Structural insights into emergent signaling modes of G protein-coupled receptors

Abstract

G protein-coupled receptors (GPCRs) represent the largest family of cell membrane proteins, with >800 GPCRs in humans alone, and recognize highly diverse ligands, ranging from photons to large protein molecules. Very important to human medicine, GPCRs are targeted by about 35% of prescription drugs. GPCRs are characterized by a seven-transmembrane α-helical structure, transmitting extracellular signals into cells to regulate major physiological processes via heterotrimeric G proteins and β-arrestins. Initially viewed as receptors whose signaling via G proteins is delimited to the plasma membrane, it is now recognized that GPCRs signal also at various intracellular locations, and the mechanisms and (patho)physiological relevance of such signaling modes are actively investigated. The propensity of GPCRs to adopt different signaling modes is largely encoded in the structural plasticity of the receptors themselves and of their signaling complexes. Here, we review emerging modes of GPCR signaling via endosomal membranes and the physiological implications of such signaling modes. We further summarize recent structural insights into mechanisms of GPCR activation and signaling. We particularly emphasize the structural mechanisms governing the continued GPCR signaling from endosomes and the structural aspects of the GPCR resensitization mechanism and discuss the recently uncovered and important roles of lipids in these processes.

Keywords: G protein-coupled receptor (GPCR); arrestin; cAMP; cyclic AMP (cAMP); endosomal membrane; endosome; environmental sensing; membrane lipid; parathyroid hormone; receptor structure-function; signal transduction; structural dynamics; structure-function; β-arrestins.

Figure 1.

Basic paradigm of GPCR signaling. Following ligand agonist binding, the receptor adopts an active-state conformation that couples to one subfamily or multiple subfamilies of heterotrimeric G proteins (Gαβγ). This interaction catalyzes the exchange of GDP for GTP on the Gα subunit. The GTP-bound Gα and Gβγ subunits dissociate and activate (→) or inhibit (—|) diverse effectors, which in turn regulate intracellular levels of second messengers, as well as activities of GIRK channels and phosphoinositide 3-kinases (PI3K) and recruitment of GRKs.

Figure 2.

General principle of GPCR signaling via cAMP. A, in the classical model, production of cAMP (1^st pool) only takes place at the cell membrane after activation of G_s by the agonist-bound receptors (step 1). This cAMP response is usually short-lived due to the action of phosphodiesterases and rapid receptor desensitization initiated by recruitment of GRKs and receptor phosphorylation (step 2), followed by recruitment of β-arrestin (βarr; step 3) driving receptor endocytosis and eventually engaging β-arrestin-dependent mitogen-activated protein kinase signaling cascades (step 4). In the more recent model, agonists—usually peptide hormones—that interact tightly with receptor in a conformationally dependent rather than G-protein–dependent manner, also induce sustained cAMP that originated from ligand–GPCR complexes in endosomes (2^nd pool) (step 4). Endosomal cAMP production continues until endosomal acidification induces the release of the agonist from the receptor (step 5) and receptor dephosphorylation (step 6), allowing receptor degradation (step 7), receptor transfer to the Golgi apparatus (step 8), and/or the receptor recycling (step 9). B, examples of cAMP time-course profiles mediated by PTH in live cells expressing PTHR and showing plasma membrane (PM; light orange) and endosomal (Endosomes; light blue) response phases for control (black), when the receptor internalization (step 4) is blocked by either dynasore or a dominant negative mutant of dynamin (purple), or when the endosomal acidification (step 5) is blocked by bafilomycin (blue). Data represent the mean ± S.E.M.

Figure 3.

Structural dynamics of GPCR signaling. A, structural features and activation hallmarks of GPCR classes. Left, comparison of inactive→active state transition between representative members of class A, B, and F GPCRs: the common activation hallmark is an outward movement of TM6. The inactive- and active-state structures are shown as semitransparent light violet and orange cartoons, respectively, with TM6 helices highlighted as an opaque cartoon and dashed lines connected with an arrow depicting transition from the inactive to the active state. The superimposed structures are as follows: inactive-state (PDB entry 2R4R) and active-state (PDB entry 3SN6) β2AR; inactive-state (PDB entry 6FJ3) and active-state (PDB entry 6NBF) PTH1R; inactive-state (PDB entry 4N4W) and active-state (PDB 6OT0) SMO. Middle, the cryo-EM structures of the mGlu5 receptor unveil the class C GPCR activation mechanism: the ligand binding to ECD induces a substantial reorganization of the ECDs, leading to repositioning of TMDs in close proximity. However, the TMDs of agonist-bound mGlu5 receptors are nearly identical to those of apo-receptors, indicating that TM6 opening likely requires the G protein presence. The protomers of mGlu5 homodimers are shown as green and wheat cartoons. The apo-receptor (PDB entry 6N52) structure is superimposed onto agonist-bound mGlu5 (PDB code 6N51) by structural alignment of green protomers. The agonist l-quisqualate is shown in magenta spheres, and the ECD-bound nanobody Nb43 in the active state is omitted for clarity. Right, putative model of an aGPCR. In addition to a GPCR-characteristic TMD (wheat cartoon, TMD of inactive glucagon receptor, PDB entry 4L6R), which has phylogenetic relation to class B GPCRs, a common feature of aGPCRs is a large, mostly multidomain ECD. With a single exception of ADGRA1, all of the aGPCRs contain a GPCR autoproteolysis–inducing (GAIN) domain N-terminal to their TMDs (the green cartoon shows the X-ray structure of a GAIN domain of latrophilin 1, PDB entry 4DLQ), which harbors a GPCR proteolysis site (GPS) with a consensus cleavage sequence H(L/I)↓(S/T). The autoproteolysis takes place in the ER, and the ECDs remain noncovalently attached to the rest of the receptor through the tight interaction of the peptide C-terminal to the GPS (called the Stachel peptide, shown in red). For many aGPCRs, the disruption of Stachel peptide interaction with the ECD results in receptor activation. B, cryo-EM structure of PTH1R in complex with LA-PTH and G_s. Two conformational states of LA-PTH binding to PTH1R were detected: state 1 (green) forms a continuous interaction with PTH1R, and in state 2 (magenta), the C-terminal tip of LA-PTH is dissociated from the receptor's ECD (LA-PTH of state 2 (PDB entry 6NBI) is superimposed onto the state 1 structure (PDB entry 6NBF)). TMD-surrounding lipids are shown as sticks; a rectangle delimits the activating portion of the LA-PTH N-terminal part inserted into the receptor's TMD core, and the interaction network in this region between LA-PTH (green) and PTH1R (orange) residues is shown on the right (polar, nonpolar, and mixed interactions are shown as yellow, blue, and green lines, respectively). C, GPCR/β-arrestin interaction plasticity. The cryo-EM structure of NTS(8-13)-bound NTS1R/β-arrestin1 complex (PDB entry 6UP7) shows β-arrestin1 binding in a tilted conformation, possibly stabilized by interaction of PIP₂ (black sticks) with the concave surface of the C-domain of β-arrestin1; the right panel shows a bottom view of a superposition of β-arrestin1 conformations bound to NTS1R (light pink) and M2R (purple; PDB entry 6U1N). D, G protein and β-arrestin can bind a GPCR simultaneously. Shown is a cryo-EM structure of a β2V2R chimera in complex with G_s (same color scheme as in A) and β-arrestin1 (combined PDB files 6NI3 and 6NI2). The flexible unresolved part of V2Rpp is shown as an orange dashed line. Gray rectangles in A–C denote putative lipid bilayer boundaries.

Figure 4.

Mechanism of PTH1R signaling via G_s and G_q proteins. A, upon PTH binding, PTH1R couples and activates heterotrimeric G_s and G_q proteins at the plasma membrane (steps 1 and 2). G_s activates adenylate cyclases (AC), leading to acute cAMP synthesis and activation of PKA. The time course of cAMP is short due to the action of phosphodiesterases (PDE) (pink box). G_q activates PLCβ, which cleaves phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) into IP₃, which diffuses through the cytosol to activate IP3-gated Ca²⁺ channels, releasing stored Ca²⁺. B, liberation of Gβγ subunits from G_q promotes PI3Kβ-dependent generation of PI(3,4,5)P₃ (step 3), which in turn promotes β-arrestin (βarr) recruitment to the PTH1R (step 4) and formation of ternary PTH1R–βarr–Gβγ complex that remains active following internalization and redistribution to early endosomes (step 5). Reassembly of the ternary PTH1R complex with Gα_s is thought to be dependent on Gα_s diffusion. C, the sustained phase of cAMP production (step 6) is due to the inhibitory action of extracellular signal-regulated protein kinase 1/2 (ERK1/2) on PDE4 (step 7) and can efficiently diffuse to the nucleus to activate nuclear PKA (step 8). D, termination of endosomal cAMP signaling is initiated by a negative feedback loop, where PKA-dependent activation of the H⁺ pump v-ATPase increases endosome acidification (step 9), which sequentially disassembles the ternary PTHR–arrestin–Gβγ signaling complex and engages retromer coupling to PTHR (step 10) and its recycling to the cell surface or redistribution in the Golgi apparatus. Adapted from Refs. and . This research was originally published in Trends in Endocrinology and Metabolism. Sutkeviciute, I., Clark, L. J., White, A. D., Gardella, T. J., and Vilardaga, J. P. PTH/PTHrP receptor signaling, allostery, and structures. Trends in Endocrinology and Metabolism. 2019; 30:860–874. © Cell Press; Proceedings of the National Academy of Sciences of the United States of America. White, A. D., Jean-Alphonse, F. G., Fang, F., Peña, K. A., Liu, S., Konig, G. M., Inoue, A., Aslanoglou, D., Gellman, S. H., Kostenis, E., Xiao, K., and Vilardaga, J. P. G_q/11-dependent regulation of endosomal cAMP generation by parathyroid hormone class B GPCR. Proceedings of the National Academy of Sciences of the United States of America. 2020; 117:7455–7460. © United States National Academy of Sciences.

Figure 5.

Structural view of retromer recruitment to endosomes. The putative engagement of the inactive PTH1R (PDB entry 6FJ3) to the retromer complex (cryo-ET structure, PDB 6H7W) through Snx27 on the endosomal membrane bilayer surface. The complex of Snx27 PDZ/PTH1R C-tail (PDB 4Z8J) was superimposed onto Vps26A/Snx27 PDZ complex (PDB entry 4P2A), which was then superimposed onto Vps26 in the retromer dimer complex (PDB entry 6H7W); the second Vps26 subunit (right side) in the retromer dimer complex was placed in a putative positioning.