SummaryArabidopsis COP1 is a negative regulator of photomorphogenesis, which targets HY5, a positive regulator of photomorphogenesis, for degradation via the proteasome pathway in the absence of light. COP1 and its interactive partner CIP8 both possess RING ®nger motifs, characteristic of some E3 ubiquitin ligases. Here we show that CIP8 promotes ubiquitin attachment to HY5 in E2-dependent fashion in vitro. CIP8 exhibits a strong interaction with the E2 enzyme AtUBC8 through its N-terminal domain. Phosphorylation of HY5 by casein kinase II requires the beta subunit 2, but does not affect HY5¢s susceptibility to ubiquitination. The RING domain of CIP8 is required but is not suf®cient for ubiquitin ligase activity. Although the RING domain of CIP8 interacts with the RING domain of COP1, addition of recombinant COP1 fails to affect CIP8¢s ubiquitin ligase activity towards HY5 in vitro. However, recombinant COP1 can pull-down native CIP8 from the extract of dark-grown seedlings, but not from the extract of light-grown seedlings in a column-binding assay, implying a requirement for light-regulated modi®cation in vivo. Our data suggest that CIP8 can form a minimal ubiquitin ligase in co-operation with the E2 enzyme AtUBC8. It is possible that the AtUBC8-CIP8 module might interact with COP1 in vivo, thereby participating in proteasome-mediated degradation of HY5.

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The constitutive photomorphogenic 1 (COP1) protein of Arabidopsis functions as a molecular switch for the seedling developmental fates: photomorphogenesis under light conditions and skotomorphogenesis in darkness. The COP1 protein contains a cysteine-rich zincbinding RING finger motif found in diverse groups of regulatory proteins. To understand the role of the COP1 RING finger in mediating protein-protein interaction, we have performed a yeast two-hybrid screen and isolated a novel protein with a RING-H2 motif, a variant type of the RING finger. This protein, designated COP1 Interacting Protein 8 (CIP8), is encoded by a single copy gene and localized to cytosol in a transient assay. In addition to the RING-H2 motif, the predicted protein has a C4 zinc finger, an acidic region, a glycine-rich cluster, and a serine-rich cluster. The COP1 RING finger and the CIP8 RING-H2 domains are sufficient for their interaction with each other both in vitro and in yeast, whereas neither motif displayed significant self-association. Moreover, site-directed mutagenesis studies demonstrated that the expected zinc-binding ligands of the RING finger and RING-H2 fingers are essential for their interaction. Our findings indicate that the RING finger motif, in this case, serves as autonomous protein-protein interaction domain. The allele specific effect of cop1 mutations on the CIP8 protein accumulation in seedlings indicates that its stability in vivo is dependent on the COP1 protein.Zinc ions provide structural integrity to many regulatory proteins, most often through cysteine and histidine ligands that form a tetrahedral geometry. The RING finger motif is a cysteine-rich zinc-binding domain defined by the consensus sequence: CX 2 CX (9 -39) CX (1-3) HX (2-3) CX 2 CX (4 -48) CX 2 C (C 3 HC 4 ). A unique feature of this motif is that the consensus residues coordinate two zinc ions in a "cross-braced" fashion (1, 2). Thus, the RING finger forms one integrated structural unit, rather than forming two tandem zinc finger modules. Proteins containing RING finger domains are found in viruses and all eukaryotes, including yeast, plants, and animals. They have diverse cellular functions, including oncogenesis, viral gene expression, signal transduction, peroxisome biogenesis, DNA repair and recombination, and membrane vesicle sorting (2). The RING finger motif has been shown in many cases to play a role in mediating protein-protein interactions. However, the precise role of the RING finger, as well as specific interactive target motifs of the RING finger, have yet to be clearly defined.The Arabidopsis COP1 1 protein serves as a repressor of photomorphogenesis. In the dark, the COP1 protein localizes to the nucleus and represses photomorphogenesis by inhibiting transcription factors that promote light-inducible gene expression (3-5). Light stimuli abrogate the nuclear localization of COP1 and allows seedlings to pursue photomorphogenic development (6). COP1 contains an N-terminal RING finger motif (7,8), which has been demonstrated to bind tw...

G protein-coupled receptors (GPCRs) are a superfamily of proteins that include some of the most important drug targets in the pharmaceutical industry. Despite the success of this group of drugs, there remains a need to identify GPCR-targeted drugs with greater selectivity, to develop screening assays for validated targets, and to identify ligands for orphan receptors. To address these challenges, the authors have created a multiplexed GPCR assay that measures greater than 3000 receptor:ligand interactions in a single microplate. The multiplexed assay is generated by combining reverse transfection in a 96-well plate format with a calcium flux readout. This assay quantitatively measures receptor activation and inhibition and permits the determination of compound potency and selectivity for entire families of GPCRs in parallel. To expand the number of GPCR targets that may be screened in this system, receptors are cotransfected with plasmids encoding a promiscuous G protein, permitting the analysis of receptors that do not normally mobilize intracellular calcium upon activation. The authors demonstrate the utility of reverse transfection cell microarrays to GPCR-targeted drug discovery with examples of ligand selectivity screening against a panel of GPCRs as well as dose-dependent titrations of selected agonists and antagonists. (Journal of Biomolecular