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Cell Communication and Signaling : CCS Sep 2023G protein-coupled receptor heteromerization is believed to exert dynamic regulatory impact on signal transduction. CXC chemokine receptor 4 (CXCR4) and its ligand...
BACKGROUND
G protein-coupled receptor heteromerization is believed to exert dynamic regulatory impact on signal transduction. CXC chemokine receptor 4 (CXCR4) and its ligand CXCL12, both of which are overexpressed in many cancers, play a pivotal role in metastasis. Likewise, lysophosphatidic acid receptor 1 (LPA) is implicated in cancer cell proliferation and migration. In our preliminary study, we identified LPA as a prospective CXCR4 interactor. In the present study, we investigated in detail the formation of the CXCR4-LPA heteromer and characterized the unique molecular features and function of this heteromer.
METHODS
We employed bimolecular fluorescence complementation, bioluminescence resonance energy transfer, and proximity ligation assays to demonstrate heteromerization between CXCR4 and LPA. To elucidate the distinctive molecular characteristics and functional implications of the CXCR4-LPA heteromer, we performed various assays, including cAMP, BRET for G protein activation, β-arrestin recruitment, ligand binding, and transwell migration assays.
RESULTS
We observed that CXCR4 forms heteromers with LPA in recombinant HEK293A cells and the human breast cancer cell line MDA-MB-231. Coexpression of LPA with CXCR4 reduced CXCL12-mediated cAMP inhibition, ERK activation, Gα activation, and β-arrestin recruitment, while CXCL12 binding to CXCR4 remained unaffected. In contrast, CXCR4 had no impact on LPA-mediated signaling. The addition of lysophosphatidic acid (LPA) further hindered CXCL12-induced Gα recruitment to CXCR4. LPA or alkyl-OMPT inhibited CXCL12-induced migration in various cancer cells that endogenously express both CXCR4 and LPA. Conversely, CXCL12-induced calcium signaling and migration were increased in LPAR1 knockout cells, and LPA-selective antagonists enhanced CXCL12-induced Gα signaling and cell migration in the parental MDA-MB-231 cells but not in LPA-deficient cells. Ultimately, complete inhibition of cell migration toward CXCL12 and alkyl-OMPT was only achieved in the presence of both CXCR4 and LPA antagonists.
CONCLUSIONS
The presence and impact of CXCR4-LPA heteromers on CXCL12-induced signaling and cell migration have been evidenced across various cell lines. This discovery provides crucial insights into a valuable regulatory mechanism of CXCR4 through heteromerization. Moreover, our findings propose a therapeutic potential in combined CXCR4 and LPA inhibitors for cancer and inflammatory diseases associated with these receptors, simultaneously raising concerns about the use of LPA antagonists alone for such conditions. Video Abstract.
Topics: Humans; Calcium Signaling; Cell Movement; Chemokine CXCL12; Ligands; Prospective Studies; Receptors, CXCR4; Receptors, Lysophosphatidic Acid
PubMed: 37749552
DOI: 10.1186/s12964-023-01261-7 -
Cell Reports Nov 2023Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Adhesion G protein-coupled receptors (aGPCRs) have attracted interest for their potential...
Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Adhesion G protein-coupled receptors (aGPCRs) have attracted interest for their potential as treatment targets. Here, we show that CD97 (ADGRE5) is the most promising aGPCR target in GBM, by virtue of its de novo expression compared to healthy brain tissue. CD97 knockdown or knockout significantly reduces the tumor initiation capacity of patient-derived GBM cultures (PDGCs) in vitro and in vivo. We find that CD97 promotes glycolytic metabolism via the mitogen-activated protein kinase (MAPK) pathway, which depends on phosphorylation of its C terminus and recruitment of β-arrestin. We also demonstrate that THY1/CD90 is a likely CD97 ligand in GBM. Lastly, we show that an anti-CD97 antibody-drug conjugate selectively kills tumor cells in vitro. Our studies identify CD97 as a regulator of tumor metabolism, elucidate mechanisms of receptor activation and signaling, and provide strong scientific rationale for developing biologics to target it therapeutically in GBM.
Topics: Humans; Glioblastoma; Phosphorylation; Receptors, G-Protein-Coupled; Signal Transduction
PubMed: 37938973
DOI: 10.1016/j.celrep.2023.113374 -
British Journal of Pharmacology Mar 2024More than 30 years after their discovery, arrestins are recognised multiprotein scaffolds that play essential roles in G protein-coupled receptor (GPCR) regulation and... (Review)
Review
More than 30 years after their discovery, arrestins are recognised multiprotein scaffolds that play essential roles in G protein-coupled receptor (GPCR) regulation and signalling. Originally named for their capacity to hinder GPCR coupling to G proteins and facilitate receptor desensitisation, arrestins have emerged as key hubs for a myriad of other functions, including receptor internalisation and scaffolding of signalling complexes. Recent structural studies have started to provide snapshots of the complexes formed by GPCRs and arrestins, supporting a wealth of biochemical data delineating the molecular determinants of such interactions. Furthermore, biophysical techniques have also provided key information with regards to the basal and active conformations of arrestins, and how these are affected upon GPCR activation. Here, we review the most recent advances on our understanding of GPCR-arrestin complexes, from structure to interactions of arrestins with the lipid bilayer and other proteins. We also present an updated view on the development of tools to study the conformational flexibility of arrestins, with the potential to provide experimental data to describe the dynamic models of arrestin activation.
PubMed: 38479842
DOI: 10.1111/bph.16331 -
Journal of Neurochemistry Jan 2024Arrestins were discovered for their role in homologous desensitization of G-protein-coupled receptors (GPCRs). Later non-visual arrestins were shown to regulate several...
Arrestins were discovered for their role in homologous desensitization of G-protein-coupled receptors (GPCRs). Later non-visual arrestins were shown to regulate several signaling pathways. Some of these pathways require arrestin binding to GPCRs, the regulation of others is receptor independent. Here, we demonstrate that arrestin-3 binds the E3 ubiquitin ligase parkin via multiple sites, preferentially interacting with its RING0 domain. Identification of the parkin domains involved suggests that arrestin-3 likely relieves parkin autoinhibition and/or stabilizes the enzymatically active "open" conformation of parkin. Arrestin-3 binding enhances ubiquitination by parkin of the mitochondrial protein mitofusin-1 and facilitates parkin-mediated mitophagy in HeLa cells. Furthermore, arrestin-3 and its mutant with enhanced parkin binding rescue mitofusin-1 ubiquitination and mitophagy in the presence of the Parkinson's disease-associated R275W parkin mutant, which is defective in both functions. Thus, modulation of parkin activity via arrestin-3 might be a novel strategy of anti-parkinsonian therapy.
PubMed: 38196269
DOI: 10.1111/jnc.16043 -
Nature May 2024The µ-opioid receptor (µOR) is an important target for pain management and molecular understanding of drug action on µOR will facilitate the development of better...
The µ-opioid receptor (µOR) is an important target for pain management and molecular understanding of drug action on µOR will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance and single-molecule fluorescence resonance energy transfer, how ligand-specific conformational changes of µOR translate into a broad range of intrinsic efficacies at the transducer level. We identify several conformations of the cytoplasmic face of the receptor that interconvert on different timescales, including a pre-activated conformation that is capable of G-protein binding, and a fully activated conformation that markedly reduces GDP affinity within the ternary complex. Interaction of β-arrestin-1 with the μOR core binding site appears less specific and occurs with much lower affinity than binding of G.
Topics: Humans; beta-Arrestin 1; Binding Sites; Fluorescence Resonance Energy Transfer; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine Diphosphate; Ligands; Models, Molecular; Protein Binding; Protein Conformation; Receptors, Opioid, mu; Single Molecule Imaging
PubMed: 38600384
DOI: 10.1038/s41586-024-07295-2 -
Current Protocols Sep 2023Purified arrestin proteins are necessary for biochemical, biophysical, and structural studies of these versatile regulators of cell signaling. Described herein is a...
Purified arrestin proteins are necessary for biochemical, biophysical, and structural studies of these versatile regulators of cell signaling. Described herein is a basic protocol for arrestin expression in Escherichia coli and purification of tag-free wild-type and mutant arrestins. The method includes ammonium sulfate precipitation of arrestins from cell lysates, followed by Heparin-Sepharose chromatography. Depending on the arrestin type and/or mutations, the next step is Q-Sepharose or SP-Sepharose chromatography. In many cases, the nonbinding column is used as a filter to bind contaminants without retaining arrestin. In some cases, both chromatographic steps must be performed sequentially to achieve high purity. Purified arrestins can be concentrated up to 10 mg/ml, remain fully functional, and withstand several cycles of freezing and thawing, provided that the overall salt concentration is maintained at or above physiological levels. © 2023 Wiley Periodicals LLC. Basic Protocol: Large-scale expression and purification of arrestins Alternate Protocol: Purification of arrestin-3 and truncated form of arrestin-1-(1-378) Support Protocol: Small-scale test expression of wild-type and mutant arrestins in E. coli.
Topics: Arrestin; Escherichia coli; Arrestins; Ammonium Sulfate; Biophysics
PubMed: 37671938
DOI: 10.1002/cpz1.832 -
Cell Death & Disease Jul 2023β-arrestin 2 (ARRB2) is functionally implicated in cancer progression via various signaling pathways. However, its role in lung cancer remains unclear. To obtain...
β-arrestin 2 (ARRB2) is functionally implicated in cancer progression via various signaling pathways. However, its role in lung cancer remains unclear. To obtain clinical insight on its function in lung cancer, microarray data from lung tumor tissues (LTTs) and matched lung normal tissues (mLNTs) of primary non-small cell lung cancer (NSCLC) patients (n = 37) were utilized. ARRB2 expression levels were markedly decreased in all 37 LTTs compared to those in matched LNTs of NSCLC patients. They were significantly co-related to enrichment gene sets associated with oncogenic and cancer genes. Importantly, Gene Set Enrichment Analysis (GSEA) between three LTTs with highly down-regulated ARRB2 and three LTTs with lowly down-regulated ARRB2 revealed significant enrichments related to toll-like receptor (TLR) signaling and autophagy genes in three LTTs with highly down-regulated ARRB2, suggesting that ARRB2 was negatively involved in TLR-mediated signals for autophagy induction in lung cancer. Biochemical studies for elucidating the molecular mechanism revealed that ARRB2 interacted with TNF receptor-associated factor 6 (TRAF6) and Beclin 1 (BECN1), thereby inhibiting the ubiquitination of TRAF6-TAB2 to activate NF-κB and TRAF6-BECN1 for autophagy stimulated by TLR3 and TLR4, suggesting that ARRB2 could inhibit the TRAF6-TAB2 signaling axis for NF-κB activation and TRAF6-BECN1 signaling axis for autophagy in response to TLR3 and TLR4. Notably, ARRB2-knockout (ARRB2KO) lung cancer cells exhibited marked enhancements of cancer migration, invasion, colony formation, and proliferation in response to TLR3 and TLR4 stimulation. Altogether, our current data suggest that ARRB2 can negatively regulate lung cancer progression by inhibiting TLR3- and TLR4-induced autophagy.
Topics: Humans; NF-kappa B; TNF Receptor-Associated Factor 6; Lung Neoplasms; Toll-Like Receptor 4; Toll-Like Receptor 3; beta-Arrestin 2; Carcinoma, Non-Small-Cell Lung; Toll-Like Receptors; Lung; Autophagy; Adaptor Proteins, Signal Transducing
PubMed: 37443143
DOI: 10.1038/s41419-023-05945-3 -
Proceedings of the National Academy of... Jul 2023E3 ubiquitin ligase Mdm2 facilitates β-arrestin ubiquitination, leading to the internalization of G protein-coupled receptors (GPCRs). In this process, β-arrestins...
E3 ubiquitin ligase Mdm2 facilitates β-arrestin ubiquitination, leading to the internalization of G protein-coupled receptors (GPCRs). In this process, β-arrestins bind to Mdm2 and recruit it to the receptor; however, the molecular architecture of the β-arrestin-Mdm2 complex has not been elucidated yet. Here, we identified the β-arrestin-binding region (ABR) on Mdm2 and solved the crystal structure of β-arrestin1 in complex with Mdm2 peptide. The acidic residues of Mdm2 bind to the positively charged concave side of the β-arrestin1 N-domain. The C-tail of β-arrestin1 is still bound to the N-domain, indicating that Mdm2 binds to the inactive state of β-arrestin1, whereas the phosphorylated C-terminal tail of GPCRs binds to activate β-arrestins. The overlapped binding site of Mdm2 and GPCR C-tails on β-arrestin1 suggests that the binding of GPCR C-tails might trigger the release of Mdm2. Moreover, hydrogen/deuterium exchange experiments further show that Mdm2 binding to β-arrestin1 induces the interdomain interface to be more dynamic and uncouples the IP-induced oligomer of β-arrestin1. These results show how the E3 ligase, Mdm2, interacts with β-arrestins to promote the internalization of GPCRs.
Topics: beta-Arrestins; Ubiquitin-Protein Ligases; Arrestins; beta-Arrestin 1; Ubiquitination; Receptors, G-Protein-Coupled; beta-Arrestin 2; Phosphorylation
PubMed: 37399373
DOI: 10.1073/pnas.2301934120 -
The Journal of Biological Chemistry Dec 2023G protein-coupled receptor (GPCR) signaling and trafficking are regulated by multiple mechanisms, including posttranslational modifications such as ubiquitination by E3...
G protein-coupled receptor (GPCR) signaling and trafficking are regulated by multiple mechanisms, including posttranslational modifications such as ubiquitination by E3 ubiquitin ligases. E3 ligases have been linked to agonist-stimulated ubiquitination of GPCRs via simultaneous binding to βarrestins. In addition, βarrestins have been suggested to assist E3 ligases for ubiquitination of key effector molecules, yet mechanistic insight is lacking. Here, we developed an in vitro reconstituted system and show that βarrestin1 (βarr1) serves as an adaptor between the effector protein signal-transducing adaptor molecule 1 (STAM1) and the E3 ligase atrophin-interacting protein 4. Via mass spectrometry, we identified seven lysine residues within STAM1 that are ubiquitinated and several types of ubiquitin linkages. We provide evidence that βarr1 facilitates the formation of linear polyubiquitin chains at lysine residue 136 on STAM1. This lysine residue is important for stabilizing the βarr1:STAM1 interaction in cells following GPCR activation. Our study identifies atrophin-interacting protein 4 as only the second E3 ligase known to conjugate linear polyubiquitin chains and a possible role for linear ubiquitin chains in GPCR signaling and trafficking.
Topics: Lysine; Polyubiquitin; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination; beta-Arrestin 1
PubMed: 37981209
DOI: 10.1016/j.jbc.2023.105474 -
Molecular Cancer Research : MCR Dec 2023Constraints on the p53 tumor suppressor pathway have long been associated with the progression, therapeutic resistance, and poor prognosis of melanoma, the most...
UNLABELLED
Constraints on the p53 tumor suppressor pathway have long been associated with the progression, therapeutic resistance, and poor prognosis of melanoma, the most aggressive form of skin cancer. Likewise, the insulin-like growth factor type 1 receptor (IGF1R) is recognized as an essential coordinator of transformation, proliferation, survival, and migration of melanoma cells. Given that β-arrestin (β-arr) system critically governs the anti/pro-tumorigenic p53/IGF1R signaling pathways through their common E3 ubiquitin-protein ligase MDM2, we explore whether unbalancing this system downstream of IGF1R can enhance the response of melanoma cells to chemotherapy. Altering β-arr expression demonstrated that both β-arr1-silencing and β-arr2-overexpression (-β-arr1/+β-arr2) facilitated nuclear-to-cytosolic MDM2 translocation accompanied by decreased IGF1R expression, while increasing p53 levels, resulting in reduced cell proliferation/survival. Imbalance towards β-arr2 (-β-arr1/+β-arr2) synergizes with the chemotherapeutic agent, dacarbazine, in promoting melanoma cell toxicity. In both 3D spheroid models and in vivo in zebrafish models, this combination strategy, through dual IGF1R downregulation/p53 activation, limits melanoma cell growth, survival and metastatic spread. In clinical settings, analysis of the TCGA-SKCM patient cohort confirms β-arr1-/β-arr2+ imbalance as a metastatic melanoma vulnerability that may enhance therapeutic benefit. Our findings suggest that under steady-state conditions, IGF1R/p53-tumor promotion/suppression status-quo is preserved by β-arr1/2 homeostasis. Biasing this balance towards β-arr2 can limit the protumorigenic IGF1R activities while enhancing p53 activity, thus reducing multiple cancer-sustaining mechanisms. Combined with other therapeutics, this strategy improves patient responses and outcomes to therapies relying on p53 or IGF1R pathways.
IMPLICATIONS
Altogether, β-arrestin system bias downstream IGF1R is an important metastatic melanoma vulnerability that may be conductive for therapeutic benefit.
Topics: Animals; Humans; beta-Arrestins; Arrestins; Tumor Suppressor Protein p53; Zebrafish; beta-Arrestin 1; Protein Isoforms; Melanoma; beta-Arrestin 2; Cell Line, Tumor; Receptor, IGF Type 1
PubMed: 37584671
DOI: 10.1158/1541-7786.MCR-22-0871