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CXCR2 Inverse Agonism Detected by Arrestin Redistribution

Institute of Zoology and Endocrinology, Ulm University Medical Center, Ulm, Germany, Institute of Pharmacology and Toxicology, Ulm University Medical Center, Ulm, Germany

Michael Wolff

Department of Integrated Lead Discovery, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany

Jörg Wiedenmann

National Oceanography Centre, University of Southampton, Southampton, UK

Barbara Moepps

Institute of Pharmacology and Toxicology, Ulm University Medical Center, Ulm, Germany

G. Ulrich Nienhaus

Institute of Biophysics, University of Ulm, Ulm, Germany, Department of Physics, University of Illinois at Urbana Champaign, Urbana, llinois, Institute of Applied Physics & Center for Functional Nanostructures (CFN), University of Karlsruhe (TH) & Karlsruhe Institute of Technology, Karlsruhe, Germany

Peter Gierschik

Institute of Pharmacology and Toxicology, Ulm University Medical Center, Ulm, Germany

Barbara Kistler

Department of Respiratory Research, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany

Ralf Heilker

Department of Integrated Lead Discovery, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany, Ralf.Heilker{at}bc.boehringer-ingelheim.com

To study CXCR2 modulated arrestin redistribution, the authors employed arrestin as a fusion protein containing either the Aequorea victoria—derived enhanced green fluorescent protein (EGFP) or a recently developed mutant of eqFP611, a red fluorescent protein derived from Entacmaea quadricolor. This mutant, referred to as RFP611, had earlier been found to assume a dimeric quarternary structure. It was therefore employed in this work as a "tandem" (td) construct for pseudo monomeric fusion protein labeling. Both arrestin fusion proteins, containing either td RFP611 (Arr td RFP611) or enhanced green fluorescent protein (EGFP; Arr EGFP), were found to colocalize with internalized fluorescently labeled Gro a few minutes after Gro addition. Intriguingly, however, Arr td RFP611 and Arr EGFP displayed distinct cellular distribution patterns in the absence of any CXCR2 activating ligand. Under these conditions, Arr td RFP611 showed a largely homoge neous cytosolic distribution, whereas Arr EGFP segregated, to a large degree, into granular spots. These observations indi cate a higher sensitivity of Arr EGFP to the constitutive activity of CXCR2 and, accordingly, an increased arrestin redistribution to coated pits and endocytic vesicles. In support of this interpretation, the authors found the known CXCR2 antagonist Sch527123 to act as an inverse agonist with respect to Arr EGFP redistribution. The inverse agonistic properties of Sch527123 were confirmed in vitro in a guanine nucleotide binding assay, revealing an IC₅₀ value similar to that observed for Arr EGFP redistribution. Thus, the redistribution assay, when based on Arr EGFP, enables the profiling of antagonistic test compounds with respect to inverse agonism. When based on Arr td RFP611, the assay may be employed to study CXCR2 agonism or neutral antagonism. ( Journal of Biomolecular Screening 2009:1076 1091)

Key Words: high content screening • CXCR2 • arrestin • high throughput screening • inverse agonism

This version was published on October 1, 2009

Journal of Biomolecular Screening, Vol. 14, No. 9, 1076-1091 (2009)
DOI: 10.1177/1087057109344616


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