Molecular mechanisms of G protein-receptor coupling

A variety of extracellular signals are transmitted into the cell interior by interactions with a superfamily of heptahelical cell surface receptors. Heterotrimeric G proteins mediate the signal transduction by coupling these receptors to intracellular effector proteins. The molecular mechanisms in G protein-receptor coupling processes are still not completely understood. In this project, two aspects of G protein-receptor coupling were examined: (1) the G protein-coupling properties of the five human muscarinic acetylcholine receptors (mAChRs); (2) a novel regulatory mechanism by a newly identified G protein signal regulator, Activator of G-protein Signaling 3 (AGS3). To study the regulation of G protein-receptor coupling, an Sf9 cell membrane-based in vitro reconstitution system was used, in which purified G protein heterotrimers were reconstituted with individually expressed membrane receptors and their coupling was assessed with radioligand binding assays.;Functional G protein coupling was successfully established for M1, M2, M4 and M5 mAChRs in urea-extracted Sf9 cell membranes. Under the same conditions, M3 mAChRs failed to couple with purified G proteins, indicating they may have a unique G protein signaling mechanism. Within the odd- or even-numbered mAChR groups similar apparent affinities for G protein interactions were observed, however, the odd-numbered mAChRs exhibited higher affinity for G protein heterotrimers than did the even-numbered mAChRs. Differences were also observed among the individual receptor subtypes in their affinity states for the agonist, Oxotremorine-M.;Studies on AGS3 revealed that cytosolic AGS3, but not membrane-associated AGS3, can interfere with receptor-Gi protein coupling. Cytosolic AGS3 can remove Gialpha subunits from the plasma membrane and sequester them in the cytosol. Each of the four AGS3 GPR (G protein regulatory) domains was able to interfere with receptor-Gi protein coupling; however, individual domains were less effective than the full-length GPR domain. None of the GPR domains distinguish among the three Gialpha subunits but they all interact more weakly with Goalpha subunits.;These studies demonstrate that five mAChRs have distinct G protein-coupling behaviors in an identical membrane environment and that AGS3 may down-regulate G protein signaling by interfering with receptor coupling. These findings contribute to the understanding of the mechanism and regulation of G protein-receptor coupling

Closely Related G-Protein-Coupled Receptors Use Multiple And Distinct Domains On G-Protein Α-Subunits For Selective Coupling

The molecular basis of selectivity in G-protein receptor coupling has been explored by comparing the abilities of G-protein heterotrimers containing chimeric Galpha subunits, comprised of various regions of Gi1alpha, Gtalpha, and Gqalpha, to stabilize the high affinity agonist binding state of serotonin, adenosine, and muscarinic receptors. The data indicate that multiple and distinct determinants of selectivity exist for individual receptors. While the A1 adenosine receptor does not distinguish between Gi1alpha and Gtalpha sequences, the 5-HT1A and 5-HT1B serotonin and M2 muscarinic receptors can couple with Gi1 but not Gt. It is possible to distinguish domains that eliminate coupling and are defined as critical, from those that impair coupling and are defined as important. Domains within the N terminus, alpha4-helix, and alpha4-helix-alpha4/beta6-loop of Gi1alpha are involved in 5-HT and M2 receptor interactions. Chimeric Gi1alpha/Gqalpha subunits verify the critical role of the Galpha C terminus in receptor coupling, however, the individual receptors differ in the C-terminal amino acids required for coupling. Furthermore, the EC50 for interactions with Gi1 differ among the individual receptors. These results suggest that coupling selectivity ultimately involves subtle and cooperative interactions among various domains on both the G-protein and the associated receptor as well as the G-protein concentration

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN

Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images

Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumorinfiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment

Dominant-negative mutations in human IL6ST underlie hyper-IgE syndrome

Autosomal dominant hyper-IgE syndrome (AD-HIES) is typically caused by dominant-negative (DN) STAT3 mutations. Patients suffer from cold staphylococcal lesions and mucocutaneous candidiasis, severe allergy, and skeletal abnormalities. We report 12 patients from 8 unrelated kindreds with AD-HIES due to DN IL6ST mutations. We identified seven different truncating mutations, one of which was recurrent. The mutant alleles encode GP130 receptors bearing the transmembrane domain but lacking both the recycling motif and all four STAT3-recruiting tyrosine residues. Upon overexpression, the mutant proteins accumulate at the cell surface and are loss of function and DN for cellular responses to IL-6, IL-11, LIF, and OSM. Moreover, the patients’ heterozygous leukocytes and fibroblasts respond poorly to IL-6 and IL-11. Consistently, patients with STAT3 and IL6ST mutations display infectious and allergic manifestations of IL-6R deficiency, and some of the skeletal abnormalities of IL-11R deficiency. DN STAT3 and IL6ST mutations thus appear to underlie clinical phenocopies through impairment of the IL-6 and IL-11 response pathways

Integrated Genomic Analysis of the Ubiquitin Pathway across Cancer Types

Protein ubiquitination is a dynamic and reversibleprocess of adding single ubiquitin molecules orvarious ubiquitin chains to target proteins. Here,using multidimensional omic data of 9,125 tumorsamples across 33 cancer types from The CancerGenome Atlas, we perform comprehensive molecu-lar characterization of 929 ubiquitin-related genesand 95 deubiquitinase genes. Among them, we sys-tematically identify top somatic driver candidates,including mutatedFBXW7with cancer-type-specificpatterns and amplifiedMDM2showing a mutuallyexclusive pattern withBRAFmutations. Ubiquitinpathway genes tend to be upregulated in cancermediated by diverse mechanisms. By integratingpan-cancer multiomic data, we identify a group oftumor samples that exhibit worse prognosis. Thesesamples are consistently associated with the upre-gulation of cell-cycle and DNA repair pathways, char-acterized by mutatedTP53,MYC/TERTamplifica-tion, andAPC/PTENdeletion. Our analysishighlights the importance of the ubiquitin pathwayin cancer development and lays a foundation fordeveloping relevant therapeutic strategies