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

Modeled membrane orientations of GRK5_1-531.

<p>Possible membrane orientations of GRK5_1-531 on POPG lipid bilayers as determined from SFG and ATR-FTIR experimental measurements by using the 2ACX crystal structure: (A) Twist=40°, Tilt=10°, (B) Twist=300°, Tilt=26°. The plane of the membrane relative to the protein is shown as a blue rectangle.</p

SFG signal of GRK5 and GRK5_1-531.

<p>No discernible SFG amide I signals were observed for 336 nM (A) GRK5 and (B) GRK5_1-531 interacting with a 9:1 POPC:POPG lipid bilayer. SFG polarized amide I signals of 336 nM (C) GRK5 and (D) GRK5_1-531 interacting with a 1:1 POPC:POPG lipid bilayer. SFG polarized amide I signals of 336 nM (E) GRK5 and (F) GRK5_1-531 interacting with a POPG lipid bilayer. SFG polarized amide I signal of 336 nM (G) GRK5_1-531 and (F) GRK5_NT interacting with a 1:1 POPC:PIP₂ lipid bilayer. The circles and squares are experimental data. The solid lines indicate the fitting results.</p

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

GRK5 in the reference orientation.

<p>The GRK5 (PDB entry: 3NYN) and definition of twist (ψ), tilt (θ) and azimuthal (ϕ) angles which rotate the protein from the molecular (x´, y´, z´) to the macroscopic (X, Y, Z) coordinate system. The regulator of G protein signaling homology (RH) domain is colored grey, the C-terminal region containing the amphipathic helix is colored red, the kinase domain yellow, and the αN helix green (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082072#B18" target="_blank">18</a>). The side chains of residues proposed to be involved in the N-terminal phospholipid binding site (K26A, K28A, K29A, K31A, K35A) are shown as purple spheres. An approximate membrane plane (defined to be consistent with Ref. 18), is shown as blue rectangle, and lies parallel to the X-Y plane. The GRK5 is depicted in the reference orientation (ψ=0°, θ=0°, ϕ=0°) used as a starting point for data analysis. In our calculation, the molecule is rotated using an Eulerian rotation scheme according to three angles: first twist (ψ) then tilt (θ) and finally azimuthal (ϕ).</p

Modeled membrane orientations of full length GRK5.

<p>Possible membrane orientations of GRK5 on POPG lipid bilayers as determined from SFG and ATR-FTIR experimental measurements using the 3NYN crystal structure: (A) twist=190°, tilt=35°, (B) twist=245°, tilt=50°. Possible membrane orientations of GRK5 as determined from SFG and ATR-FTIR experimental measurements by using the 2ACX crystal structure: (C) twist=70°, tilt=2°, (D) twist=340°, tilt=10°. The plane of the membrane relative to the protein is shown as a blue rectangle.</p

ATR-FTIR spectra of GRK5 and GRK5_1-531.

<p>Experimental ATR-FTIR spectra of 336 nM (A) GRK5 and (B) GRK5_1-531 on POPG lipid bilayers for the p and s polarizations. The circles and crosses are experimental data. The solid lines are fitting results.</p

Possible orientations of GRK5_1-531.

<p>(A) The possible orientations of GRK5_1-531 on POPG lipid bilayers determined by combination of SFG and ATR-FTIR measurements (Figure S6 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 2ACX crystal structure. (B) The same plot as panel A, but only showing orientations with a score ≥ 70% (red). (C) The possible orientations of GRK5_1-531 on a 1:1 POPC:PIP₂ lipid bilayer determined by SFG measurement (</p><p></p><p></p><p></p><p><mi>χ</mi></p><p><mi>z</mi><mi>z</mi><mi>z</mi></p><p><mo>(</mo><mn>2</mn><mo>)</mo></p><p></p><mo>/</mo><p><mi>χ</mi></p><p><mi>x</mi><mi>x</mi><mi>z</mi></p><p><mo>(</mo><mn>2</mn><mo>)</mo></p><p></p><p></p><p></p><p></p>= 0.87±30%) using the 2ACX crystal structure.<p></p

Unveiling the Membrane-Binding Properties of N‑Terminal and C‑Terminal Regions of G Protein-Coupled Receptor Kinase 5 by Combined Optical Spectroscopies

G protein-coupled receptor kinase 5 (GRK5) is thought to associate with membranes in part via N- and C-terminal segments that are typically disordered in available high-resolution crystal structures. Herein we investigate the interactions of these regions with model cell membrane using combined sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflectance–Fourier transform infrared (ATR-FTIR) spectroscopy. It was found that both regions associate with POPC lipid bilayers but adopt different structures when doing so: GRK5 residues 2–31 (GRK5_2–31) was in random coil whereas GRK5_546–565 was partially helical. When the subphase for the GRK5_2–31 peptide was changed to 40% TFE/60% 10 mM phosphate pH 7.4 buffer, a large change in the SFG amide I signal indicated that GRK5_2–31 became partially helical. By inspecting the membrane behavior of two different segments of GRK5_2–31, namely, GRK5_2–24 and GRK5_25–31, we found that residues 25–31 are responsible for membrane binding, whereas the helical character is imparted by residues 2–24. With SFG, we deduced that the orientation angle of the helical segment of GRK5_2–31 is 46 ± 1° relative to the surface normal in 40% TFE/60% 10 mM phosphate pH = 7.4 buffer but increases to 78 ± 11° with higher ionic strength. We also investigated the effect of PIP₂ in the model membrane and concluded that the POPC:PIP₂ (9:1) lipid bilayer did not change the behavior of either peptide compared to a pure POPC lipid bilayer. With ATR-FTIR, we also found that Ca²⁺·calmodulin is able to extract both peptides from the POPC lipid bilayer, consistent with the role of this protein in disrupting GRK5 interactions with the plasma membrane in cells

Novel Irreversible Agonists Acting at the A₁ Adenosine Receptor

The A₁ adenosine receptor (A₁AR) is an important G protein-coupled receptor that regulates a range of physiological functions. Herein we report the discovery of novel irreversible agonists acting at the A₁AR, which have the potential to serve as useful research tools for studying receptor structure and function. A series of novel adenosine derivatives bearing electrophilic substituents was synthesized, and four compounds, <b>8b</b>, <b>15a</b>, <b>15b</b>, and <b>15d</b>, were shown to possess similar potency and efficacy to the reference high efficacy agonist, NECA, in an assay of ERK1/2 phosphorylation assay. Insensitivity to antagonist addition in a real-time, label-free, xCELLigence assay was subsequently used to identify compounds that likely mediated their agonism through an irreversible interaction with the A₁AR. Of these compounds, <b>15b</b> and <b>15d</b> were more directly validated as irreversible agonists of the A₁AR using membrane-based [³H]­DPCPX and [³⁵S]­GTPγS binding experiments