DRY Motif Research Articles

Divergent roles of DRY and NPxxY motifs in selective activation of downstream signalling by the apelin receptor.

G protein-coupled receptors (GPCRs) serve as critical communication hubs, translating a wide range of extracellular signals into intracellular responses that govern numerous physiological processes. In class-A GPCRs, conserved motifs mediate conformational changes of the active states of the receptor, and signal transduction is achieved by selectively binding to Gα proteins and/or adapter protein, arrestin. Apelin receptor (APJR) is a class-A GPCR that regulates a wide range of intracellular signalling cascades in response to apelin and elabela peptide ligands. Understanding how conserved motifs within APJR mediate activation and signal specificity remains unexplored. This study focuses on the functional roles of the DRY and NPxxY motifs within APJR by analyzing their impact on downstream signaling pathways across the receptor's conformational ensembles. Our findings provide compelling evidence that mutations within the conserved DRY and NPxxY motifs of APJR significantly alter its conformational preferences where modification of DRY motif leads to abrogation of G-protein coupling and mutation of NPxxY motif causing abolition of β-arrestin-2 recruitment. These observations shed light on the importance of these motifs in APJR activation and its potential for functional selectivity, highlighting the role of DRY/NPxxY as conformational switches of APJR signalling.

Evolutionary diversity of CXCL16-CXCR6: Convergent substitutions and recurrent gene loss in sauropsids.

The CXCL16-CXCR6 axis is crucial for regulating the persistence of CD8 tissue-resident memory T cells (TRM). CXCR6 deficiency lowers TRM cell numbers in the lungs and depletes ILC3s in the lamina propria, impairing mucosal defence. This axis is linked to diseases like HIV/SIV, cancer, and COVID-19. Together, these highlight that the CXCL16-CXCR6 axis is pivotal in host immunity. Previous studies of the CXCL16-CXCR6 axis found genetic variation among species but were limited to primates and rodents. To understand the evolution and diversity of CXCL16-CXCR6 across vertebrates, we compared approximately 400 1-to-1 CXCR6 orthologs spanning diverse vertebrates. The unique DRF motif of CXCR6 facilitates leukocyte adhesion by interacting with cell surface-expressed CXCL16 and plays a key role in G-protein selectivity during receptor signalling; however, our findings show that this motif is not universal. The DRF motif is restricted to mammals, turtles, and frogs, while the DRY motif, typical in other CKRs, is found in snakes and lizards. Most birds exhibit the DRL motif. These substitutions at the DRF motif affect the receptor-Gi/o protein interaction. We establish recurrent CXCR6 gene loss in 10 out of 36 bird orders, including Galliformes and Passeriformes, Crocodilia, and Elapidae, attributed to segmental deletions and/or frame-disrupting changes. Notably, single-cell RNA sequencing of the lung shows a drop in TRM cells in species with CXCR6 loss, suggesting a possible link. The concurrent loss of ITGAE, CXCL16, and CXCR6 in chickens may have altered CD8 TRM cell abundance, with implications for immunity against viral diseases and vaccines inducing CD8 TRM cells.

The repertoire of G-protein-coupled receptor variations in the Japanese population 54KJPN.

G-protein-coupled receptors (GPCRs) are the largest superfamily in the human genome and the major targets for the market drugs. Recent massive genomics studies revealed numerous natural variations in the general population. 54KJPN is the most extensive Japanese population genomics study, curating the whole genome sequences from about 54,000 individuals. Here, by analyzing 390 non-olfactory GPCR genes in the 54KJPN dataset, we annotated 25,443 missense single-nucleotide variations. Among them, we found 120 major variations that appear with an allele frequency greater than 0.5, including variations that occurred on posttranslational modification sites. Structural alignment of GPCRs using the generic numbering system in the GPCRdb reveals enrichment of alterations in the conserved arginine residue within the DRY motif, which contributes to downstream G-protein signaling. A comparison with the worldwide 1000 Genomes Project (1KGP) dataset found 23 variations that were present exclusively in the 54KJPN dataset. This study will be the basis for future pharmacogenomics studies for the Japanese population.

Conformational dynamics underlying atypical chemokine receptor 3 activation

Atypical Chemokine Receptor 3 (ACKR3) belongs to the G protein-coupled receptor family but it does not signal through G proteins. The structural properties that govern the functional selectivity and the conformational dynamics of ACKR3 activation are poorly understood. Here, we combined hydrogen/deuterium exchange mass spectrometry, site-directed mutagenesis, and molecular dynamics simulations to examine the binding mode and mechanism of action of ACKR3 ligands of different efficacies. Our results show that activation or inhibition of ACKR3 is governed by intracellular conformational changes of helix 6, intracellular loop 2, and helix 7, while the DRY motif becomes protected during both processes. Moreover, we identified the binding sites and the allosteric modulation of ACKR3 upon β-arrestin 1 binding. In summary, this study highlights the structure-function relationship of small ligands, the binding mode of β-arrestin 1, the activation dynamics, and the atypical dynamic features in ACKR3 that may contribute to its inability to activate G proteins.

Effects of an external static EF on the conformational transition of 5-HT1A receptor: A molecular dynamics simulation study

The serotonin receptor subtype 1A (5-HT1AR), one of the G-protein-coupled receptor (GPCR) family, has been implicated in several neurological conditions. Understanding the activation and inactivation mechanism of 5-HT1AR at the molecular level is critical for discovering novel therapeutics in many diseases. Recently there has been a growing appreciation for the role of external electric fields (EFs) in influencing the structure and activity of biomolecules. In this study, we used molecular dynamics (MD) simulations to examine conformational features of active states of 5-HT1AR and investigate the effect of an external static EF with 0.02 V/nm applied on the active state of 5-HT1AR. Our results showed that the active state of 5-HT1AR maintained the native structure, while the EF led to structural modifications in 5-HT1AR, particularly inducing the inward movement of transmembrane helix 6 (TM6). Furthermore, it disturbed the conformational switches associated with activation in the CWxP, DRY, PIF, and NPxxY motifs, consequently predisposing an inclination towards the inactive-like conformation. We also found that the EF led to an overall increase in the dipole moment of 5-HT1AR, encompassing TM6 and pivotal amino acids. The analyses of conformational properties of TM6 showed that the changed secondary structure and decreased solvent exposure occurred upon the EF condition. The interaction of 5-HT1AR with the membrane lipid bilayer was also altered under the EF. Our findings reveal the molecular mechanism underlying the transition of 5-HT1AR conformation induced by external EFs, which offer potential novel insights into the prospect of employing structure-based EF applications for GPCRs.

Evaluating conserved domains and motifs of decapod gonadotropin-releasing hormone G protein-coupled receptor superfamily.

G protein-coupled receptors (GPCRs) are an ancient family of signal transducers that are both abundant and consequential in metazoan endocrinology. The evolutionary history and function of the GPCRs of the decapod superfamilies of gonadotropin-releasing hormone (GnRH) are yet to be fully elucidated. As part of which, the use of traditional phylogenetics and the recycling of a diminutive set of mis-annotated databases has proven insufficient. To address this, we have collated and revised eight existing and three novel GPCR repertoires for GnRH of decapod species. We developed a novel bioinformatic workflow that included clustering analysis to capture likely GnRH receptor-like proteins, followed by phylogenetic analysis of the seven transmembrane-spanning domains. A high degree of conservation of the sequences and topology of the domains and motifs allowed the identification of species-specific variation (up to ~70%, especially in the extracellular loops) that is thought to be influential to ligand-binding and function. Given the key functional role of the DRY motif across GPCRs, the classification of receptors based on the variation of this motif can be universally applied to resolve cryptic GPCR families, as was achieved in this work. Our results contribute to the resolution of the evolutionary history of invertebrate GnRH receptors and inform the design of bioassays in their deorphanization and functional annotation.

Full-length transcriptome reveals the circularly polarized light response-related molecular genetic characteristics of Oratosquilla oratoria

The mantis shrimp is the only animal that can recognize circularly polarized light (CPL), but its molecular genetic characteristics are unclear. Multi-tissue level full-length (FL) transcriptome sequencing of Oratosquilla oratoria, a representative widely distributed mantis shrimp, was performed in the present study. We used comparative transcriptomics to explore the critical genes of O. oratoria selected by CPL and the GNβ gene associated with CPL signal transduction was hypothesized to be positively selected. Furthermore, the FL transcriptomes of O. oratoria compound eyes under five light conditions were sequenced and used to detect alternative splicing (AS). The ASs associated with CPL recognition mainly occurred in the LWS, ARR and TRPC regions. The number of FL transcripts with AS events and annotation information also provided evidence that O. oratoria could recognize LCPL. Additionally, 51 sequences belonging to the LWS, UV and Peropsin gene families were identified based on conserved 7tm domains. The LWS, UV and Peropsin opsins have similar 3D structures with seven domains across the cell membrane and conserved KSLRTPSN, DRY, and QAKK motifs. In conclusion, these results are undoubtedly valuable for perfecting the vision theory of O. oratoria and other mantis shrimp.

Cholesterol Biases the Conformational Landscape of the Chemokine Receptor CCR3: A MAS SSNMR-Filtered Molecular Dynamics Study.

Cholesterol directs the pathway of ligand-induced G protein-coupled receptor (GPCR) signal transduction. The GPCR C-C motif chemokine receptor 3 (CCR3) is the principal chemotactic receptor for eosinophils, with roles in cancer metastasis and autoinflammatory conditions. Recently, we discovered a direct correlation between bilayer cholesterol and increased agonist-triggered CCR3 signal transduction. However, the allosteric molecular mechanism escalating ligand affinity and G protein coupling is unknown. To study cholesterol-guided CCR3 conformational selection, we implement comparative, objective measurement of protein architectures by scoring shifts (COMPASS) to grade model structures from molecular dynamics simulations. In this workflow, we scored predicted chemical shifts against 2-dimensional solid-state NMR 13C-13C correlation spectra of U-15N,13C-CCR3 samples prepared with and without cholesterol. Our analysis of trajectory model structures uncovers that cholesterol induces site-specific conformational restraint of extracellular loop (ECL) 2 and conserved motion in transmembrane helices and ECL3 not observed in simulations of bilayers with only phosphatidylcholine lipids. PyLipID analysis implicates direct cholesterol agency in CCR3 conformational selection and dynamics. Residue-residue contact scoring shows that cholesterol biases the conformational selection of the orthosteric pocket involving Y411.39, Y1133.32, and E2877.39. Lastly, we observe contact remodeling in activation pathway residues centered on the initial transmission switch, Na+ pocket, and R3.50 in the DRY motif. Our observations have unique implications for understanding of CCR3 ligand recognition and specificity and provide mechanistic insight into how cholesterol functions as an allosteric regulator of CCR3 signal transduction.

D121 Located within the DRY Motif of P2Y12 Is Essential for P2Y12-Mediated Platelet Function.

Platelets are anucleate cells that mediate hemostasis. This occurs via a primary signal that is reinforced by secreted products such as ADP that bind purinergic receptors (P2Y1 and P2Y12) on the platelet surface. We recently identified a human subject, whom we termed platelet defect subject 25 (PDS25) with a platelet functional disorder associated with the P2Y12 receptor. PDS25 has normal blood cell counts and no history of bleeding diathesis. However, platelets from PDS25 have virtually no response to 2-MeSADP (a stable analogue of ADP). Genetic analysis of P2Y12 from PDS25 revealed a heterozygous mutation of D121N within the DRY motif. Rap1b activity was reduced in platelets from PDS25, while VASP phosphorylation was enhanced, suggesting that signaling from the P2Y12 receptor was interrupted by the heterozygous mutation. To explore this further, we produced knock-in mice that mimic our subject. Bleeding failed to cease in homozygous KI mice during tail bleeding assays, while tail bleeding times did not differ between WT and heterozygous KI mice. Furthermore, occlusions failed to form in most homozygous KI mice following carotid artery injury via FeCl3. These data indicate that the aspartic acid residue found in the DRY motif of P2Y12 is essential for P2Y12 function.

Cryo-EM structures of human bradykinin receptor-Gq proteins complexes

The type 2 bradykinin receptor (B2R) is a G protein-coupled receptor (GPCR) in the cardiovascular system, and the dysfunction of B2R leads to inflammation, hereditary angioedema, and pain. Bradykinin and kallidin are both endogenous peptide agonists of B2R, acting as vasodilators to protect the cardiovascular system. Here we determine two cryo-electron microscopy (cryo-EM) structures of human B2R-Gq in complex with bradykinin and kallidin at 3.0 Å and 2.9 Å resolution, respectively. The ligand-binding pocket accommodates S-shaped peptides, with aspartic acids and glutamates as an anion trap. The phenylalanines at the tail of the peptides induce significant conformational changes in the toggle switch W2836.48, the conserved PIF, DRY, and NPxxY motifs, for the B2R activation. This further induces the extensive interactions of the intracellular loops ICL2/3 and helix 8 with Gq proteins. Our structures elucidate the molecular mechanisms for the ligand binding, receptor activation, and Gq proteins coupling of B2R.

Unique features of different classes of G-protein-coupled receptors revealed from sequence coevolutionary and structural analysis.

G-protein-coupled receptors (GPCRs) are the largest family of human membrane proteins and represent the primary targets of about one third of currently marketed drugs. Despite the critical importance, experimental structures have been determined for only a limited portion of GPCRs and functional mechanisms of GPCRs remain poorly understood. Here, we have constructed novel sequence coevolutionary models of the A and B classes of GPCRs and compared them with residue contact frequency maps generated with available experimental structures. Significant portions of structural residue contacts were successfully detected in the sequence-based covariational models. "Exception" residue contacts predicted from sequence coevolutionary models but not available structures added missing links that were important for GPCR activation and allosteric modulation. Moreover, we identified distinct residue contacts involving different sets of functional motifs for GPCR activation, such as the Na+ pocket, CWxP, DRY, PIF, and NPxxY motifs in the class A and the HETx and PxxG motifs in the class B. Finally, we systematically uncovered critical residue contacts tuned by allosteric modulation in the two classes of GPCRs, including those from the activation motifs and particularly the extracellular and intracellular loops in class A GPCRs. These findings provide a promising framework for rational design of ligands to regulate GPCR activation and allosteric modulation.

Genome-wide identification and analysis of chemokine receptor superfamily in miiuy croaker, Miichthys miiuy

The chemokine receptor (ChemR) superfamily, which is divided into 4 subfamilies (CXCR, CCR, XCR, and CX3CR), is the main receptors of chemokines in innate immune responses. In the current study, we have identified 27 ChemRs in miiuy croaker: 13 CCR genes, 11 CXCR genes, and 3 XCR genes. Multiple characteristics of these genes, including phylogeny, gene structures, conserved motifs, chromosome locations, evolutionary mechanism, and expression levels upon the bacterial challenge were analyzed. Gene structure and location analysis showed that all ChemR genes contain fewer introns (≤4) and they are unevenly distributed on the 12 chromosomes. And the XCR subfamily of miiuy croaker don't have the DRY motif of ChemR. Phylogenetic and synteny analysis showed that these genes experienced tandem and segmental duplication event in several species, and tandem duplication might be the main expansion way in miiuy croaker. The major ChemRs of each orthologous group in vertebrates were selected for molecular evolution analysis, the results of which indicated that compared with vertebrates, ChemRs of teleost fishes may have a relatively high evolutionary dynamic. In addition, a total of 21 positively selected codons were detected in vertebrate ChemRs under Model 8. RNA-Seq analysis and qRT-PCR verification demonstrated that CXCR3.2, CXCR5, and XCR1 genes were up-regulated significantly upon the Vibrio harveyi infection. These results provide valuable information for investigating the evolutionary relationships of chemokine receptor superfamily in miiuy croaker and laid the basis for further functional analysis.

Living-Cell Diffracted X-ray Tracking Analysis Confirmed Internal Salt Bridge Is Critical for Ligand-Induced Twisting Motion of Serotonin Receptors.

Serotonin receptors play important roles in neuronal excitation, emotion, platelet aggregation, and vasoconstriction. The serotonin receptor subtype 2A (5-HT2AR) is a Gq-coupled GPCR, which activate phospholipase C. Although the structures and functions of 5-HT2ARs have been well studied, little has been known about their real-time dynamics. In this study, we analyzed the intramolecular motion of the 5-HT2AR in living cells using the diffracted X-ray tracking (DXT) technique. The DXT is a very precise single-molecular analytical technique, which tracks diffraction spots from the gold nanocrystals labeled on the protein surface. Trajectory analysis provides insight into protein dynamics. The 5-HT2ARs were transiently expressed in HEK 293 cells, and the gold nanocrystals were attached to the N-terminal introduced FLAG-tag via anti-FLAG antibodies. The motions were recorded with a frame rate of 100 μs per frame. A lifetime filtering technique demonstrated that the unliganded receptors contain high mobility population with clockwise twisting. This rotation was, however, abolished by either a full agonist α-methylserotonin or an inverse agonist ketanserin. Mutation analysis revealed that the “ionic lock” between the DRY motif in the third transmembrane segment and a negatively charged residue of the sixth transmembrane segment is essential for the torsional motion at the N-terminus of the receptor.

The TRY‐motif in the Relaxin Family Peptide Receptor 3 (RXFP3) functions as a Molecular Switch Between DNA Damage Response and Cell Cycle Control

The majority of Rhodopsin-like Class A G protein-coupled receptors (GPCRs) contain the conserved Aspartate–Arginine–Tyrosine (DRY) motif in transmembrane helix 3 (TM3). This DRY-motif is responsible for the presence of an ionic lock, consisting of a salt bridge between the positively charged R3.50 in TM3 and the negatively charged glutamic acid (E6.30) on TM6 in typical rhodopsin-like GPCRs. While the R3.50 is 96% conserved across all class A GPCRs, D3.49 and Y3.51 are 66, and 67% conserved respectively. This variance in D3.49 and Y3.51 residues potential identifies a locus for evolutionary modification of GPCR signaling. The relaxin family peptide receptor 3 (RXFP3), a Class A GPCR, which plays a crucial role in the aging process via providing resilience to stress, possesses a natural variant of the DRY motif, where Aspartate is replaced by Threonine, creating a TRY motif. This variation enhances the ligand-independent activity of the RXFP3 receptor. We investigated how this natural variation may be implicated in its stress-resistance role through selective mutation of the TRY motif back to a canonical DRY sequence. Using an unbiased quantitative proteomic perturbation response approach, we found that protein network analysis revealed that DNA damage repair functions are supported by the presence of a TRY motif while reversion to a DRY sequence in the RXFP3 diverts signaling activity instead to cell-cycle/cellular senescence control. Thus it is likely that the RXFP3 has been naturally mutated to a TRY sequence to promote its anti-aging stress sensor role.

A75 ON THE TRAIL OF ALTERNATIVE LIGANDS AND SIGNALING PATHWAYS FOR LGR5

Abstract Background Leucine-rich G protein coupled receptor-5 (LGR5) is a GPCR originally identified as a marker for intestinal stem cells but is now associated to stemness in numerous other tissues. R-spondins (RSPO) are the only reported ligands of LGR5, which upon receptor binding, potentiates the canonical Wnt/b-catenin pathway. Surprisingly, despite the presence of classical GPCR features such as conserved DRY and NPXXY motifs RSPO binding to LGR5 does not induce classical GPCR behaviors such as coupling to heterotrimeric G-proteins or engagement of b-arrestins. For this reason, LGR5 is still considered to be an orphan receptor. Aims Aberrant expression of LGR5 is a common feature of many cancers, including gastrointestinal cancers, but it is still unclear why LGR5 is pro-tumorigenic in some cancers, while anti-tumorigenic in others. One potential explanation is that LGR5 does not only signals through the Wnt/b-catenin pathway, but also to alternative pathways. Thus, the goals of this study were 1), to elucidate LGR5 signaling networks and 2), to identify new LGR5 ligands and activity modulators. We suggested that the intestinal lumen content could be a source of bioactive molecules that could act as alternative LGR5 ligands Methods All experiments were performed on HEK293 cells expressing recombinant LGR5 (HEK293/LGR5) and compared to control cells expressing empty vector. Characterization of LGR5 signaling network was initiated with LC MS/MS spectrometric analyses using cells stimulated with Wnt3a and RSPO1. To identify new LGR5 modulators, commercially available bacteria-derived metabolites (BDM) were tested on HEK293/LGR5 cells stimulated with Wnt3a and RSPO1 and assessed using the TOP-Flash luciferase system. Finally, a mouse intestinal lumen extract (ILE) was investigated in whole-cell impedance assay to detect if it contained native BDM or potential host-derived LGR5 ligands. Results Mass spectrometric analyses revealed distinct protein expression and phosphorylation profiles in HEK293/LGR5 cells stimulated with Wnt3a and RSPO1. The TOP/Flash luciferase assays showed that the flavonoid derivative 3,4-dihydroxyphenylacetic acid (DOPAC) and lipopolysaccharide (LPS) acted as negative regulators of LGR5, while butyrate had no effect. Interestingly, stimulation of HEK293/LGR5 with ILE induced a Gαq-like response in the impedance assay, while control cells did not respond. Conclusions Stimulation of LGR5 with Wnt and RSPO triggered a complex signaling network. In agreement with our hypothesis, some BDM such as DOPAC and LPS were indeed activity modulators of LGR5. Moreover, a still unidentified compound present in the ILE triggered a selective GPCR-like response in HEK293/LGR5 cells. Future works aim at identifying this putative LGR5 ligand(s) and establish the LGR5 interactome using proximity labeling and mass spectrometry. Funding Agencies NSERC, Université de Sherbrooke, FRQS

Functional analysis of the CXCR1a gene response to SGIV viral infection in grouper

Chemokine receptors are a superfamily of seven-transmembrane domain G-coupled receptors and have important roles in immune surveillance, inflammation, and development. In previous studies, a series of CXCRs in grouper (Epinephelus coioides) was identified; however, the function of CXCR in viral infection has not been studied. To better understand the effect of the CXCR family on the fish immune response, full-length CXCR1a was cloned, and its immune response to Singapore grouper iridovirus (SGIV) was investigated. Grouper CXCR1a shared a seven-transmembrane (7-TM) region and a G protein-coupled receptor (GPCR) family 1 that contained a triaa stretch (DRY motif). Phylogenetic analysis indicated that CXCR1a showed the nearest relationship to Takifugu rubripes, followed by other fish, bird and mammal species. Fluorescence microscopy revealed that CXCR1a was expressed predominantly in the cytoplasm. Overexpression of CXCR1a in grouper cells significantly inhibited the replication of SGIV, demonstrating that CXCR1a delayed the occurrence of cytopathic effects (CPE) induced by SGIV infection and inhibited viral gene transcription. Furthermore, our results also showed that CXCR1a overexpression significantly increased the expression of interferon-related cytokines and activated ISRE and IFN promoter activities. Taken together, the results demonstrated that CXCR1a might have an antiviral function against SGIV infection.

Severely Impaired Platelet Function Due to a Novel Mutation of P2Y12 from a Human Subject with No History of Bleeding

Platelets mediate hemostasis through amplifying an initial stimulus and aggregating at a site of injury. The identification of bleeding phenotypes in humans has the potential to reveal new insight into the mechanisms behind platelet signaling, as well as pinpoint new potential therapeutic targets. In fact, the study of bleeding disorders in human subjects has led to the identification of a number of disease states and proteins important to platelet functional responsiveness.We have recently identified a human subject (PDS25) with a platelet functional disorder that has a normal CBC, and no history of bleeding diathesis. However, platelets from PDS25 have virtually no response to even high concentrations of 2-MeSADP, even though P2Y12 protein expression appears unaltered (Figure 1). Further, platelet reactivity to high doses of agonist for other receptors, such as PAR-4 and GPVI, is normal. Low concentrations of these agonists, which typically rely on feedback for platelet reactivity, elicit an inhibited response using platelets from PDS25 compared to platelets from a healthy control. Consistently, Rap1b activity (Figure 2) was reduced in platelets from PDS25, while VASP phosphorylation was enhanced, suggesting that signaling from the P2Y12 receptor was interrupted by the heterozygous mutation. To determine whether it is the receptor or a downstream signaling component that is dysfunctional and because shape change in response to 2-MeSADP is normal in platelets from PDS25, we co-stimulated 2-MeSADP treated platelets with epinephrine to initiate signaling through Gz, which is very similar to Gi. We found that the response of platelets from PDS25 to 2-MeSADP can be rescued with the addition of epinephrine, suggesting that the signaling components downstream of Gi/zare intact. These data suggest that the aberrant reactivity observed in platelets from PDS25 is due to the P2Y12 receptor. Therefore, we performed a genetic analysis of P2Y12 from PDS25, which revealed a heterozygous mutation of D121N within the conserved DRY motif (Figure 3). We are currently evaluating how this heterozygous mutation confers such a strong phenotype. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Molecular characterization and expression analysis of Asian swamp eel (Monopterus albus) CXC chemokine receptor (CXCR) 1a, CXCR1b, CXCR2, CXCR3a, CXCR3b, and CXCR4 after bacteria and poly I:C challenge

The CXC chemokine receptors (CXCRs) play critical roles in innate and adaptive immune systems. In this study, six Asian swamp eel (Monopterus albus) CXCRs (MaCXCR1–4) were identified and their molecular characterization and expression patterns were analyzed. The open reading frames (ORFs) of MaCXCR1a, MaCXCR1b, MaCXCR2, MaCXCR3a, MaCXCR3b, and MaCXCR4 were 1074 bp (base pairs), 1080 bp, 1125 bp, 1146 bp, 1083 bp, and 1140 bp, and encoded proteins of 357 aa (amino acids), 359 aa, 374 aa, 381 aa, 360 aa, and 379 aa, respectively. All these CXCRs have seven conserved transmembrane domains and four cysteines (with the exception of MaCXCR3b). Multiple sequence alignment revealed that the MaCXCRs possess a typical G-protein receptor family 1 signature and a DRY motif. There are also one to four potential N-glycosylation sites in the extracellular regions of the MaCXCRs, mainly distributed in the N-terminus and extracellular hydrophilic loop (ECL) 2 region. Phylogenetic analysis demonstrated that the MaCXCRs were clustered together with homologous proteins from other fish. Taken together with the amino acid identity and similarity analysis, these results suggested that the MaCXCRs are conserved with other homologous genes, in which CXCR4 is more conserved than CXCR1–3. The MaCXCRs loci showed conserved synteny among teleost fish, and we found that human CXCR1 shares a common ancestor with fish CXCR1a. MaCXCRs were constitutively expressed in a wide range of tissues (especially in immune-related tissues) with different expression levels, suggesting that the MaCXCRs have different roles in un-stimulated tissues, and may play vital roles under normal conditions. MaCXCRs showed different fold changes in the spleen after Aeromonas veronii and polyinosinic-polycytidylic acid (poly I:C) challenge, which suggested that MaCXCR1a and MaCXCR3a have longer antiviral activities compared with their antibacterial functions, and that MaCXCR1b possesses stronger antiviral than antibacterial activity. MaCXCR4 may play vital roles during bacterial and viral infection; however, MaCXCR2 has relatively small effect in antibacterial and antiviral responses. The differential responses of these genes to bacteria and poly I:C implied the differences in the mechanisms of defense against viruses and bacteria.

Characterization and expression of a long neuropeptide F (NPF) receptor in the Chagas disease vector Rhodnius prolixus.

In this study, a long neuropeptide F receptor of the blood-feeding hemipteran, Rhodnius prolixus (RhoprNPFR) has been cloned and characterized. Approximately 70% of the RhoprNPFR deduced protein sequence is identical to that of other hemipteran NPFRs. RhoprNPFR has seven highly-conserved transmembrane domains, two cysteine residues in the 2nd and 3rd extracellular loops that likely form a disulfide bond integral for maintaining the structure of the receptor, and a conserved DRY motif after the third transmembrane domain. All of these characteristics are typical of class A rhodopsin-like GPCRs. The receptor transcript is predominantly expressed in the central nervous system (CNS) and gut of both fifth instar and adult R. prolixus. Using fluorescent in situ hybridization (FISH), we identified six bilaterally-paired large median neurosecretory cells (approximately 30μm in diameter) in the brain that express the RhoprNPFR mRNA transcript. We also found RhoprNPFR transcript expression in endocrine cells in the anterior midgut of fifth instars, as well as in putative pre-follicular cells present in the germarium and between developing oocytes, and in the nutritive cord. These results suggest that RhoprNPFR may play a role within the CNS, and in digestion and possibly egg production and/or egg development in R. prolixus.

Modeling Angiotensin II‐mediated activation of the Angiotensin II Type 1 Receptor

It is well known that agonist binding to GPCRs engages a structural rearrangement, leading to GDP disassociation from the G‐alpha subunit and subsequent GTP association and activation of the Gα subunit. However, the structural mechanism mediating intramolecular molecular communication between the agonist binding pocket and G‐alpha subunit remains unknown. A meta‐analysis of 27 crystal structures of inactive and active state GPCRs demonstrated a common structural rearrangement of the GPCRs; specifically, a convergence of residue rearrangement near the G protein‐receptor interface. In an inactive state, residues 3.46 and 1.53 are in proximity to 6.37 and 7.53, respectively; whereas in an active state, a rearrangement pairs residues 5.55 and 3.46 with 6.41 and 7.53, respectively. In order to understand the structural mechanism via which Angiotensin (Ang) II induces G‐alpha activation of the Angiotensin II Type 1 Receptor (AT1R), triplicate 300 ns molecular dynamic (MD) simulations of an ‘empty’‐AT1R, constitutively active (CA) AT1R, and Ang II bound to both the wild‐type and CA AT1Rs were conducted and analyzed. After measuring RMSD of the helical residues, Ang II, and the distances of the common intra‐receptor contact residues, it was determined that the Ang II‐AT1R structures were still inactive and Ang II was still unstable. A C‐terminus Gαq fragment, containing the last 17 residues, as well as a mini‐Gαq, modeled from the mini‐Gαs crystal structure, was then inserted into the bottom of the Ang II‐AT1R complex in two separate positions (aligned and fitted) and additional MD simulations were run. RMSD of the fitted mini‐Gq systems revealed a stable structure with intra‐contact residues in mid‐transition to the active state at 300 ns of simulation. Ang II binding in each system was analyzed via MM/GBSA; additionally, the binding energy was compared to the relative stability of Ang II in the binding pocket as well as the structural changes in the AT1R to identify correlations between the binding affinity and specific structural states of Ang II within the AT1R. Additionally, common GPCR motifs: PIF (P5.50, V3.40, F6.44), DRY (D3.49, R3.40, Y3.51), and NPxxY (N7.51, P7.52, x7.53, x7.54, Y7.55) were examined in each simulation. In all Ang II‐AT1R‐Gαq models, the DRY motif salt bridge did not separate by more than 5 Å, but the PIF and NPxxY were in the active conformation states as defined by crystal structures of activated GPCRs. However, one of the triplicate MD simulation of Ang II‐CA AT1R displayed separation of the DRY with partial movement of the PIF, but no change in the NPxxY and common intra‐receptor contact residues. Taken together the data obtained thus far indicate that the simulations are in a transition state and that G proteins are required for movement of the common intra‐receptor contact residues. Further, ongoing, simulation is expected to reveal a fully active AT1R. Moreover, subsequent analysis will likely suggest the mechanism via which Ang II and Gαq binding induce structural rearrangement of the AT1R leading to an active state. Unraveling this mechanism will allow for hypothesis directed experiments to test the models and potentially elucidate novel targets to control AT1R function and understand how polymorphisms affect the AT1R.Support or Funding InformationSupport for this work is from WesternU MSPS program funds.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.