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.

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.

G Protein-Coupled Receptors: A Century of Research and Discovery.

GPCRs (G protein-coupled receptors), also known as 7 transmembrane domain receptors, are the largest receptor family in the human genome, with ≈800 members. GPCRs regulate nearly every aspect of human physiology and disease, thus serving as important drug targets in cardiovascular disease. Sharing a conserved structure comprised of 7 transmembrane α-helices, GPCRs couple to heterotrimeric G-proteins, GPCR kinases, and ß-arrestins, promoting downstream signaling through second messengers and other intracellular signaling pathways. GPCR drug development has led to important cardiovascular therapies, such as antagonists of ß-adrenergic and angiotensin II receptors for heart failure and hypertension, and agonists of the glucagon-like peptide-1 receptor for reducing adverse cardiovascular events and other emerging indications. There continues to be a major interest in GPCR drug development in cardiovascular and cardiometabolic disease, driven by advances in GPCR mechanistic studies and structure-based drug design. This review recounts the rich history of GPCR research, including the current state of clinically used GPCR drugs, and highlights newly discovered aspects of GPCR biology and promising directions for future investigation. As additional mechanisms for regulating GPCR signaling are uncovered, new strategies for targeting these ubiquitous receptors hold tremendous promise for the field of cardiovascular medicine.

ß-Arrestins as Important Regulators of Glucose and Energy Homeostasis.

ß-Arrestin-1 and -2 (also known as arrestin-2 and -3, respectively) are ubiquitously expressed cytoplasmic proteins that dampen signaling through G protein-coupled receptors. However, ß-arrestins can also act as signaling molecules in their own right. To investigate the potential metabolic roles of the two ß-arrestins in modulating glucose and energy homeostasis, recent studies analyzed mutant mice that lacked or overexpressed ß-arrestin-1 and/or -2 in distinct, metabolically important cell types. Metabolic analysis of these mutant mice clearly demonstrated that both ß-arrestins play key roles in regulating the function of most of these cell types, resulting in striking changes in whole-body glucose and/or energy homeostasis. These studies also revealed that ß-arrestin-1 and -2, though structurally closely related, clearly differ in their metabolic roles under physiological and pathophysiological conditions. These new findings should guide the development of novel drugs for the treatment of various metabolic disorders, including type 2 diabetes and obesity.

Art2 mediates selective endocytosis of methionine transporters during adaptation to sphingolipid depletion.

Accumulating evidence in several model organisms indicates that reduced sphingolipid biosynthesis promotes longevity, although underlying mechanisms remain unclear. In yeast, sphingolipid depletion induces a state resembling amino acid restriction, which we hypothesized might be due to altered stability of amino acid transporters at the plasma membrane. To test this, we measured surface abundance for a diverse panel of membrane proteins in the presence of myriocin, a sphingolipid biosynthesis inhibitor, in Saccharomyces cerevisiae. Unexpectedly, we found that surface levels of most proteins examined were either unaffected or increased during myriocin treatment, consistent with an observed decrease in bulk endocytosis. In contrast, sphingolipid depletion triggered selective endocytosis of the methionine transporter Mup1. Unlike methionine-induced Mup1 endocytosis, myriocin triggered Mup1 endocytosis that required the Rsp5 adaptor Art2, C-terminal lysine residues of Mup1 and the formation of K63-linked ubiquitin polymers. These findings reveal cellular adaptation to sphingolipid depletion by ubiquitin-mediated remodeling of nutrient transporter composition at the cell surface.

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.

G protein-receptor kinases 5/6 are the key regulators of G protein-coupled receptor 35-arrestin interactions.

Human G protein-coupled receptor 35 is regulated by agonist-mediated phosphorylation of a set of five phospho-acceptor amino acids within its C-terminal tail. Alteration of both Ser300 and Ser303 to alanine in the GPR35a isoform greatly reduces the ability of receptor agonists to promote interactions with arrestin adapter proteins. Here, we have integrated the use of cell lines genome edited to lack expression of combinations of G protein receptor kinases (GRKs), selective small molecule inhibitors of subsets of these kinases, and antisera able to specifically identify either human GPR35a or mouse GPR35 only when Ser300 and Ser303 (orce; the equivalent residues in mouse GPR35) have become phosphorylated to demonstrate that GRK5 and GRK6 cause agonist-dependent phosphorylation of these residues. Extensions of these studies demonstrated the importance of the GRK5/6-mediated phosphorylation of these amino acids for agonist-induced internalization of the receptor. Homology and predictive modeling of the interaction of human GPR35 with GRKs showed that the N terminus of GRK5 is likely to dock in the same methionine pocket on the intracellular face of GPR35 as the C terminus of the α5 helix of Gα13 and, that while this is also the case for GRK6, GRK2 and GRK3 are unable to do so effectively. These studies provide unique and wide-ranging insights into modes of regulation of GPR35, a receptor that is currently attracting considerable interest as a novel therapeutic target in diseases including ulcerative colitis.

ß-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium.

Postoperative delirium (POD) is a frequent and debilitating complication, especially amongst high risk procedures, such as orthopedic surgery. This kind of neurocognitive disorder negatively affects cognitive domains, such as memory, awareness, attention, and concentration after surgery; however, its pathophysiology remains unknown. Multiple lines of evidence supporting the occurrence of inflammatory events have come forward from studies in human patients' brain and bio-fluids (CSF and serum), as well as in animal models for POD. ß-arrestins are downstream molecules of guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs). As versatile proteins, they regulate numerous pathophysiological processes of inflammatory diseases by scaffolding with inflammation-linked partners. Here we report that ß-arrestin1, one type of ß-arrestins, decreases significantly in the reactive astrocytes of a mouse model for POD. Using ß-arrestin1 knockout (KO) mice, we find aggravating effect of ß-arrestin1 deficiency on the cognitive dysfunctions and inflammatory phenotype of astrocytes in POD model mice. We conduct the in vitro experiments to investigate the regulatory roles of ß-arrestin1 and demonstrate that ß-arrestin1 in astrocytes interacts with the dynamin-related protein 1 (Drp1) to regulate mitochondrial fusion/fission process. ß-arrestin1 deletion cancels the combination of ß-arrestin1 and cellular Drp1, thus promoting the translocation of Drp1 to mitochondrial membrane to provoke the mitochondrial fragments and the subsequent mitochondrial malfunctions. Using ß-arrestin1-biased agonist, cognitive dysfunctions of POD mice and pathogenic activation of astrocytes in the POD-linked brain region are reduced. We, therefore, conclude that ß-arrestin1 is a promising target for the understanding of POD pathology and development of POD therapeutics.

Arrestin domain-containing 3 (Arrdc3) modulates insulin action and glucose metabolism in liver.

Insulin action in the liver is critical for glucose homeostasis through regulation of glycogen synthesis and glucose output. Arrestin domain-containing 3 (Arrdc3) is a member of the α-arrestin family previously linked to human obesity. Here, we show that Arrdc3 is differentially regulated by insulin in vivo in mice undergoing euglycemic-hyperinsulinemic clamps, being highly up-regulated in liver and down-regulated in muscle and fat. Mice with liver-specific knockout (KO) of the insulin receptor (IR) have a 50% reduction in Arrdc3 messenger RNA, while, conversely, mice with liver-specific KO of Arrdc3 (L-Arrdc3 KO) have increased IR protein in plasma membrane. This leads to increased hepatic insulin sensitivity with increased phosphorylation of FOXO1, reduced expression of PEPCK, and increased glucokinase expression resulting in reduced hepatic glucose production and increased hepatic glycogen accumulation. These effects are due to interaction of ARRDC3 with IR resulting in phosphorylation of ARRDC3 on a conserved tyrosine (Y382) in the carboxyl-terminal domain. Thus, Arrdc3 is an insulin target gene, and ARRDC3 protein directly interacts with IR to serve as a feedback regulator of insulin action in control of liver metabolism.

Emerging Paradigm of Intracellular Targeting of G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) recognize a diverse array of extracellular stimuli, and they mediate a broad repertoire of signaling events involved in human physiology. Although the major effort on targeting GPCRs has typically been focused on their extracellular surface, a series of recent developments now unfold the possibility of targeting them from the intracellular side as well. Allosteric modulators binding to the cytoplasmic surface of GPCRs have now been described, and their structural mechanisms are elucidated by high-resolution crystal structures. Furthermore, pepducins, aptamers, and intrabodies targeting the intracellular face of GPCRs have also been successfully utilized to modulate receptor signaling. Moreover, small molecule compounds, aptamers, and synthetic intrabodies targeting ß-arrestins have also been discovered to modulate GPCR endocytosis and signaling. Here, we discuss the emerging paradigm of intracellular targeting of GPCRs, and outline the current challenges, potential opportunities, and future outlook in this particular area of GPCR biology.

Direct evidence that the GPCR CysLTR2 mutant causative of uveal melanoma is constitutively active with highly biased signaling.

Uveal melanoma is the most common eye cancer in adults and is clinically and genetically distinct from skin cutaneous melanoma. In a subset of cases, the oncogenic driver is an activating mutation in CYSLTR2, the gene encoding the G protein-coupled receptor cysteinyl-leukotriene receptor 2 (CysLTR2). The mutant CYSLTR2 encodes for the CysLTR2-L129Q receptor, with the substitution of Leu to Gln at position 129 (3.43). The ability of CysLTR2-L129Q to cause malignant transformation has been hypothesized to result from constitutive activity, but how the receptor could escape desensitization is unknown. Here, we characterize the functional properties of CysLTR2-L129Q. We show that CysLTR2-L129Q is a constitutively active mutant that strongly drives Gq/11 signaling pathways. However, CysLTR2-L129Q only poorly recruits ß-arrestin. Using a modified Slack-Hall operational model, we quantified the constitutive activity for both pathways and conclude that CysLTR2-L129Q displays profound signaling bias for Gq/11 signaling pathways while escaping ß-arrestin-mediated downregulation. CYSLTR2 is the first known example of a G protein-coupled receptor driver oncogene that encodes a highly biased constitutively active mutant receptor. These results provide new insights into the mechanism of CysLTR2-L129Q oncoprotein signaling and suggest CYSLTR2 as a promising potential therapeutic target in uveal melanoma.

Deciphering the signaling mechanisms of ß-arrestin1 and ß-arrestin2 in regulation of cancer cell cycle and metastasis.

ß-Arrestins are ubiquitously expressed intracellular proteins with many functions which interact directly and indirectly with a wide number of cellular partners and mediate downstream signaling. Originally, ß-arrestins were identified for their contribution to GPCR desensitization to agonist-mediated activation, followed by receptor endocytosis and ubiquitylation. However, current investigations have now recognized that in addition to GPCR arresting (hence the name arrestin). ß-Arrestins are adaptor proteins that control the recruitment, activation, and scaffolding of numerous cytoplasmic signaling complexes and assist in G-protein receptor signaling, thus bringing them into close proximity. They have participated in various cellular processes such as cell proliferation, migration, apoptosis, and transcription via canonical and noncanonical pathways. Despite their significant recognition in several physiological processes, these activities are also involved in the onset and progression of various cancers. This review delivers a concise overview of the role of ß-arrestins with a primary emphasis on the signaling processes which underlie the mechanism of ß-arrestins in the onset of cancer. Understanding these processes has important implications for understanding the therapeutic intervention and treatment of cancer in the future.

Pituitary Tumors: Genetic and Molecular Factors Underlying Pathogenesis and Clinical Behavior.

Pituitary neuroendocrine tumors (PitNETs) are the most common intracranial neoplasms. Although generally benign, they can show a clinically aggressive course, with local invasion, recurrences, and resistance to medical treatment. No universally accepted biomarkers of aggressiveness are available yet, and predicting clinical behavior of PitNETs remains a challenge. In rare cases, the presence of germline mutations in specific genes predisposes to PitNET formation, as part of syndromic diseases or familial isolated pituitary adenomas, and associates to more aggressive, invasive, and drug-resistant tumors. The vast majority of cases is represented by sporadic PitNETs. Somatic mutations in the α subunit of the stimulatory G protein gene (gsp) and in the ubiquitin-specific protease 8 (USP8) gene have been recognized as pathogenetic factors in sporadic GH- and ACTH-secreting PitNETs, respectively, without an association with a worse clinical phenotype. Other molecular factors have been found to significantly affect PitNET drug responsiveness and invasive behavior. These molecules are cytoskeleton and/or scaffold proteins whose alterations prevent proper functioning of the somatostatin and dopamine receptors, targets of medical therapy, or promote the ability of tumor cells to invade surrounding tissues. The aim of the present review is to provide an overview of the genetic and molecular alterations that can contribute to determine PitNET clinical behavior. Understanding subcellular mechanisms underlying pituitary tumorigenesis and PitNET clinical phenotype will hopefully lead to identification of new potential therapeutic targets and new markers predicting the behavior and the response to therapeutic treatments of PitNETs.

Exploring GPCR-arrestin interfaces with genetically encoded crosslinkers.

ß-arrestins (ßarr1 and ßarr2) are ubiquitous regulators of G protein-coupled receptor (GPCR) signaling. Available data suggest that ß-arrestins dock to different receptors in different ways. However, the structural characterization of GPCR-arrestin complexes is challenging and alternative approaches to study GPCR-arrestin complexes are needed. Here, starting from the finger loop as a major site for the interaction of arrestins with GPCRs, we genetically incorporate non-canonical amino acids for photo- and chemical crosslinking into ßarr1 and ßarr2 and explore binding topologies to GPCRs forming either stable or transient complexes with arrestins: the vasopressin receptor 2 (rhodopsin-like), the corticotropin-releasing factor receptor 1, and the parathyroid hormone receptor 1 (both secretin-like). We show that each receptor leaves a unique footprint on arrestins, whereas the two ß-arrestins yield quite similar crosslinking patterns. Furthermore, we show that the method allows defining the orientation of arrestin with respect to the GPCR. Finally, we provide direct evidence for the formation of arrestin oligomers in the cell.

Key phosphorylation sites in GPCRs orchestrate the contribution of ß-Arrestin 1 in ERK1/2 activation.

ß-arrestins (ßarrs) are key regulators of G protein-coupled receptor (GPCR) signaling and trafficking, and their knockdown typically leads to a decrease in agonist-induced ERK1/2 MAP kinase activation. Interestingly, for some GPCRs, knockdown of ßarr1 augments agonist-induced ERK1/2 phosphorylation although a mechanistic basis for this intriguing phenomenon is unclear. Here, we use selected GPCRs to explore a possible correlation between the spatial positioning of receptor phosphorylation sites and the contribution of ßarr1 in ERK1/2 activation. We discover that engineering a spatially positioned double-phosphorylation-site cluster in the bradykinin receptor (B2 R), analogous to that present in the vasopressin receptor (V2 R), reverses the contribution of ßarr1 in ERK1/2 activation from inhibitory to promotive. An intrabody sensor suggests a conformational mechanism for this role reversal of ßarr1, and molecular dynamics simulation reveals a bifurcated salt bridge between this double-phosphorylation site cluster and Lys294 in the lariat loop of ßarr1, which directs the orientation of the lariat loop. Our findings provide novel insights into the opposite roles of ßarr1 in ERK1/2 activation for different GPCRs with a direct relevance to biased agonism and novel therapeutics.

Acute and chronic metabolic effects of carvedilol in high-fructose, high-fat diet-fed mice: implication of ß-arrestin2 pathway.

We aimed to investigate the acute and chronic effects of carvedilol on insulin resistance in high-fructose, high-fat diet (HFrHFD) - fed mice and the implication of the ß-arrestin2 pathway. The acute effect of carvedilol (10 mg/kg, i.p.) on glucose tolerance and hepatic lipid signaling in normal and insulin resistant mice was investigated. Then, the chronic effect of carvedilol on insulin resistance and dyslipidemia in HFrHFD-fed mice was examined. Changes in ß-arrestin2 and its downstream signals in liver, skeletal muscle, and adipose tissue were measured. This involved measuring phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) levels and protein kinase B (AKT) activity. Carvedilol acutely reduced fasting blood glucose levels in both normal and insulin resistant mice without significantly affecting the glucose tolerance. These acute effects were associated with increased hepatic PIP2 but decreased hepatic DAG levels. Chronic administration of carvedilol significantly ameliorated insulin resistance and dyslipidemia in HFrHFD-fed mice. These chronic effects were associated with increased ß-arrestin2, PIP2, and AKT activity levels but decreased DAG levels in the classical insulin target tissues. In conclusion, carvedilol acutely maintains glucose homeostasis and chronically ameliorates insulin resistance and dyslipidemia in HFrHFD-fed mice. The insulin sensitizing effects of carvedilol are highly correlated with the upregulation of ß-arrestin2 pathway.

How Arrestins and GRKs Regulate the Function of Long Chain Fatty Acid Receptors.

FFA1 and FFA4, two G protein-coupled receptors that are activated by long chain fatty acids, play crucial roles in mediating many biological functions in the body. As a result, these fatty acid receptors have gained considerable attention due to their potential to be targeted for the treatment of type-2 diabetes. However, the relative contribution of canonical G protein-mediated signalling versus the effects of agonist-induced phosphorylation and interactions with ß-arrestins have yet to be fully defined. Recently, several reports have highlighted the ability of ß-arrestins and GRKs to interact with and modulate different functions of both FFA1 and FFA4, suggesting that it is indeed important to consider these interactions when studying the roles of FFA1 and FFA4 in both normal physiology and in different disease settings. Here, we discuss what is currently known and show the importance of understanding fully how ß-arrestins and GRKs regulate the function of long chain fatty acid receptors.

The Two ß-Arrestins Regulate Distinct Metabolic Processes: Studies with Novel Mutant Mouse Models.

The two ß-arrestins (ß-arrestin-1 and -2; alternative names: arrestin-2 and -3, respectively) are well known for their ability to inhibit signaling via G protein-coupled receptors. However, ß-arrestins can also act as signaling molecules in their own right. Although the two proteins share a high degree of sequence and structural homology, early studies with cultured cells indicated that ß-arrestin-1 and -2 are not functionally redundant. Recently, the in vivo metabolic roles of the two ß-arrestins have been studied using mutant mice selectively lacking either ß-arrestin-1 or -2 in cell types that are of particular relevance for regulating glucose and energy homeostasis. These studies demonstrated that the ß-arrestin-1 and -2 mutant mice displayed distinct metabolic phenotypes in vivo, providing further evidence for the functional heterogeneity of these two highly versatile signaling proteins.

Endocytosis at the Crossroad of Polarity and Signaling Regulation: Learning from Drosophila melanogaster and Beyond.

Cellular trafficking through the endosomal-lysosomal system is essential for the transport of cargo proteins, receptors and lipids from the plasma membrane inside the cells and across membranous organelles. By acting as sorting stations, vesicle compartments direct the fate of their content for degradation, recycling to the membrane or transport to the trans-Golgi network. To effectively communicate with their neighbors, cells need to regulate their compartmentation and guide their signaling machineries to cortical membranes underlying these contact sites. Endosomal trafficking is indispensable for the polarized distribution of fate determinants, adaptors and junctional proteins. Conversely, endocytic machineries cooperate with polarity and scaffolding components to internalize receptors and target them to discrete membrane domains. Depending on the cell and tissue context, receptor endocytosis can terminate signaling responses but can also activate them within endosomes that act as signaling platforms. Therefore, cell homeostasis and responses to environmental cues rely on the dynamic cooperation of endosomal-lysosomal machineries with polarity and signaling cues. This review aims to address advances and emerging concepts on the cooperative regulation of endocytosis, polarity and signaling, primarily in Drosophila melanogaster and discuss some of the open questions across the different cell and tissue types that have not yet been fully explored.