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 α a few minutes after Gro α 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)
Fluorescently tagged canine adenovirus via modification with protein IX–enhanced green fluorescent protein
