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Low input RNA sequencing of GPR35-positive and negative intestinal macrophages

ABSTRACT: Low input RNA sequencing of GPR35-positive and negative intestinal macrophages

PROVIDER: PRJNA545130 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR9137282.fastq.gz	Fastqsanger.gz
	SRR9137283.fastq.gz	Fastqsanger.gz
	SRR9137284.fastq.gz	Fastqsanger.gz
	SRR9137285.fastq.gz	Fastqsanger.gz
	SRR9137286.fastq.gz	Fastqsanger.gz

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Low input RNA sequencing of GPR35-positive and negative intestinal macrophages

Project description:Aim: We aimed to determine differences in transcriptional profiles of GPR35-positive and negative macrophages in the steady state intestine. Method: Corresponding subpopulations were sorted by FACS from intestinal lamina propria cells of unmanipulated wild-type C56BL/6 mice. RNA from 4 replicates of each group were sequenced and analyzed. Result: GPR35 positive macrophages are transcriptionally distinct from GPR35 negative macrophages overall with 2798 downregulated and 2773 upregulated genes, and regarding cytokine expression profile with higher Il1b, Tnf and Il10 mRNA expressions. Conclusion: Our study reports the first analysis on transcriptional charasteristics of intestinal macrophages depending on their GPR35 expression providing insights into the function of GPR35 in these cells.

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Project description:Mutations in GPR35 (G protein coupled receptor 35) are linked to ulcerative colitis and primary sclerosing cholangitis. How GPR35, which interacts with the Na/K-ATPase to determine ion transport, influences the transcriptome of macrophages and the ileum is unknown. Here compare transcript expression in bone marrow-derived macrophages and isolated ileum from mice deficient in GPR35 (knockout) with wild type mice by bulk RNA sequencing.

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Project description:Purpose: Our previous experiments showed that LPA activated inhibitory G-protein signaling in human GPR35-transfected cells. We wanted to test the LPA signaling in presence or absence of GPR35. Methods: We isolated RNA from bone-marrow derived macrophages (BMDMs) from WT or GPR35-deficient mice that were treated with LPA or LPS, and left untreated for control. Results: We have found that GPR35-deficiency alters the transcriptional profiles in response to LPA in BMDMs. On the other hand, LPS-treated WT and GPR35-deficient BMDMs showed similar transcriptional changes. Conclusion: GPR35-deficientcy alters the LPA but not LPS signaling.

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Agonist-induced phosphorylation of orthologues of the orphan receptor GPR35 as an activation biomarker

Project description:Mass spectrometry, mutagenesis and labelling with [32P] orthophosphate identified that each of the five hydroxy-amino acids in the intracellular C-terminal tail of human GPR35a became phosphorylated in response to agonist occupancy of the receptor and that, apart from Ser294, each of these contributed to the effectiveness of interaction of the receptor with arrestin-3. Key to such interactions was Ser303. Despite there being a greater number of hydroxy-amino acids in the C-terminal tail of both mouse and rat GPR35 the serine corresponding to residue 303 in human GPR35a also played a dominant role in arrestin-3 interactions for both rodent orthologues. Fully phospho-site deficient mutants of human GPR35a and mouse GPR35 failed to interact effectively with arrestin-3 and the human phospho-deficient variant was not internalized from the surface of cells in response to agonist treatment. Even in cells stably expressing species orthologues of GPR35 a substantial proportion of the expressed protein(s) was, however, immature. Phospho-site specific antisera targeting the region encompassing Ser303 in human (Ser301 in mouse) GPR35 identified only the mature forms of GPR35 and provided effective biomarkers of the activation status of the receptors both in immunoblotting and immunocytochemical studies. Such antisera may be useful tools to evaluate target engagement in drug discovery and target validation programmes.

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