Phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) is a major mechanism of desensitization of these receptors. GPCR activation of GRKs involves an allosteric site on GRKs distinct from the catalytic site. Although recent studies have suggested an important role of the N-and C-termini and domains surrounding the kinase active site in allosteric activation, the nature of that site and the relative roles of the RH domain in particular remain unknown. Based on evolutionary trace analysis of both the RH and kinase domains of the GRK family, we identified an important cluster encompassing helices 3, 9, and 10 in the RH domain in addition to sites in the kinase domain. To define its function, a panel of GRK5 and -6 mutants was generated and screened by intactcell assay of constitutive GRK phosphorylation of the ␤ 2 -adrenergic receptor (␤2AR), in vitro GRK phosphorylation of lightactivated rhodopsin, and basal catalytic activity measured by tubulin phosphorylation and autophosphorylation. A number of double mutations within helices 3, 9, and 10 reduced phosphorylation of the ␤2AR and rhodopsin by 50 to 90% relative to wild-type GRK, as well as autophosphorylation and tubulin phosphorylation. Based on these results, helix 9 peptide mimetics were designed, and several were found to inhibit rhodopsin phosphorylation by GRK5 with an IC 50 of ϳ30 M. In summary, our studies have uncovered previously unrecognized functionally important sites in the regulator of G-protein signaling homology domain of GRK5 and -6 and identified a peptide inhibitor with potential for specific blockade of GRK-mediated phosphorylation of receptors.To understand the mechanism underlying the activation of GRKs by the GPCRs, it is essential to identify functional sites in the GRKs involved in their activation. The serine/ threonine GRK family includes seven members, GRK1-7, classified into three subfamilies on the basis of their sequence homology: the rhodopsin kinase subfamily (GRK1 and -7), activities of which are restricted to the visual system; the ␤-adrenergic receptor kinase subfamily (GRK2 and -3); and the GRK4 subfamily (GRK4 -6) (Krupnick and Benovic, 1998;Pitcher et al., 1998). Crystal structures have been determined for GRK2 in complex with the ␤␥ and G␣ q subunits of G proteins (Lodowski et al., 2003(Lodowski et al., , 2005(Lodowski et al., , 2006Tesmer et al., 2005), for GRK6 bound to 5Ј-adenylylimidodiphosphate (Lodowski et al., 2006), and recently for six crystal structures of rhodopsin kinase (Singh et al., 2008). These structures all seem to be in the inactive state. Thus, neither the activestate conformation of GRKs nor how they interface with GPCRs is known.Membrane localization and activation of GRKs are complex, involving several domains within the kinase. It was shown that the GRK2 N-terminal fragment (residues 45-178) coimmunoprecipitated with metabotropic glutamate receptor 1 (Dhami et al., 2002) and that a single mutation in

SummaryFF domains are small protein-protein interaction modules that have two flanking conserved phenylalanine residues. They are present in proteins involved in transcription, RNA splicing, and signal transduction, and often exist in tandem arrays. Although several individual FF domain structures have been determined by NMR, the tandem nature of most FF domains has not been revealed. Here we report the 2.7 Å resolution crystal structure of the first three FF domains of human transcription elongation factor CA150. Each FF domain is composed of three a-helices and a 3 10 helix between α-helices 2 and 3. The most striking feature of the structure is that an FF domain is connected to the next by an α-helix that continues from helix 3 to helix 1 of the next. The consequent elongated arrangement allows exposure of many charged residues within the region that can be engaged in interaction with other molecules. Binding studies using a peptide ligand suggests that a specific conformation of the FF domains might be required to achieve higher affinity binding. Additionally, we explore potential DNA binding of the FF construct used in this study. Overall we provide the first crystal structure of a FF domain and insights into the tandem nature of the FF domains and suggest that in addition to protein binding, FF domains might be involved in DNA binding.

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Refluxing a solution of chromones in methanol containing ammonium formate and Pd-C leads to facile hydrogenation to chromanones.