Structure-guided engineering of biased-agonism in the human niacin receptor via single amino acid substitution.

The Hydroxycarboxylic acid receptor 2 (HCA2), also known as the niacin receptor or GPR109A, is a prototypical GPCR that plays a central role in the inhibition of lipolytic and atherogenic activities. Its activation also results in vasodilation that is linked to the side-effect of flushing associated with dyslipidemia drugs such as niacin. GPR109A continues to be a target for developing potential therapeutics in dyslipidemia with minimized flushing response. Here, we present cryo-EM structures of the GPR109A in complex with dyslipidemia drugs, niacin or acipimox, non-flushing agonists, MK6892 or GSK256073, and recently approved psoriasis drug, monomethyl fumarate (MMF). These structures elucidate the binding mechanism of agonists, molecular basis of receptor activation, and insights into biased signaling elicited by some of the agonists. The structural framework also allows us to engineer receptor mutants that exhibit G-protein signaling bias, and therefore, our study may help in structure-guided drug discovery efforts targeting this receptor.

Molecular insights into atypical modes of ß-arrestin interaction with seven transmembrane receptors.

ß-arrestins (ßarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven primarily by agonist-induced receptor activation and phosphorylation. Here, we present seven cryo-electron microscopy structures of ßarrs either in the basal state, activated by the muscarinic receptor subtype 2 (M2R) through its third intracellular loop, or activated by the ßarr-biased decoy D6 receptor (D6R). Combined with biochemical, cellular, and biophysical experiments, these structural snapshots allow the visualization of atypical engagement of ßarrs with 7TMRs and also reveal a structural transition in the carboxyl terminus of ßarr2 from a ß strand to an α helix upon activation by D6R. Our study provides previously unanticipated molecular insights into the structural and functional diversity encoded in 7TMR-ßarr complexes with direct implications for exploring novel therapeutic avenues.

Molecular basis of anaphylatoxin binding, activation, and signaling bias at complement receptors.

The complement system is a critical part of our innate immune response, and the terminal products of this cascade, anaphylatoxins C3a and C5a, exert their physiological and pathophysiological responses primarily via two GPCRs, C3aR and C5aR1. However, the molecular mechanism of ligand recognition, activation, and signaling bias of these receptors remains mostly elusive. Here, we present nine cryo-EM structures of C3aR and C5aR1 activated by their natural and synthetic agonists, which reveal distinct binding pocket topologies of complement anaphylatoxins and provide key insights into receptor activation and transducer coupling. We also uncover the structural basis of a naturally occurring mechanism to dampen the inflammatory response of C5a via proteolytic cleavage of the terminal arginine and the G-protein signaling bias elicited by a peptide agonist of C3aR identified here. In summary, our study elucidates the innerworkings of the complement anaphylatoxin receptors and should facilitate structure-guided drug discovery to target these receptors in a spectrum of disorders.

Structural snapshots uncover a key phosphorylation motif in GPCRs driving ß-arrestin activation.

Agonist-induced GPCR phosphorylation is a key determinant for the binding and activation of ß-arrestins (ßarrs). However, it is not entirely clear how different GPCRs harboring divergent phosphorylation patterns impart converging active conformation on ßarrs leading to broadly conserved functional responses such as desensitization, endocytosis, and signaling. Here, we present multiple cryo-EM structures of activated ßarrs in complex with distinct phosphorylation patterns derived from the carboxyl terminus of different GPCRs. These structures help identify a P-X-P-P type phosphorylation motif in GPCRs that interacts with a spatially organized K-K-R-R-K-K sequence in the N-domain of ßarrs. Sequence analysis of the human GPCRome reveals the presence of this phosphorylation pattern in a large number of receptors, and its contribution in ßarr activation is demonstrated by targeted mutagenesis experiments combined with an intrabody-based conformational sensor. Taken together, our findings provide important structural insights into the ability of distinct GPCRs to activate ßarrs through a significantly conserved mechanism.

Allosteric modulation of GPCR-induced ß-arrestin trafficking and signaling by a synthetic intrabody.

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of ß-arrestin (ßarr) recruitment and trafficking. For several GPCRs such as the vasopressin receptor subtype 2 (V2R), agonist-stimulation first drives the translocation of ßarrs to the plasma membrane, followed by endosomal trafficking, which is generally considered to be orchestrated by multiple phosphorylation sites. We have previously shown that mutation of a single phosphorylation site in the V2R (i.e., V2RT360A) results in near-complete loss of ßarr translocation to endosomes despite robust recruitment to the plasma membrane, and compromised ERK1/2 activation. Here, we discover that a synthetic intrabody (Ib30), which selectively recognizes activated ßarr1, efficiently rescues the endosomal trafficking of ßarr1 and ERK1/2 activation for V2RT360A. Molecular dynamics simulations reveal that Ib30 enriches active-like ßarr1 conformation with respect to the inter-domain rotation, and cellular assays demonstrate that it also enhances ßarr1-ß2-adaptin interaction. Our data provide an experimental framework to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can be potentially integrated in the paradigm of GPCR-targeted drug discovery.

Emerging structural insights into GPCR-ß-arrestin interaction and functional outcomes.

Agonist-induced recruitment of ß-arrestins (ßarrs) to G protein-coupled receptors (GPCRs) plays a central role in regulating the spatio-temporal aspects of GPCR signaling. Several recent studies have provided novel structural and functional insights into our understanding of GPCR-ßarr interaction, subsequent ßarr activation and resulting functional outcomes. In this review, we discuss these recent advances with a particular emphasis on recognition of receptor-bound phosphates by ßarrs, the emerging concept of spatial positioning of key phosphorylation sites, the conformational transition in ßarrs during partial to full-engagement, and structural differences driving functional outcomes of ßarr isoforms. We also highlight the key directions that require further investigation going forward to fully understand the structural mechanisms driving ßarr activation and functional responses.

Intrinsic bias at non-canonical, ß-arrestin-coupled seven transmembrane receptors.

G-protein-coupled receptors (GPCRs), also known as seven transmembrane receptors (7TMRs), typically interact with two distinct signal-transducers, i.e., G proteins and ß-arrestins (ßarrs). Interestingly, there are some non-canonical 7TMRs that lack G protein coupling but interact with ßarrs, although an understanding of their transducer coupling preference, downstream signaling, and structural mechanism remains elusive. Here, we characterize two such non-canonical 7TMRs, namely, the decoy D6 receptor (D6R) and the complement C5a receptor subtype 2 (C5aR2), in parallel with their canonical GPCR counterparts. We discover that D6R and C5aR2 efficiently couple to ßarrs, exhibit distinct engagement of GPCR kinases (GRKs), and activate non-canonical downstream signaling pathways. We also observe that ßarrs adopt distinct conformations for D6R and C5aR2, compared to their canonical GPCR counterparts, in response to common natural agonists. Our study establishes D6R and C5aR2 as ßarr-coupled 7TMRs and provides key insights into their regulation and signaling with direct implication for biased agonism.

Distinct phosphorylation sites in a prototypical GPCR differently orchestrate ß-arrestin interaction, trafficking, and signaling.

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a key determinant for their interaction with ß-arrestins (ßarrs) and subsequent functional responses. Therefore, it is important to decipher the contribution and interplay of different receptor phosphorylation sites in governing ßarr interaction and functional outcomes. Here, we find that several phosphorylation sites in the human vasopressin receptor (V2R), positioned either individually or in clusters, differentially contribute to ßarr recruitment, trafficking, and ERK1/2 activation. Even a single phosphorylation site in V2R, suitably positioned to cross-talk with a key residue in ßarrs, has a decisive contribution in ßarr recruitment, and its mutation results in strong G-protein bias. Molecular dynamics simulation provides mechanistic insights into the pivotal role of this key phosphorylation site in governing the stability of ßarr interaction and regulating the interdomain rotation in ßarrs. Our findings uncover important structural aspects to better understand the framework of GPCR-ßarr interaction and biased signaling.

Crystal Structure of ß-Arrestin 2 in Complex with CXCR7 Phosphopeptide.

ß-Arrestins (ßarrs) critically regulate G-protein-coupled receptor (GPCR) signaling and trafficking. ßarrs have two isoforms, ßarr1 and ßarr2. Receptor phosphorylation is a key determinant for the binding of ßarrs, and understanding the intricate details of receptor-ßarr interaction is the next frontier in GPCR structural biology. The high-resolution structure of active ßarr1 in complex with a phosphopeptide derived from GPCR has been revealed, but that of ßarr2 remains elusive. Here, we present a 2.3-Å crystal structure of ßarr2 in complex with a phosphopeptide (C7pp) derived from the carboxyl terminus of CXCR7. The structural analysis of C7pp-bound ßarr2 reveals key differences from the previously determined active conformation of ßarr1. One of the key differences is that C7pp-bound ßarr2 shows a relatively small inter-domain rotation. Antibody-fragment-based conformational sensor and hydrogen/deuterium exchange experiments further corroborated the structural features of ßarr2 and suggested that ßarr2 adopts a range of inter-domain rotations.

Emerging Insights into the Structure and Function of Complement C5a Receptors.

Complement factor C5a is an integral constituent of the complement cascade critically involved in the innate immune response, and it exerts its functions via two distinct receptors, C5aR1 and C5aR2. While C5aR1 is a prototypical G-protein-coupled receptor (GPCR), C5aR2 lacks functional coupling to heterotrimeric G proteins, although both receptors efficiently recruit ß arrestins (ßarrs). Here, we discuss the recent studies providing direct structural details of ligand-receptor interactions, and a framework of functional bias in this system, including the differences in terms of structural motifs and transducer coupling. We also discuss the functional analogy of C5aR2 with the atypical chemokine receptors (ACKRs), and highlight the future directions to elucidate the mechanistic basis of the functional divergence of these receptors activated by a common natural agonist.

Terminating G-Protein Coupling: Structural Snapshots of GPCR-ß-Arrestin Complexes.

ß-arrestins (ßarrs) play multifaceted roles in the signaling and regulation of G-protein-coupled receptors (GPCRs) including their desensitization and endocytosis. Recently determined cryo-EM structures of two different GPCRs in complex with ßarr1 provide the first glimpse of GPCR-ßarr engagement and a structural framework to understand their interaction.

Conformational Sensors and Domain Swapping Reveal Structural and Functional Differences between ß-Arrestin Isoforms.

Desensitization, signaling, and trafficking of G-protein-coupled receptors (GPCRs) are critically regulated by multifunctional adaptor proteins, ß-arrestins (ßarrs). The two isoforms of ßarrs (ßarr1 and 2) share a high degree of sequence and structural similarity; still, however, they often mediate distinct functional outcomes in the context of GPCR signaling and regulation. A mechanistic basis for such a functional divergence of ßarr isoforms is still lacking. By using a set of complementary approaches, including antibody-fragment-based conformational sensors, we discover structural differences between ßarr1 and 2 upon their interaction with activated and phosphorylated receptors. Interestingly, domain-swapped chimeras of ßarrs display robust complementation in functional assays, thereby linking the structural differences between receptor-bound ßarr1 and 2 with their divergent functional outcomes. Our findings reveal important insights into the ability of ßarr isoforms to drive distinct functional outcomes and underscore the importance of integrating this aspect in the current framework of biased agonism.

The Gut Feeling: GPCRs Enlighten the Way.

Host-microbiome interactions affect host physiology, but the underlying mechanisms are not well understood. Recent papers from Chen et al. (2019) and Colosimo et al. (2019) in this issue of Cell Host & Microbe demonstrate that metabolites produced by several members of the gut microbiota can efficiently activate host G protein-coupled receptors and influence host physiology.