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Functional Characterization of the Human Fractalkine Receptor CX3CR1 Using Photina™, a new Highly Sensitive Photoprotein
EP20272
Poster Title: Functional Characterization of the Human Fractalkine Receptor CX3CR1 Using Photina™, a new Highly Sensitive Photoprotein
Submitted on 19 Dec 2013
Author(s): Maria Grazia Giribaldi, Anna Della Bella, Viviana Agus and Lia Scarabottolo
Affiliations: Axxam srl
Poster Views: 1,625
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Poster Information
Abstract: G-protein-coupled-receptors (GPCRs) are the largest family of cell surface receptors with an estimate total of 1000 members encoded in the human genome. These seven transmembrane domain receptors can be activated by a vast diversity of extracellular inputs
such as amino acids, lipids, ions, proteases, nucleotides, peptides, large polypeptides, odorants, biogenic amines, and even photons. A wide variety of physiological functions are regulated by GPCRs: metabolism, secretion, neurotransmission, infl ammatory and
immune responses.

GPCRs currently represent the most important targets for drug development. High throughput screening (HTS) and ultra-HTS (uHTS) for human GPCRs are major goals for pharmaceutical companies.

CX3CR1 is a GPCR expressed in leukocytes and in myeloid cell lines. The receptor mediates a pathway for leukocyte traffi cking upon activation by fractalkine (FKN) also called CX3CL1. FKN is a unique CX3C chemokine that functions not only as a chemoattractant but also as an adhesion molecule and it is expressed on endothelial cells. Soluble FKN causes migration of NK cells, cytotoxic T lymphocytes and macrophages; whereas the membrane-bound form captures and enhances the subsequent migration of these cells
in response to secondary stimulation with other chemokines. Furthermore, stimulation through membrane-bound FKN activates NK cells, leading to increased cytotoxicity and interferon-ψ production.

Recently, accumulating evidence shows that FKN is involved in the pathogenesis of various diseases, such as atherosclerosis, lomerulonephritis, cardiac allograft rejection, and rheumatoid arthritis. In addition, polymorphisms in CX3CR1, which reduce its binding activity to FKN, have been reported to increase the risk of HIV disease and to reduce the risk of coronary artery disease.

CX3CR1 is a Gi-coupled and weakly Gq-coupled receptor able to inhibit adenylate cyclase and to stimulate intracellular Ca2+ mobilization. We have generated a cell line expressing the recombinant human CX3CR1 receptor in a stable way, in order to test its functionality following both the Gi- and Gq-coupled pathways.

Two different luminescence detection systems have been used: glow luminescence via a cAMP responsiveluciferase reporter vector and fl ash luminescence with a novel improved Ca2+-sensitive photoprotein, PhotinaTM. The scheme of this reporter cell line developed at Axxam is reported below.
Summary: CX3CR1 has been stably transfected into CHOmitoPhotina™- MRE-CRE-Luciferase cell line and assays suitable for HTS and uHTS have been developed. Report abuse »
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