Unraveling allostery within the angiotensin II type 1 receptor for Gαq and ß-arrestin coupling.

G protein-coupled receptors engage both G proteins and ß-arrestins, and their coupling can be biased by ligands and mutations. Here, to resolve structural elements and mechanisms underlying effector coupling to the angiotensin II (AngII) type 1 receptor (AT1R), we combined alanine scanning mutagenesis of the entire sequence of the receptor with pharmacological profiling of Gαq and ß-arrestin engagement to mutant receptors and molecular dynamics simulations. We showed that Gαq coupling to AT1R involved a large number of residues spread across the receptor, whereas fewer structural regions of the receptor contributed to ß-arrestin coupling regulation. Residue stretches in transmembrane domain 4 conferred ß-arrestin bias and represented an important structural element in AT1R for functional selectivity. Furthermore, we identified allosteric small-molecule binding sites that were enclosed by communities of residues that produced biased signaling when mutated. Last, we showed that allosteric communication within AT1R emanating from the Gαq coupling site spread beyond the orthosteric AngII-binding site and across different regions of the receptor, including currently unresolved structural regions. Our findings reveal structural elements and mechanisms within AT1R that bias Gαq and ß-arrestin coupling and that could be harnessed to design biased receptors for research purposes and to develop allosteric modulators.

Reversal of obesity development in Ceacam1-/- male mice by bone marrow transplantation or introduction of the human CEACAM1 gene.

OBJECTIVE: Although Ceacam1-/- male mice become obese on normal chow, the effect of bone marrow transplantation or introduction of the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) gene has not been studied, to the knowledge of the authors. METHODS: This study analyzed Ceacam1-/- mice on normal diet or high-fat diet (HFD), including effects of bone marrow transplantation or introduction of the CEACAM1 gene. RESULTS: Male Ceacam1-/- mice on normal diet versus HFD for 24 weeks gained significantly more weight than controls, and Ceacam1-/- mice aged up to 2 years had a high frequency of liver cancer. Transplantation of wild-type bone marrow into Ceacam1-/- mice or introduction of the human CEACAM1 gene fully or partially reversed the obesity phenotype. Liver lipidomics on Ceacam1-/- versus wild-type controls on an HFD revealed a significant increase in diacyl glycerides. An increase in fatty acid transporter CD36 levels further suggests that loss of Ceacam1 leads to a major dysregulation of free fatty acid uptake. CONCLUSIONS: CEACAM1 expression in both the liver and immune cells regulates obesity and lipid storage pathways in the liver. Bone marrow reconstitution of the immune system or introduction of the human CEACAM1 gene can fully or partially reverse the phenotype.

CXCR2 specific endocytosis of immunomodulatory peptide LL-37 in human monocytes and formation of LL-37 positive large vesicles in differentiated monoosteophils.

Immunomodulatory peptide cathelicidin/LL-37 induces human monocyte differentiation into a novel bone repair cell, the monoosteophil. We now demonstrate that LL-37 is endocytosed by monocytes over a period of 6 days producing large (10 × 2 µm), specialized LL-37 and integrin α3 positive vesicles. CXCR2, a membrane receptor previously associated with the binding of LL-37 to neutrophils, was co-endocytosed with LL-37 where both markers remained within the cytosol over a 16 h observation period. Endocytosis of LL-37 was mediated by a clathrin- and cavoelin/lipid raft-dependent pathway into early Rab5+ endosomes expressing APPL1 and EEA1. From 4 to 16 h, LL-37 vesicles co-localized with the Golgi, mitochondria, and to a lesser extent lysosomes and ER. By day 6, LL-37 was associated with large (>10 µm) vesicles, adjacent to Golgi, mitochondria, ER and lysosomes. LL-37 co-stained with integrin α3, tetraspanin CD9, GPI-linked CD59 and costimulatory molecule CD276 (B7-H3) in these vesicles. Continuous tracking of LL-37 with its associated vesicles over 6 days indicates that LL-37 is an extremely stable, membrane-associated peptide that plays a critical role in the differentiation of monocytes into monoosteophils.

CEACAM1 regulates the IL-6 mediated fever response to LPS through the RP105 receptor in murine monocytes.

BACKGROUND: Systemic inflammation and the fever response to pathogens are coordinately regulated by IL-6 and IL-1ß. We previously showed that CEACAM1 regulates the LPS driven expression of IL-1ß in murine neutrophils through its ITIM receptor. RESULTS: We now show that the prompt secretion of IL-6 in response to LPS is regulated by CEACAM1 expression on bone marrow monocytes. Ceacam1-/- mice over-produce IL-6 in response to an i.p. LPS challenge, resulting in prolonged surface temperature depression and overt diarrhea compared to their wild type counterparts. Intraperitoneal injection of a 64Cu-labeled LPS, PET imaging agent shows confined localization to the peritoneal cavity, and fluorescent labeled LPS is taken up by myeloid splenocytes and muscle endothelial cells. While bone marrow monocytes and their progenitors (CD11b+Ly6G-) express IL-6 in the early response (< 2 h) to LPS in vitro, these cells are not detected in the bone marrow after in vivo LPS treatment perhaps due to their rapid and complete mobilization to the periphery. Notably, tissue macrophages are not involved in the early IL-6 response to LPS. In contrast to human monocytes, TLR4 is not expressed on murine bone marrow monocytes. Instead, the alternative LPS receptor RP105 is expressed and recruits MD1, CD14, Src, VAV1 and ß-actin in response to LPS. CEACAM1 negatively regulates RP105 signaling in monocytes by recruitment of SHP-1, resulting in the sequestration of pVAV1 and ß-actin from RP105. CONCLUSION: This novel pathway and regulation of IL-6 signaling by CEACAM1 defines a novel role for monocytes in the fever response of mice to LPS.

SNPs in inflammatory genes CCL11, CCL4 and MEFV in a fibromyalgia family study.

BACKGROUND: Fibromyalgia (FM) is a chronic pain syndrome with a high incidence in females that may involve activation of the immune system. We performed exome sequencing on chemokine genes in a region of chromosome 17 identified in a genome-wide family association study. METHODS AND FINDINGS: Exome sequence analysis of 100 FM probands was performed at 17p13.3-q25 followed by functional analysis of SNPs found in the chemokine gene locus. Missense SNPs (413) in 17p13.3-q25 were observed in at least 10 probands. SNPs rs1129844 in CCL11 and rs1719152 in CCL4 were associated with elevated plasma chemokine levels in FM. In a transmission disequilibrium test (TDT), rs1129844 was unequally transmitted from parents to their affected children (p< 0.0074), while the CCL4 SNP was not. The amino acid change (Ala23Thr), resulting from rs1129844 in CCL11, predicted to alter processing of the signal peptide, led to reduced expression of CCL11. The variant protein from CCL4 rs1719152 exhibited protein aggregation and a potent down-regulation of its cognate receptor CCR5, a receptor associated with hypotensive effects. Treatment of skeletal muscle cells with CCL11 produced high levels of CCL4 suggesting CCL11 regulates CCL4 in muscle. The immune association of FM with SNPs in MEFV, a chromosome 16 gene associated with recurrent fevers, had a p< 0.008 TDT for a combined 220 trios. CONCLUSIONS: SNPs with significant TDTs were found in 36% of the cohort for CCL11 and 12% for MEFV, along with a protein variant in CCL4 (41%) that affects CCR5 down-regulation, supporting an immune involvement for FM.

CD5 Binds to Interleukin-6 and Induces a Feed-Forward Loop with the Transcription Factor STAT3 in B Cells to Promote Cancer.

The participation of a specific subset of B cells and how they are regulated in cancer is unclear. Here, we demonstrate that the proportion of CD5(+) relative to interleukin-6 receptor α (IL-6Rα)-expressing B cells was greatly increased in tumors. CD5(+) B cells responded to IL-6 in the absence of IL-6Rα. IL-6 directly bound to CD5, leading to activation of the transcription factor STAT3 via gp130 and its downstream kinase JAK2. STAT3 upregulated CD5 expression, thereby forming a feed-forward loop in the B cells. In mouse tumor models, CD5(+) but not CD5(-) B cells promoted tumor growth. CD5(+) B cells also showed activation of STAT3 in multiple types of human tumor tissues. Thus, our findings demonstrate a critical role of CD5(+) B cells in promoting cancer.

Discovery of potential new gene variants and inflammatory cytokine associations with fibromyalgia syndrome by whole exome sequencing.

Fibromyalgia syndrome (FMS) is a chronic musculoskeletal pain disorder affecting 2% to 5% of the general population. Both genetic and environmental factors may be involved. To ascertain in an unbiased manner which genes play a role in the disorder, we performed complete exome sequencing on a subset of FMS patients. Out of 150 nuclear families (trios) DNA from 19 probands was subjected to complete exome sequencing. Since >80,000 SNPs were found per proband, the data were further filtered, including analysis of those with stop codons, a rare frequency (<2.5%) in the 1000 Genomes database, and presence in at least 2/19 probands sequenced. Two nonsense mutations, W32X in C11orf40 and Q100X in ZNF77 among 150 FMS trios had a significantly elevated frequency of transmission to affected probands (p = 0.026 and p = 0.032, respectively) and were present in a subset of 13% and 11% of FMS patients, respectively. Among 9 patients bearing more than one of the variants we have described, 4 had onset of symptoms between the ages of 10 and 18. The subset with the C11orf40 mutation had elevated plasma levels of the inflammatory cytokines, MCP-1 and IP-10, compared with unaffected controls or FMS patients with the wild-type allele. Similarly, patients with the ZNF77 mutation have elevated levels of the inflammatory cytokine, IL-12, compared with controls or patients with the wild type allele. Our results strongly implicate an inflammatory basis for FMS, as well as specific cytokine dysregulation, in at least 35% of our FMS cohort.

Direct interaction of tumor suppressor CEACAM1 with beta catenin: identification of key residues in the long cytoplasmic domain.

CEACAM1-4L (carcinoembryonic antigen cell adhesion molecule 1, with 4 extracellular Ig-like domains and a long, 71 amino acid cytoplasmic domain) is expressed in epithelial cells and activated T-cells, but is down-regulated in most epithelial cell cancers and T-cell leukemias. A highly conserved sequence within the cytoplasmic domain has ca 50% sequence homology with Tcf-3 and -4, transcription factors that bind beta-catenin, and to a lesser extent (32% homology), with E-cadherin that also binds beta-catenin. We show by quantitative yeast two-hybrid, BIAcore, GST-pull down, and confocal analyses that this domain directly interacts with beta-catenin, and that H-469 and K-470 are key residues that interact with the armadillo repeats of beta-catenin. Jurkat cells transfected with CEACAM1-4L have 2-fold less activity in the TOPFLASH reporter assay, and in MCF7 breast cancer cells that fail to express CEACAM1, transfection with CEACAM1 and growth in Ca2+ media causes redistribution of beta-catenin from the cytoplasm to the cell membrane, demonstrating a functional role for the long cytoplasmic domain of CEACAM1 in regulation of beta-catenin activity.

Detecting tissue-specific regulation of alternative splicing as a qualitative change in microarray data.

Alternative splicing has recently emerged as a major mechanism of regulation in the human genome, occurring in perhaps 40-60% of human genes. Thus, microarray studies of functional regulation could, in principle, be extended to detect not only the changes in the overall expression of a gene, but also changes in its splicing pattern between different tissues. However, since changes in the total expression of a gene and changes in its alternative splicing can be mixed in complex ways among a set of samples, separating these effects can be difficult, and is essential for their accurate assessment. We present a simple and general approach for distinguishing changes in alternative splicing from changes in expression, based on detecting systematic anti-correlation between the log-ratios of two different samples versus a pool containing both samples. We have tested this analysis method on microarray data for five human tissues, generated using a standard microarray platform and experimental protocols shown previously to be sensitive to alternative splicing. Our automatic analysis was able to detect a wide variety of tissue-specific alternative splicing events, such as exon skipping,mutually exclusive exons, alternative 3' and alternative 5' splicing, alternative initiation and alternative termination, all of which were validated by independent reverse-transcriptase PCR experiments, with validation rates of 70-85%. Our analysis method also enables hierarchical clustering of genes and samples by the level of similarity to their alternative splicing patterns, revealing patterns of tissue-specific regulation that are distinct from those obtained by hierarchical clustering of gene expression from the same microarray data. Our data and analysis source code are available from http://www.bioinformatics.ucla.edu/ASAP.