A GAIN in understanding autoproteolytic G protein‐coupled receptors and polycystic kidney disease proteins

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102153/1/embj201251.pd

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

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Structure of a monomeric variant of rhodopsin kinase at 2.5 Å resolution

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92137/1/S1744309112017435.pd

Using iterative fragment assembly and progressive sequence truncation to facilitate phasing and crystal structure determination of distantly related proteins

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/119121/1/ayd2rr5107.pd

Molecular basis for activation of G protein-coupled receptor kinases

The analysis presents the structure of the G protein-coupled receptor (GPCR) kinase 6 (GRK6) in an active conformation. A model of the GRK–GPCR complex is provided and this combined with mutational analysis explains how GRKs are able to selectively recognize activated GPCRs

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

Membrane Orientation and Binding Determinants of G Protein-Coupled Receptor Kinase 5 as Assessed by Combined Vibrational Spectroscopic Studies

<div><p>G-protein coupled receptors (GPCRs) are integral membrane proteins involved in a wide variety of biological processes in eukaryotic cells, and are targeted by a large fraction of marketed drugs. GPCR kinases (GRKs) play important roles in feedback regulation of GPCRs, such as of β-adrenergic receptors in the heart, where GRK2 and GRK5 are the major isoforms expressed. Membrane targeting is essential for GRK function in cells. Whereas GRK2 is recruited to the membrane by heterotrimeric Gβγ subunits, the mechanism of membrane binding by GRK5 is not fully understood. It has been proposed that GRK5 is constitutively associated with membranes through elements located at its N-terminus, its C-terminus, or both. The membrane orientation of GRK5 is also a matter of speculation. In this work, we combined sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) to help determine the membrane orientation of GRK5 and a C-terminally truncated mutant (GRK5_1-531) on membrane lipid bilayers. It was found that GRK5 and GRK5_1-531 adopt a similar orientation on model cell membranes in the presence of PIP₂ that is similar to that predicted for GRK2 in prior studies. Mutation of the N-terminal membrane binding site of GRK5 did not eliminate membrane binding, but prevented observation of this discrete orientation. The C-terminus of GRK5 does not have substantial impact on either membrane binding or orientation in this model system. Thus, the C-terminus of GRK5 may drive membrane binding in cells via interactions with other proteins at the plasma membrane or bind in an unstructured manner to negatively charged membranes.</p> </div

Possible orientations of full length GRK5 by using 3NYN or 2ACX crystal structures.

<p>(A) The determined possible orientations of GRK5 on POPG lipid bilayers by combination of SFG and ATR-FTIR measurements (Figure S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082072#pone.0082072.s001" target="_blank">File S1</a>) by using the 3NYN crystal structure. The effect of experimental errors (such as uncertainty in the Fresnel coefficients) is accounted for using a coloring scheme based on how well the calculated and experimentally measured quantities agree for each possible orientation. The total score is calculated as the product of the scores for all individual criteria. A score of 100% indicates an exact match for all experimental measurements. (B) The same plot as panel A, but only showing orientation areas with a score ≥ 70% (red). (C) The possible orientations of GRK5 on POPG lipid bilayers determined by combination of SFG and ATR-FTIR measurements (Figure S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082072#pone.0082072.s001" target="_blank">File S1</a>) using the crystal structure of 2ACX. (D) The same plot as panel C, but only showing orientation areas with a score ≥ 70% (red).</p