Insights into congenital stationary night blindness based on the structure of G90D rhodopsin

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102109/1/embr201344.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102109/2/embr201344.reviewer_comments.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102109/3/embr201344-sup-0001.pd

Identification and evaluation of inhibitors for the human cytoplasmic protein tyrosine phosphatase

Transient phosphorylation of the cytoplasmic residues of integral membrane proteins is a common mechanism for the transmission of extracellular signals to intracellular components. The EphA2 Receptor Tyrosine Kinase (EphA2) regulates normal cell growth through maintenance of cell-cell contact mediated signaling. However, EphA2 is found under-phosphorylated in transformed cell culture, which is correlated with the disruption of normal cell-cell contacts. EphA2 is the cellular substrate for the Human Cytoplasmic Protein Tyrosine Phosphatase (HCPTP), which is found overexpressed in these transformed cell lines. General inhibition of phosphatases in cellular studies has restored EphA2 phosphorylation state and the cells to a non-transformed phenotype. Thus the selective inhibition of HCPTP presents a novel therapeutic route for the treatment of metastatic transformation. Initial attempts at inhibitor design have focused on both rationally designed inhibitors, which are based upon a cocrystal structure of a homologous low molecular weight phosphatase from yeast, and in silico screening of small molecule libraries. Low micromolar inhibitors, which are among the most effective small molecule inhibitors reported to date, have been identified and kinetically characterized. Crystallographic experiments were undertaken to provide a detailed atomic description of the binding of small molecules to HCPTP. Cocrystal complexes between HCPTP and small molecules have identified a secondary binding site distant from the active site, which presents the possibility of identifying interactions at either site. Solution state NMR experiments have been used to confirm the presence of this secondary site and attempt to characterize the interactions of the previously identified strong inhibitors with HCPTP. The kinetic results, cocrystallographic structures, and NMR experiments present more robust, dynamic models for micromolar inhibitors of HCPTP that may led to the next generation of potent, selective inhibitors

Identification and characterization of amlexanox as a G protein-coupled receptor kinase 5 inhibitor

G protein-coupled receptor kinases (GRKs) have been implicated in human diseases ranging from heart failure to diabetes. Previous studies have identified several compounds that selectively inhibit GRK2, such as paroxetine and balanol. Far fewer selective inhibitors have been reported for GRK5, a target for the treatment of cardiac hypertrophy, and the mechanism of action of reported compounds is unknown. To identify novel scaffolds that selectively inhibit GRK5, a differential scanning fluorometry screen was used to probe a library of 4480 compounds. The best hit was amlexanox, an FDA-approved anti-inflammatory, anti-allergic immunomodulator. The crystal structure of amlexanox in complex with GRK1 demonstrates that its tricyclic aromatic ring system forms ATP-like interactions with the hinge of the kinase domain, which is likely similar to how this drug binds to IκB kinase ε (IKKε), another kinase known to be inhibited by this compound. Amlexanox was also able to inhibit myocyte enhancer factor 2 transcriptional activity in neonatal rat ventricular myocytes in a manner consistent with GRK5 inhibition. The GRK1 amlexanox structure thus serves as a springboard for the rational design of inhibitors with improved potency and selectivity for GRK5 and IKKε

G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs)*

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β2-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling

Effect of Lipid Composition on the Membrane Orientation of the G Protein-Coupled Receptor Kinase 2–Gβ₁γ₂ Complex

Interactions between proteins and cell membranes are critical for biological processes such as transmembrane signaling, and specific components of the membrane may play roles in helping to organize or mandate particular conformations of both integral and peripheral membrane proteins. One example of a signaling enzyme whose function is dependent on membrane binding and whose activity is affected by specific lipid components is G protein-coupled receptor (GPCR) kinase 2 (GRK2). Efficient GRK2-mediated phosphorylation of activated GPCRs is dependent not only on its recruitment to the membrane by heterotrimeric Gβγ subunits but also on the presence of highly negatively charged lipids, in particular phosphatidylinositol 4′,5′-bisphosphate (PIP₂). We hypothesized that PIP₂ may favor a distinct orientation of the GRK2–Gβγ complex on the membrane that is more optimal for function. In this study, we compared the possible orientations of the GRK2–Gβγ complex and Gβγ alone on model cell membranes prepared with various anionic phospholipids as deduced from sum frequency generation vibrational and attenuated total reflectance Fourier transform infrared spectroscopic methods. Our results indicate that PIP₂ affects the membrane orientation of the GRK2–Gβ₁γ₂ complex but not that of complexes formed with anionic phospholipid binding deficient mutations in the GRK2 pleckstrin homology (PH) domain. Gβ₁γ₂ exhibits a similar orientation on the lipid bilayer regardless of its lipid composition. The PIP₂-induced orientation of the GRK2–Gβ₁γ₂ complex is therefore most likely caused by specific interactions between PIP₂ and the GRK2 PH domain. Thus, PIP₂ not only helps recruit GRK2 to the membrane but also “fine tunes” the orientation of the GRK2–Gβγ complex so that it is better positioned to phosphorylate activated GPCRs

Identification and Structure–Function Analysis of Subfamily Selective G Protein-Coupled Receptor Kinase Inhibitors

Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors

Crystal structure of G protein-coupled receptor kinase 5 in complex with a rationally designed inhibitor

G protein-coupled receptor kinases (GRKs) regulate cell signaling by initiating the desensitization of active G protein-coupled receptors. The two most widely expressed GRKs (GRK2 and GRK5) play a role in cardiovascular disease and thus represent important targets for the development of novel therapeutic drugs. In the course of a GRK2 structure-based drug design campaign, one inhibitor (CCG215022) exhibited nanomolar IC(50) values against both GRK2 and GRK5 and good selectivity against other closely related kinases such as GRK1 and PKA. Treatment of murine cardiomyocytes with CCG215022 resulted in significantly increased contractility at 20-fold lower concentrations than paroxetine, an inhibitor with more modest selectivity for GRK2. A 2.4 Å crystal structure of the GRK5·CCG215022 complex was determined and revealed that the inhibitor binds in the active site similarly to its parent compound GSK180736A. As designed, its 2-pyridylmethyl amide side chain occupies the hydrophobic subsite of the active site where it forms three additional hydrogen bonds, including one with the catalytic lysine. The overall conformation of the GRK5 kinase domain is similar to that of a previously determined structure of GRK6 in what is proposed to be its active state, but the C-terminal region of the enzyme adopts a distinct conformation. The kinetic properties of site-directed mutants in this region are consistent with the hypothesis that this novel C-terminal structure is representative of the membrane-bound conformation of the enzyme

G Protein-Coupled Receptor Kinase 2 (GRK2) and 5 (GRK5) Exhibit Selective Phosphorylation of the Neurotensin Receptor <i>in Vitro</i>

G protein-coupled receptor kinases (GRKs) play an important role in the desensitization of G protein-mediated signaling of G protein-coupled receptors (GPCRs). The level of interest in mapping their phosphorylation sites has increased because recent studies suggest that the differential pattern of receptor phosphorylation has distinct biological consequences. <i>In vitro</i> phosphorylation experiments using well-controlled systems are useful for deciphering the complexity of these physiological reactions and understanding the targeted event. Here, we report on the phosphorylation of the class A GPCR neurotensin receptor 1 (NTSR1) by GRKs under defined experimental conditions afforded by nanodisc technology. Phosphorylation of NTSR1 by GRK2 was agonist-dependent, whereas phosphorylation by GRK5 occurred in an activation-independent manner. In addition, the negatively charged lipids in the immediate vicinity of NTSR1 directly affect phosphorylation by GRKs. Identification of phosphorylation sites in agonist-activated NTSR1 revealed that GRK2 and GRK5 target different residues located on the intracellular receptor elements. GRK2 phosphorylates only the C-terminal Ser residues, whereas GRK5 phosphorylates Ser and Thr residues located in intracellular loop 3 and the C-terminus. Interestingly, phosphorylation assays using a series of NTSR1 mutants show that GRK2 does not require acidic residues upstream of the phospho-acceptors for site-specific phosphorylation, in contrast to the β₂-adrenergic and μ-opioid receptors. Differential phosphorylation of GPCRs by GRKs is thought to encode a particular signaling outcome, and our <i>in vitro</i> study revealed NTSR1 differential phosphorylation by GRK2 and GRK5