Regulator of G protein Signaling, or RGS, proteins serve an important regulatory role in signaling mediated by G protein-coupled receptors (GPCRs). They all share a common RGS domain that directly interacts with active, GTP-bound Gα subunits of heterotrimeric G proteins. RGS proteins stabilize the transition state for GTP hydrolysis on Gα and thus induce a conformational change in the Gα subunit that accelerates GTP hydrolysis, thereby effectively turning off signaling cascades mediated by GPCRs. This GTPase accelerating protein (GAP) activity is the canonical mechanism of action for RGS proteins, although many also possess additional functions and domains. RGS proteins are divided into four families, R4, R7, R12 and RZ based on sequence homology, domain structure as well as specificity towards Gα subunits. For reviews on RGS proteins and their potential as therapeutic targets, see e.g. [225, 529, 578, 583, 584, 742, 753, 444, 10].
The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands ( www.guidetopharmacology.org ), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point‐in‐time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16176 . In addition to this overview, in which are identified ‘Other protein targets’ which fall outside of the subsequent categorisation, there are six areas of focus: G protein‐coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid‐2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC‐IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
G‐protein Coupled Receptors (GPCRs) are common targets for therapeutic drugs; however, unwanted side effects arise from the variety of functions GPCRs can serve in different tissues. Regulator of G protein Signaling (RGS) proteins are tissue‐selective negative modulators of the initial effector of GPCR activation, the Gα subunit of heterotrimeric G‐proteins. Targeting RGS proteins instead of GPCRs provides a novel way to address disease while minimize off‐target effects. RGS2 is a Gα q ‐specific GTP‐ase activating protein, reduced levels of which are implicated in cardiovascular disease, anxiety, and various types of cancers. A key regulatory mechanism for RGS2 is its rapid degradation via the ubiquitin‐proteasomal pathway. We previously demonstrated that RGS2 is targeted for proteasomal degradation via a novel Cullin‐RING E3 ligase complex. The substrate recognition component of this E3 ligase is F‐box Only Protein 44 (FBXO44) and inhibiting the interaction with FBXO44 would represent a novel strategy to stabilize RGS2 levels. For this study, we hypothesized that the N‐terminal of RGS2 binds FBXO44. To investigate this hypothesis we utilized western blotting, siRNA knock‐down, co‐immunoprecipitation, and a β‐galactosidase‐dependent chemiluminescent assay. We examined N‐terminally truncated mutants of RGS2 based on previously reported alternative translation initiation sites at methionine 5, 16 and 33 (M5, M16 and M33, respectively). We found that truncating the N‐terminal of RGS2 resulted in reduced ubiquitination when treated with the proteasome inhibitor MG‐132 in addition to preventing co‐immunoprecipitation with FBXO44. In addition, siRNA knockdown of FBXO44 only stabilized full‐length RGS2 as well. However, MG‐132 treatment stabilized full‐length RGS2 as well as the M5 variant, but not M16 or M33, as demonstrated by both western blotting and a chemiluminescent enzyme complementation assay. To explain the differences between full‐length and M5 RGS2, we mutated Serine 3 to Alanine (S3A, phospho‐dead) or Aspartic acid (S3D, phospho‐mimetic), hypothesizing that loss of a phosphorylation site would destabilize M5 if it retained the FBXO44‐interaction site. We found that WT and S3A RGS2 were stabilized by MG‐132 treatment, but S3D was not. In addition, less S3D RGS2 co‐immunoprecipitated with FBXO44 compared to WT or S3A indicating that phosphorylation at Ser3 prevents RGS2 from binding FBXO44. Altogether our data indicate that RGS2 binds FBXO44 through an N‐terminal motif located between residues 5 and 16, and that phosphorylation at Ser3 can inhibit this interaction and prevent RGS2 degradation. Ongoing studies aims to further define the interaction site between RGS2 and FBXO44, as well as developing high‐throughput screening strategies to identify small molecule RGS2‐FBXO44 interaction inhibitors. These would be promising leads in disease states associated with low RGS2 protein levels. Support or Funding Information American Heart Association (15SDG21630002) Ralph W. and Grace M. Showalter Research Trust
Regulator of G protein signaling (RGS) proteins are negative modulators of G protein signaling that have emerged as promising drug targets to improve specificity and reduce side effects of G protein–coupled receptor–related therapies. Several small molecule RGS protein inhibitors have been identified; however, enhancing RGS protein function is often more clinically desirable but presents a challenge. Low protein levels of RGS2 are associated with various pathologies, including hypertension and heart failure. For this reason, RGS2 is a prominent example wherein enhancing its function would be beneficial. RGS2 is rapidly ubiquitinated and proteasomally degraded, providing a point of intervention for small molecule RGS2-stabilizing compounds. We previously identified a novel cullin-RING E3 ligase utilizing F-box only protein 44 (FBXO44) as the substrate recognition component. Here, we demonstrate that RGS2 associates with FBXO44 through a stretch of residues in its N terminus. RGS2 contains four methionine residues close to the N terminus that can act as alternative translation initiation sites. The shorter translation initiation variants display reduced ubiquitination and proteasomal degradation as a result of lost association with FBXO44. In addition, we show that phosphorylation of Ser3 may be an additional mechanism to protect RGS2 from FBXO44-mediated proteasomal degradation. These findings contribute to elucidating mechanisms regulating steady state levels of RGS2 protein and will inform future studies to develop small molecule RGS2 stabilizers. These would serve as novel leads in pathologies associated with low RGS2 protein levels, such as hypertension, heart failure, and anxiety.
SIGNIFICANCE STATEMENT
E3 ligases provide a novel point of intervention for therapeutic development, but progress is hindered by the lack of available information about specific E3-substrate pairs. Here, we provide molecular detail on the recognition of regulator of G protein signaling protein 2 (RGS2) by its E3 ligase, increasing the potential for rational design of small molecule RGS2 protein stabilizers. These would be clinically useful in pathologies associated with low RGS2 protein levels, such as hypertension, heart failure, and anxiety.
Abstract ID 128664Poster Board 163 Regulator of G protein Signaling (RGS) proteins negatively modulate signaling downstream of G protein-coupled receptors (GPCRs) by accelerating GTP hydrolysis at G α subunits of heterotrimeric G proteins. As such, they represent novel alternatives to current approaches targeting GPCR signaling. While research on RGS proteins and how they are regulated has expanded rapidly in recent years, large gaps still remain regarding specific mechanisms of regulation for the majority of RGS proteins. As a multitude of pathologies have been linked to reduced expression and/or function of one or more RGS proteins, identifying selective mechanisms that regulate individual RGS proteins will open for novel therapeutic strategies. One example is RGS2, which is selective for G αq signaling. Decreased RGS2 levels are implicated in numerous diseases, including cardiovascular disease, asthma, and several types of cancer. Thus, identifying selective means of enhancing RGS2 protein levels would be a viable therapeutic strategy. RGS2 is rapidly degraded through the ubiquitin-proteasomal pathway, and we previously identified F-box only protein 44 (FBXO44) as the substrate recognition component of the E3 ligase responsible for facilitating RGS2 degradation. As such, the RGS2-FBXO44 interaction is a potential target for pharmacological intervention. Detailed information on the FBXO44 recognition site (degron) in RGS2 will aid in structure-based small molecule inhibitor design, as well as in identifying additional FBXO44 targets, which would help predict possible side effects of targeting this interaction. We previously determined that FBXO44 binds RGS2 through a short degron motif near the RGS2 N-terminus. The degron in the only other known FBXO44 substrate, BRCA1, remains unidentified, only narrowed down to the first 167 N-terminal residues. The exact sequence of the RGS2 degron is absent within this region in BRCA1. However, identifying what chemical properties are important for FBXO44-mediated degradation of RGS2 may provide predictive tools for identifying the degron sequence of BRCA1, as well as other potential FBXO44 substrates. Thus, the goal of this study was to dissect the molecular properties for FBXO44 binding of the RGS2 degron. First, we used an in vitro peptide array utilizing systemic residue substitution and identified several amino acid changes that altered binding both positively and negatively. In the absence of structural data, we then created an in silico model using AlphaFold prediction and used molecular dynamics simulations to further examine how this interaction occurs and generate further hypotheses for testing. Finally, we experimentally confirmed our results in cells through co-immunoprecipitation and proteasomal inhibition, using mutated full-length RGS2. Several substitutions were fairly tolerated and resulted in little to no effect on the RGS2-FBXO44 interaction. However, Cys13 is a crucial component of the degron and mutation to a Ser completely abolished the interaction. Furthermore, Arg substitutions at several positions significantly enhanced the interaction with FBXO44, indicating that a positive charge is beneficial. These results provide structural insights into RGS2-FBXO44 binding, which will aid in structure-based drug discovery efforts. It also provides a framework for building a consensus recognition motif for FBXO44, which could aid in identifying more substrates for this understudied F-box protein. R01GM143493
Abstract ID 16497Poster Board 121 Regulators of G protein Signaling (RGS) proteins are negative regulators of G-protein-coupled receptors (GPCRs). They reduce the amplitude and duration of GPCR signaling by accelerating the hydrolysis of GTP to GDP through their GTPase-accelerating protein (GAP) activity. Altered RGS protein function is involved in numerous diseased states. However, the therapeutic regulation of RGS proteins is challenging as they lack binding pockets for small molecules. Therefore, identifying mechanisms that control RGS protein activity and expression could be a potential targeting strategy. A notable example where altering RGS protein activity would be beneficial is asthma, a long-term respiratory disease, characterized by inflammation of the airways, resulting in the obstruction of airflow to the lungs. Aberrant activation of Gαq and downstream signaling contribute to chronic symptoms of asthma. RGS2 is known to be selective for Gαq over other G protein subtypes and has reduced expression levels in asthmatic patients. Low RGS2 protein levels are also implicated in many other diseases such as hypertension, cancer, and heart failure. Indeed, we previously showed that pharmacologically stabilizing RGS2 protein levels inhibits Gαq-mediated signaling and are cardioprotective in a mouse model of cardiac injury, suggesting that enhanced RGS2 protein levels correlate with increased function. Therefore, targeting the mechanisms that regulate RGS2 protein levels would be a feasible therapeutic strategy. RGS2 is rapidly degraded through the ubiquitin-proteasomal system (UPS). In the UPS, ubiquitin molecules are covalently linked to the substrate through the cascade of enzymatic reactions (facilitated by E1: ubiquitin-activating enzyme; E2: ubiquitin-conjugating enzyme; E3 ligase). The ubiquitinated substrate is then recognized and degraded by the 26S proteasomal complex. We recently identified the E3 ligase that recognizes RGS2. This E3 ligase complex consists of Cullin 4B (Cul4B), DNA Damage Binding Protein 1 (DDB1), and F-box Only Protein 44 (FBXO44), where FBXO44 acts as the substrate recognition site for RGS2. Therefore, we hypothesize that inhibiting the RGS2-FBXO44 interaction will lead to enhanced RGS2 levels. To identify small molecule RGS2-FBXO44 interaction inhibitors, we utilized NanoLuc® Binary Technology (NanoBiT) to detect the interaction between RGS2 and FBXO44 in a high-throughput screen (HTS). We developed a HEK-293T cell line stably expressing the RGS2-SmBit and LgBit-FBXO44 and optimized the NanoBit assay for HTS. Using this assay, we screened 1600 compounds (Life Chemicals PPI fragment library) at the Purdue Chemicals Genomics Facility. Following hit confirmation and chemical clustering, the top 20 hits that inhibited the RGS2-FBXO44 protein-protein interaction at least 50% were selected for follow-up. The concentration-dependent activity of these 20 hits was performed to only select compounds inhibiting at least 50% of the interaction and having acceptable inhibition curves (Hill slope 0.5 – 2.0) and potencies (IC50<50μM). We are currently working to further validate these compounds as RGS2 stabilizers via inhibiting the RGS2-FBXO44 protein-protein interaction. We will also detect the effects of the selected hits on Gq-mediated signaling. These hit compounds will be optimized by applying iterative rational drug-design strategies to deliver future lead candidates to stabilize RGS2 protein levels.
Since their discovery in the mid-1990s, regulator of G protein signaling (RGS) proteins have emerged as key regulators of signaling through G protein–coupled receptors. Among the over 20 known RGS proteins, RGS2 has received increasing interest as a potential therapeutic drug target with broad clinical implications. RGS2 is a member of the R4 subfamily of RGS proteins and is unique in that it is selective for Gαq. Despite only having an RGS domain, responsible for the canonical GTPase activating protein activity, RGS2 can regulate additional processes, such as protein synthesis and adenylate cyclase activity, through protein-protein interactions. Here we provide an update of the current knowledge of RGS2 function as it relates to molecular mechanisms of regulation as well as its potential role in regulating a number of physiologic systems and pathologies, including cardiovascular disease and central nervous system disorders, as well as various forms of cancer.
SIGNIFICANCE STATEMENT
Regulator of G protein signaling (RGS) proteins represent an exciting class of novel drug targets. RGS2, in particular, could have broad clinical importance. As more details are emerging on the regulation of RGS2 in various physiological systems, the potential utility of this small protein in therapeutic development is increasing.
The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15537. In addition to this overview, in which are identified 'Other protein targets' which fall outside of the subsequent categorisation, there are six areas of focus: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
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