Membrane-mediated oligomerization of G protein coupled receptors and its implications for GPCR function

The dimerization or even oligomerization of G protein coupled receptors (GPCRs) causes ongoing, controversial debates about its functional role and the coupled biophysical, biochemical or biomedical implications. A continously growing number of studies hints to a relation between oligomerization and function of GPCRs and strengthens the assumption that receptor assembly plays a key role in the regulation of protein function. Additionally, progress in the structural analysis of GPCR-G protein and GPCR-ligand interactions allows to distinguish between actively functional and non-signalling complexes. Recent findings further suggest that the surrounding membrane, i.e. its lipid composition may modulate the preferred dimerization interface and as a result the abundance of distinct dimeric conformations. In this review, the association of GPCRs and the role of the membrane in oligomerization will be discussed. An overview of the different reported oligomeric interfaces is provided and their capability for signaling discussed. The currently available data is summarized with regard to the formation of GPCR oligomers, their structures and dependency on the membrane microenvironment as well as the coupling of oligomerization to receptor function

Closely related, yet unique: Distinct homo- and heterodimerization patterns of G protein coupled chemokine receptors and their fine-tuning by cholesterol

Chemokine receptors, a subclass of G protein coupled receptors (GPCRs), play essential roles in the human immune system, they are involved in cancer metastasis as well as in HIV-infection. A plethora of studies show that homo- and heterodimers or even higher order oligomers of the chemokine receptors CXCR4, CCR5, and CCR2 modulate receptor function. In addition, membrane cholesterol affects chemokine receptor activity. However, structural information about homo- and heterodimers formed by chemokine receptors and their interplay with cholesterol is limited. Here, we report homo- and heterodimer configurations of the chemokine receptors CXCR4, CCR5, and CCR2 at atomistic detail, as obtained from thousands of molecular dynamics simulations. The observed homodimerization patterns were similar for the closely related CC chemokine receptors, yet they differed significantly between the CC receptors and CXCR4. Despite their high sequence identity, cholesterol modulated the CC homodimer interfaces in a subtype-specific manner. Chemokine receptor heterodimers display distinct dimerization patterns for CXCR4/CCR5 and CXCR4/CCR2. Furthermore, associations between CXCR4 and CCR5 reveal an increased cholesterol-sensitivity as compared to CXCR4/CCR2 heterodimerization patterns. This work provides a first comprehensive structural overview over the complex interaction network between chemokine receptors and indicates how heterodimerization and the interaction with the membrane environment diversifies the function of closely related GPCRs

Involvement of individual transmembrane helices at homodimer interfaces.

<p><b>a</b> Homodimer binding position densities (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006062#sec011" target="_blank">Materials and methods</a>), i.e. involvement of individual transmembrane helices at chemokine receptor homodimer interfaces obtained after 3 <i>μs</i> in pure POPC membranes (bottom-up), and after 8 <i>μs</i> (6 <i>μs</i> in case of CXCR4) in POPC membranes containing 30% cholesterol (top-down). The data for CXCR4 were taken from our previous study [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006062#pcbi.1006062.ref047" target="_blank">47</a>]. Binding position angles are assigned to individual transmembrane helices according to <b>b</b> (sample coordinate system based on the principal axes of CCR5). Transmembrane helices are colored according to the scheme established in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006062#pcbi.1006062.g002" target="_blank">Fig 2</a>. Bleached transmembrane segments (on TM2 and TM3) at the x-axis label correspond to TM areas that are not exposed at the protein surface.</p

CXCR4/CC chemokine receptor heterodimer interfaces.

<p><b>a</b> Populations of CXCR4/CC chemokine receptor heterodimers after 3 <i>μs</i> in pure POPC membranes (light colors), and after 8 <i>μs</i> in POPC membranes containing 30% cholesterol (dark colors). <b>b</b> Heterodimer binding position densities as introduced in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006062#pcbi.1006062.g003" target="_blank">Fig 3</a>. Binding position densities on CXCR4 are shown in red, on CCR5 in green, and on CCR2 in blue. <b>c</b> Representative chemokine receptor heterodimer structures for the most populated interfaces. The protein surface is colored red for CXCR4, green for CCR5, and blue for CCR2. Transmembrane helices are colored consistent with <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006062#pcbi.1006062.g002" target="_blank">Fig 2</a>. <b>d</b> Interactions between CXCR4/CC chemokine receptor heterodimers and cholesterol. Cholesterol molecules are shown as sticks and colored in cyan and the density distribution of cholesterol around the protein is colored in light orange. Residues interacting at the TM4,5/TM1,H8 CXCR4/CCR5 interface are enframed according to the transmembrane helix color they are located on. The density distribution of cholesterol at the main binding spot on CXCR4 between TM1 and TM7, impeding the formation of CXCR4/CCR5 heterodimers involving TM1 of CXCR4, is highlighted in cyan.</p

Comparison of protein root mean square deviations (RMSD) between coarse-grained and atomistic simulations.

<p>RMSDs of transmembrane helix backbone beads determined from 100 coarse-grained (CG) homodimerization simulations (initial 500 ns) in POPC membranes (for systems where no interaction energies between both monomers were observed until 1 <i>μs</i>, shown in light grey). The average RMSDs of CG simulations are colored in red for CXCR4, green for CCR5, and blue for CCR2. The RMSD curves for atomistic simulations (CHARMM36 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006062#pcbi.1006062.ref111" target="_blank">111</a>] for CXCR4 and CHARMM36m [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006062#pcbi.1006062.ref116" target="_blank">116</a>] for CC chemokine receptors) of corresponding chemokine receptor monomers in POPC membranes, shown in black, were calculated by converting the atomistic simulation frame-by-frame (time step of 100 ps) to the Martini2.2 force field in order to enable a direct comparison to the CG simulations. The data for CXCR4 was taken from our previous study [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006062#pcbi.1006062.ref047" target="_blank">47</a>].</p

CCR5/CCR2 heterodimer interfaces.

<p><b>a</b> Populations of CCR5/CCR2 chemokine receptor heterodimers after 3 <i>μs</i> in pure POPC membranes (light colors), and after 8 <i>μs</i> in POPC membranes containing 30% cholesterol (dark colors). <b>b</b> Representative chemokine receptor heterodimer structures for the most populated interfaces. The protein surface of CCR5 is colored in green and in blue for CCR2. Transmembrane segments are colored according to <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006062#pcbi.1006062.g002" target="_blank">Fig 2</a>. <b>c</b> Heterodimer binding position densities as introduced in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006062#pcbi.1006062.g003" target="_blank">Fig 3</a>. Binding positions on CCR5 are colored in green and on CCR2 in blue. <b>d</b> The spatial distribution of cholesterol around the protein is colored in light orange. Cholesterol-free dimers (taken from the simulations in pure POPC) are shown in light grey.</p

Dimerization and dissociation propensities of the studied chemokine receptors.

<p><b>a</b> The dimerization propensity is given as the relative number of dimers formed per microsecond and was calculated as the ratio between the final number of dimers divided by the total simulation time in microseconds. <b>b</b> Dissociation propensities were computed by dividing the total number of dissociation events (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006062#pcbi.1006062.s002" target="_blank">S2d Fig</a>) by the total number of dimerization events for most populated dimer interfaces (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006062#sec011" target="_blank">Materials and methods</a>).</p

Relative orientation angles at dimer interfaces.

<p>Three relative angles are calculated to describe dimer configurations: <i>β</i> describes the position of binding of monomer B to the reference structure monomer A given as the rotation of monomer B around the z-axis of monomer A. The angle <i>ϕ</i> defines the rotation of monomer B around its’ own z-axis (z’) and <i>χ</i> = (180° + <i>β</i> − <i>ϕ</i>)<i>mod</i>360 describes the position of binding of monomer A to monomer B.</p

Cholesterol binding to CXCR4, CCR5, and CCR2 chemokine receptor monomers.

<p>The spatial distributions of the five closest cholesterol molecules around the chemokine receptors (<b>a</b>) CXCR4, (<b>b</b>) CCR5, and (<b>c</b>) CCR2, are shown in light orange, the spatial distributions of the polar headgroup of cholesterol (ROH beads) are shown in dark red. Receptor helices are colored according to the following scheme: TM1: red, TM2: light blue, TM3: yellow, TM4: grey, TM5: purple, TM6: dark blue, TM7: green, H8: orange. The helix thickness encodes the residue-resolved cholesterol-occupancy. Residues showing high cholesterol occupancies are explicitely listed and enframed according to the transmembrane helix they are located on. Residues previously identified in experiments as important for chemokine receptor function [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006062#pcbi.1006062.ref032" target="_blank">32</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006062#pcbi.1006062.ref068" target="_blank">68</a>] are highlighted in bold. The data for CXCR4 (<b>a</b>) was taken from our previous study [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006062#pcbi.1006062.ref047" target="_blank">47</a>] and included for comparison.</p