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CXCR2 Inverse Agonism Detected by Arrestin Redistribution

^* To whom correspondence should be addressed. E-mail: ralf.heilker{at}bc.boehringer-ingelheim.com.

	Abstract

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 homogeneous cytosolic distribution, whereas Arr EGFP segregated, to a large degree, into granular spots. These observations indicate 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 2009:000-000)

First published on September 22, 2009, doi:10.1177/1087057109344616

Journal of Biomolecular Screening 2009;14:1076.

A more recent version of this article appeared on October 1, 2009


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