Dataset Information

Global interactomics analysis of the human Duffy antigen receptor for chemokines

ABSTRACT: The Duffy antigen receptor for chemokines or DARC is a seven trans-membrane glycoprotein that is expressed primarily on the surface of red blood cells. Additionally, it is also expressed on the surface of post-venular endothelial cells, Purkinje cells of the cerebellum, inner ear hair cells, cells of the respiratory pathway and kidney epithelium. DARC binds to more than twenty different chemokines – belonging to both C-C and C-X-C subfamilies – making it the most promiscuous chemokine receptor. However, despite binding to such a large number of chemokines, no G-protein or β-arrestin signaling has been reported downstream to this receptor thus far. While canonical downstream signaling is not likely in erythrocytes considering their anucleate nature, DARC is also expressed in endothelial cells, and therefore, it is plausible that it signals through non-canonical signaling pathways. Considering that DARC does not couple to the canonical GPCR partners, we carried out a global interactome analysis upon stimulation of DARC-expressing HEK-293 cells with CCL7. We first performed a co-immunoprecipitation step under cross-linking condition to enrich DARC-interacting proteins followed by their detection using mass-spectrometry. In these experiments, we also used unstimulated cells as a reference, which were lysed and immunoprecipitated in parallel to subtract non-specific proteins. We carried out these experiments as four independent biological replicates.

INSTRUMENT(S): Orbitrap Exploris 240

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: gagan jhingan

LAB HEAD: Arun Shukla

PROVIDER: PXD051602 | Pride | 2024-10-17

REPOSITORIES: Pride

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Publications

Molecular mechanism of distinct chemokine engagement and functional divergence of the human Duffy antigen receptor.

Saha Shirsha S Khanppnavar Basavraj B Maharana Jagannath J Kim Heeryung H Carino Carlo Marion C CMC Daly Carole C Houston Shane S Sharma Saloni S Zaidi Nashrah N Dalal Annu A Mishra Sudha S Ganguly Manisankar M Tiwari Divyanshu D Kumari Poonam P Jhingan Gagan Deep GD Yadav Prem N PN Plouffe Bianca B Inoue Asuka A Chung Ka Young KY Banerjee Ramanuj R Korkhov Volodymyr M VM Shukla Arun K AK

Cell 20240731 17

The Duffy antigen receptor is a seven-transmembrane (7TM) protein expressed primarily at the surface of red blood cells and displays strikingly promiscuous binding to multiple inflammatory and homeostatic chemokines. It serves as the basis of the Duffy blood group system in humans and also acts as the primary attachment site for malarial parasite Plasmodium vivax and pore-forming toxins secreted by Staphylococcus aureus. Here, we comprehensively profile transducer coupling of this receptor, disc ...[more]

PMID: 39089252

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Project description:Sphingosine 1-phosphate (S1P) influences T cell migration into and out of secondary lymphoid organs; however, its mechanism of action remains uncertain. Our previous research shows that agonism of the S1P receptor S1P1 inhibits the egress of T lymphocytes from the peripheral tissues into afferent lymphatics. To better define the mechanism of inhibition, we developed an in vitro model to characterize T cell transendothelial migration across lymphatics. Two commercially available endothelial cell lines (MS-1 and SVEC4-10) were characterized by flow cytometry, real time RT-PCR, and Affymetrix Gene Array. These cell lines were grown to confluent monolayers in transwell systems, on either the upper or lower surface of the transwell insert. T cells were isolated from the spleens of (C57BL/6 x C3H/HeJ)F1, S1P1 KO, or S1P1 KO littermate controls, and either treated with the S1P receptor modulator FTY720 or left untreated. Cells were migrated to chemokines (CCL19 or CCL21) for 4 hours, and migration quantified. Flow cytometry, RT-PCR, and array results identified MS-1 as a blood vascular endothelial cell line, expressing high levels of CD31, CD34, and ICAM-1 as well as other endothelial cell markers; while SVEC4-10 closely resemble a lymphatic phenotype, expressing LYVE-1, VEGFR-3, and podoplanin. T cells efficiently migrate across MS-1, whether grown on the upper or lower surface; whereas migration across SVEC4-10 only occurs when cells are grown on the lower surface of the transwell (iSVEC), recapitulating basal (abluminal) to apical (luminal) migration that occurs in vivo. FTY720 inhibits T cell migration across iSVEC, but not across MS-1. Inhibition is due to drug effects only on T cells but not endothelial cells. S1P1 KO T cells treated with FTY720 are not inhibited in their migration across the iSVEC line, showing that S1P1 stimulation is required for migration inhibition. The in vitro model developed here is the first to use endothelial cell lines to analyze the regulation of T cell migration across lymphatic endothelium. The results show there is directional control of T cell migration across lymphatic cells, such that T cells only migrate from a basal to apical direction. Agonism of S1P1 specifically inhibits migration, while absence of the receptor does not. These findings have important implications for the use of S1P1 agonists in transplantation, as inhibition of cell entry into afferent lymphatics and lymph nodes could impede the development of graft rejection. We used microarrays to detail the global gene expression of these two cell lines in order to beter determine their phenotype as blood vascular or lymphatic endothelial cell line for use in our newly-developed in vitro model. Experiment Overall Design: MS-1 or SVEC4-10 endothelial cell lines were grown in culture flasks for 3-5 days in standard culture medium (see protocol below) until reaching confluence. Cells were then gently dissociated from flask and cells were placed in Trizol reagent for RNA isolation.

			Action	DRS
	DR_ARUN_IITK_ANALYSIS.pdResult	Other
	S_S1_E1.raw	Raw
	S_S2_E1.raw	Raw
	S_S3_E1.raw	Raw
	S_S4_E1.raw	Raw