ala7, Phe11, and Pro13 residues of SG2A. Our results explain how SG2A achieves selective interaction with GALR2 and inhibits interaction with GALR3. The results described here can be used broadly for in silico virtual screening of small molecules for the development of GALR subtype-specific agonists and/or antagonists.

Monitoring GPCR-ß-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery.

Interactions between G-protein coupled receptors (GPCRs) and ß-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with ß-arrestins and other cytosolic proteins is extremely valuable in the field of GPCR drug discovery particularly during the study of GPCR biased agonism. Here, we show the application of a novel structural complementation assay to accurately monitor receptor-ß-arrestin interactions in real time living systems. This method is simple, accurate and can be easily extended to any GPCR of interest and also it has the advantage that it overcomes unspecific interactions due to the presence of a low expression promoter present in each vector system. This structural complementation assay provides key features that allow an accurate and precise monitoring of receptor-ß-arrestin interactions, making it suitable in the study of biased agonism of any GPCR system as well as GPCR c-terminus 'phosphorylation codes' written by different GPCR-kinases (GRKs) and post-translational modifications of arrestins that stabilize or destabilize the receptor-ß-arrestin complex.

Spexin-Based Galanin Receptor Type 2 Agonist for Comorbid Mood Disorders and Abnormal Body Weight.

Despite the established comorbidity between mood disorders and abnormal eating behaviors, the underlying molecular mechanism and therapeutics remain to be resolved. Here, we show that a spexin-based galanin receptor type 2 agonist (SG2A) simultaneously normalized mood behaviors and body weight in corticosterone pellet-implanted (CORTI) mice, which are underweight and exhibit signs of anhedonia, increased anxiety, and depression. Administration of SG2A into the lateral ventricle produced antidepressive and anxiolytic effects in CORTI mice. Additionally, SG2A led to a recovery of body weight in CORTI mice while it induced significant weight loss in normal mice. In Pavlovian fear-conditioned mice, SG2A decreased contextual and auditory fear memory consolidation but accelerated the extinction of acquired fear memory without altering innate fear and recognition memory. The main action sites of SG2A in the brain may include serotonergic neurons in the dorsal raphe nucleus for mood control, and proopiomelanocortin/corticotropin-releasing hormone neurons in the hypothalamus for appetite and body weight control. Furthermore, intranasal administration of SG2A exerted the same anxiolytic and antidepressant-like effects and decreased food intake and body weight in a dose-dependent manner. Altogether, these results indicate that SG2A holds promise as a clinical treatment for patients with comorbid mood disorders and abnormal appetite/body weight.

Conformational signatures in ß-arrestin2 reveal natural biased agonism at a G-protein-coupled receptor.

Discovery of biased ligands and receptor mutants allows characterization of G-protein- and ß-arrestin-mediated signaling mechanisms of G-protein-coupled receptors (GPCRs). However, the structural mechanisms underlying biased agonism remain unclear for many GPCRs. We show that while Galanin induces the activation of the galanin receptor 2 (Galr2) that leads to a robust stimulation toward Gαq-protein and ß-arrestin1/2, an alternative ligand Spexin and its analog have biased agonism toward G-protein signaling relative to Galanin. We used intramolecular fluorescein arsenical hairpin bioluminescence resonance energy transfer-based biosensors of ß-arrestin2 combined with NanoBit technology to measure ß-arrestin2-Galr2 interactions in real-time living systems. We found that Spexin and Galanin induce specific active conformations of Galr2, which may lead to different internalization rates of the receptor as well as different signaling outputs. This work represents an additional pharmacological evidence of endogenous G-protein-biased agonism at a GPCR.

Development of Spexin-based Human Galanin Receptor Type II-Specific Agonists with Increased Stability in Serum and Anxiolytic Effect in Mice.

The novel neuropeptide spexin (SPX) was discovered to activate galanin receptor 2 (GALR2) and 3 (GALR3) but not galanin receptor 1 (GALR1). Although GALR2 is known to display a function, particularly in anxiety, depression, and appetite regulation, the further determination of its function would benefit from a more stable and selective agonist that acts only at GALR2. In the present study, we developed a GALR2-specific agonist with increased stability in serum. As galanin (GAL) showed a low affinity to GALR3, the residues in SPX were replaced with those in GAL, revealing that particular mutations such as Gln5 → Asn, Met7 → Ala, Lys11 → Phe, and Ala13 → Pro significantly decreased potencies toward GALR3 but not toward GALR2. Quadruple (Qu) mutation of these residues still retained potency to GALR2 but totally abolished the potency to both GALR3 and GALR1. The first amino acid modifications or D-Asn1 substitution significantly increased the stability when they are incubated in 100% fetal bovine serum. Intracerebroventricular administration of the mutant peptide with D-Asn1 and quadruple substitution (dN1-Qu) exhibited an anxiolytic effect in mice. Taken together, the GALR2-specific agonist with increased stability can greatly help delineation of GALR2-mediated functions and be very useful for treatments of anxiety disorder.

Prevertebrate Local Gene Duplication Facilitated Expansion of the Neuropeptide GPCR Superfamily.

In humans, numerous genes encode neuropeptides that comprise a superfamily of more than 70 genes in approximately 30 families and act mainly through rhodopsin-like G protein-coupled receptors (GPCRs). Two rounds of whole-genome duplication (2R WGD) during early vertebrate evolution greatly contributed to proliferation within gene families; however, the mechanisms underlying the initial emergence and diversification of these gene families before 2R WGD are largely unknown. In this study, we analyzed 25 vertebrate rhodopsin-like neuropeptide GPCR families and their cognate peptides using phylogeny, synteny, and localization of these genes on reconstructed vertebrate ancestral chromosomes (VACs). Based on phylogeny, these GPCR families can be divided into five distinct clades, and members of each clade tend to be located on the same VACs. Similarly, their neuropeptide gene families also tend to reside on distinct VACs. Comparison of these GPCR genes with those of invertebrates including Drosophila melanogaster, Caenorhabditis elegans, Branchiostoma floridae, and Ciona intestinalis indicates that these GPCR families emerged through tandem local duplication during metazoan evolution prior to 2R WGD. Our study describes a presumptive evolutionary mechanism and development pathway of the vertebrate rhodopsin-like GPCR and cognate neuropeptide families from the urbilaterian ancestor to modern vertebrates.

Ligand binding pocket formed by evolutionarily conserved residues in the glucagon-like peptide-1 (GLP-1) receptor core domain.

Glucagon-like peptide-1 (GLP-1) plays a pivotal role in glucose homeostasis through its receptor GLP1R. Due to its multiple beneficial effects, GLP-1 has gained great attention for treatment of type 2 diabetes and obesity. However, little is known about the molecular mechanism underlying the interaction of GLP-1 with the heptahelical core domain of GLP1R conferring high affinity ligand binding and ligand-induced receptor activation. Here, using chimeric and point-mutated GLP1R, we determined that the evolutionarily conserved amino acid residue Arg(380) flanked by hydrophobic Leu(379) and Phe(381) in extracellular loop 3 (ECL3) may have an interaction with Asp(9) and Gly(4) of the GLP-1 peptide. The molecular modeling study showed that Ile(196) at transmembrane helix 2, Met(233) at ECL1, and Asn(302) at ECL2 of GLP1R have contacts with His(1) and Thr(7) of GLP-1. This study may shed light on the mechanism underlying high affinity interaction between the ligand and the binding pocket that is formed by these conserved residues in the GLP1R core domain.

Salt effects on the conformational stability of the visual G-protein-coupled receptor rhodopsin.

Membrane protein stability is a key parameter with important physiological and practical implications. Inorganic salts affect protein stability, but the mechanisms of their interactions with membrane proteins are not completely understood. We have undertaken the study of a prototypical G-protein-coupled receptor, the α-helical membrane protein rhodopsin from vertebrate retina, and explored the effects of inorganic salts on the thermal decay properties of both its inactive and photoactivated states. Under high salt concentrations, rhodopsin significantly increased its activation enthalpy change for thermal bleaching, whereas acid denaturation affected the formation of a denatured loose-bundle state for both the active and inactive conformations. This behavior seems to correlate with changes in protonated Schiff-base hydrolysis. However, chromophore regeneration with the 11-cis-retinal chromophore and MetarhodopsinII decay kinetics were slower only in the presence of sodium chloride, suggesting that in this case, the underlying phenomenon may be linked to the activation of rhodopsin and the retinal release processes. Furthermore, the melting temperature, determined by means of circular dichroism and differential scanning calorimetry measurements, was increased in the presence of high salt concentrations. The observed effects on rhodopsin could indicate that salts favor electrostatic interactions in the retinal binding pocket and indirectly favor hydrophobic interactions at the membrane protein receptor core. These effects can be exploited in applications where the stability of membrane proteins in solution is highly desirable.