Structural Insights into the Intrinsically Disordered GPCR C-Terminal Region, Major Actor in Arrestin-GPCR Interaction

Abstract

Arrestin-dependent pathways are a central component of G protein-coupled receptor (GPCRs) signaling. However, the molecular processes regulating arrestin binding are to be further illuminated, in particular with regard to the structural impact of GPCR C-terminal disordered regions. Here, we used an integrated biophysical strategy to describe the basal conformations of the C-terminal domains of three class A GPCRs, the vasopressin V2 receptor (V2R), the growth hormone secretagogue or ghrelin receptor type 1a (GHSR) and the β2-adernergic receptor (β2AR). By doing so, we revealed the presence of transient secondary structures in these regions that are potentially involved in the interaction with arrestin. These secondary structure elements differ from those described in the literature in interaction with arrestin. This suggests a mechanism where the secondary structure conformational preferences in the C-terminal regions of GPCRs could be a central feature for optimizing arrestins recognition.

Keywords: GPCR; NMR; arrestin; intrinsically disordered proteins or regions (IDPs/IDRs).

Figure 1

Schematic representation of a GPCR. GPCRs share a common core domain with seven transmembrane helices (7TM) linked by extracellular (ECL) and intracellular (ICL) loops. These loops, as well as the N- and C-terminal domains, are variable in length and sequence. The sequences of the three peptides used in this study are indicated just below.

Figure 2

The C-terminal domain of V2R-Cter (blue), GHSR-Cter (green), and β2AR-Cter (purple) are monomeric and disordered. The color code is maintained for all panels. (a) SEC-MALS curves (colored dashed lines). Molar masses derived from MALS are indicated by thick black line (right axis); (b) far-UV circular dichroism (CD) spectra (colored lines) present a minimum around 200 nm (black dashed line) and a shoulder at 220 nm (black dashed and dotted line) characteristic of disordered proteins with transient secondary structure content; (c) ¹⁵N-HSQC spectra display a low proton spectral dispersion (~1 ppm) typical of IDPs. HSQCs were recorded on 300 µM samples at 700 MHz and 20 °C, in 50 mM Bis-Tris pH 6.7, 150 mM NaCl buffer.

Figure 3

Bioinformatics predictions, secondary structure propensity, backbone dynamics, and RDC conformational profile of the disordered V2R-Cter (blue), GHSR-Cter (green), and β2AR-Cter (purple). (a) GPCR C-termini are composed of more than 50% disorder-promoting residues (in orange). Ordered-promoting residues are indicated in purple. Secondary structures obtained by the consensus of all NMR data are indicated under the sequence and highlighted according to their respective color code. Helix, β-strand, extended conformation (β-strand or polyproline helix 2, PPII), and turns are represented as red cylinder, blue arrow, purple box, and green bars, respectively; (b) disorder prediction by SPOT-Disorder 2 (red), SPOT-Disorder Single (black), PONDR-FIT (purple), PrDOS (green), DisPro (orange), DISOPRED 3 (grey), and Espritz-NMR (blue). The disorder/order threshold (0.5) is indicated in black line; (c) secondary structure prediction by SOPMA, PSIPRED, JPRED4, PSSpred, SPOT 1D, and SPIDER 3 web-servers are represented for helices (red cylinder), strands (blue arrow), and turns (green); (d) computed secondary structure SCS Cα-SCS Cβ using random coil chemical shifts from POTENCI; (e) heteronuclear ¹⁵N{¹H}-NOEs are represented with colored curves and the average with dash colored lines; (f) back-calculated and experimental ¹D_HN RDCs. Comparison of experimental RDC (colored according to the GPCR-Cter color code) and back-calculated values computed with FM on a random-coil ensemble (dash color line) and biased ensemble (red line) and populated as indicated in the figure for helical structure (highlighted in red), extended structure (highlighted in blue), or turn (highlighted in green). Spectra were recorded at 700 MHz and 20 °C, in 50 mM Bis-Tris pH 6.7, 150 mM NaCl buffer.

Figure 4

The C-terminal regions of β2AR-Cter (purple), GHSR-Cter (green), and V2R-Cter (blue) revealed different transient secondary structures, which could be involved in arrestin binding. GRK2 (red) and GRK6 (blue) phosphorylated sites according to [9] for β2AR, [72] for GHSR, and [73] for V2R are indicated in the sequence. Secondary structures obtained by NMR secondary structure consensus are indicated under the sequence, according to Figure 3.

Figure 5

A conformational change must occur in the C-terminal region of V2R (blue) upon binding to arrestin and/or phosphorylation. Comparison of the free, in solution state of V2R-Cter to its bound state identified in complexes between a fully phosphorylated phospho-peptide of vasopressin V2 C-terminus (V2Rpp) and arrestin-2. On top is represented the sequence of human V2R C-terminus. Residues known to be phosphorylated by GRKs are colored in green [73]. Below, V2R-Cter sequences used in each work are represented in dashed lines. Residues encompassing the binding regions are identified in red and phosphorylated residues are noted as p. Stars (*) indicate that the V2R C-terminus is fused to another receptor (PDB: 6U1N, muscarinic receptor; 6TKO: β1-adrenergic receptor). Helices and β-strands are represented as red cylinders and blue arrows, respectively. In the frame, the PDB structure of V2R:arrestin-2 complexes is shown [21]. Phosphorylated residues of V2Rpp (in blue) are highlighted as sticks.