Visualization of arrestin recruitment by a G-protein-coupled receptor

Abstract

G Protein Coupled Receptors (GPCRs) are critically regulated by β-arrestins (βarrs), which not only desensitize G protein signaling but also initiate a G protein independent wave of signaling1-5. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)-G protein complex, has provided novel insights into the structural basis of receptor activation6-11. Lacking however has been complementary information on recruitment of βarrs to activated GPCRs primarily due to challenges in obtaining stable receptor-βarr complexes for structural studies. Here, we devised a strategy for forming and purifying a functional β2AR-βarr1 complex that allowed us to visualize its architecture by single particle negative stain electron microscopy (EM) and to characterize the interactions between β2AR and βarr1 using hydrogen-deuterium exchange mass spectrometry (HDXMS) and chemical cross-linking. EM 2D averages and 3D reconstructions reveal bimodal binding of βarr1 to the β2AR, involving two separate sets of interactions, one with the phosphorylated carboxy-terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of cross-linked residues suggest engagement of the finger loop of βarr1 with the seven-transmembrane core of the receptor. In contrast, focal areas of increased HDX indicate regions of increased dynamics in both N and C domains of βarr1 when coupled to the β2AR. A molecular model of the β2AR-βarr signaling complex was made by docking activated βarr1 and β2AR crystal structures into the EM map densities with constraints provided by HDXMS and cross-linking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented herein provides a framework for better understanding the basis of GPCR regulation by arrestins.

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11

“…HDX‐MS has also been used as a powerful tool to map out protein‐protein interfaces both in solution and on membrane surfaces 15, 16, 17, 18, 19, 20. Herein we describe an experimental approach to use HDX‐MS to define dynamic regions within a protein complex, in this case the type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11, and use this information to generate optimized constructs for X‐ray crystallography.…”

β-arrestin–biased signaling through the β 2 -adrenergic receptor promotes cardiomyocyte contraction

“…Recently, visualization of the GPCR-arrestin interface was achieved by serial femtosecond X-ray laser crystallography of a rhodopsin/arrestin complex (24). Additional insight was gained through cocrystallization studies of an arrestin finger loop peptide and rhodopsin (25), and by electron microscopy and deuterium exchange analysis of a β-arrestin1 complex with a β 2 AR-vasopressin 2 receptor C-terminal tail fusion (26) (25). This conformational stabilization is similar to that induced by G s interaction with the β 2 AR (27).…”

Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein

“…Nevertheless, high resolution cryo-EM on asymmetric and relatively small membrane proteins (<200 kDa) remains challenging due to the low signal-to-noise ratio that hampers the accuracy of angular determination for 3D reconstructions. This problem is compounded by the intrinsically dynamic character of the 7TM bundle 14, 15 and the relative instability of GPCR complexes, such as with G proteins 16 or arrestins 17 , often resulting in conformational variability or even dissociation during cryo-EM specimen preparation. Notwithstanding these challenges, cryo-EM visualization for GPCR complexes holds tremendous potential for uncovering the various molecular mechanisms involved in signal transduction and regulation of GPCRs and their effector proteins.…”