- A calcium-sensing receptor mutation causing hypocalcemia disrupts a transmembrane salt bridge to activate β-arrestin-biased signaling. [Journal Article]
- SSSci Signal 2018 Feb 20; 11(518)
- The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that signals through Gq/11and Gi/oto stimulate cytosolic calcium (Ca2+i) and mitogen-activated protein kinase (MAPK) signali...
The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that signals through Gq/11and Gi/oto stimulate cytosolic calcium (Ca2+i) and mitogen-activated protein kinase (MAPK) signaling to control extracellular calcium homeostasis. Studies of loss- and gain-of-functionCASRmutations, which cause familial hypocalciuric hypercalcemia type 1 (FHH1) and autosomal dominant hypocalcemia type 1 (ADH1), respectively, have revealed that the CaSR signals in a biased manner. Thus, some mutations associated with FHH1 lead to signaling predominantly through the MAPK pathway, whereas mutations associated with ADH1 preferentially enhance Ca2+iresponses. We report a previously unidentified ADH1-associated R680G CaSR mutation, which led to the identification of a CaSR structural motif that mediates biased signaling. Expressing CaSRR680Gin HEK 293 cells showed that this mutation increased MAPK signaling without altering Ca2+iresponses. Moreover, this gain of function in MAPK activity occurred independently of Gq/11and Gi/oand was mediated instead by a noncanonical pathway involving β-arrestin proteins. Homology modeling and mutagenesis studies showed that the R680G CaSR mutation selectively enhanced β-arrestin signaling by disrupting a salt bridge formed between Arg680and Glu767, which are located in CaSR transmembrane domain 3 and extracellular loop 2, respectively. Thus, our results demonstrate CaSR signaling through β-arrestin and the importance of the Arg680-Glu767salt bridge in mediating signaling bias.
- Endothelial cell protein C receptor-dependent signaling. [Journal Article]
- COCurr Opin Hematol 2018 Feb 15
- CONCLUSIONS: Recent studies provide a mechanistic basis to how EPCR contributes to PAR1-mediated biased signaling. EPCR may play a role in influencing a wide array of biological functions by binding to diverse ligands.
- Arrestins: structural disorder creates rich functionality. [Review]
- PCProtein Cell 2018 Feb 16
- Arrestins are soluble relatively small 44-46 kDa proteins that specifically bind hundreds of active phosphorylated GPCRs and dozens of non-receptor partners. There are binding partners that demonstra...
Arrestins are soluble relatively small 44-46 kDa proteins that specifically bind hundreds of active phosphorylated GPCRs and dozens of non-receptor partners. There are binding partners that demonstrate preference for each of the known arrestin conformations: free, receptor-bound, and microtubule-bound. Recent evidence suggests that conformational flexibility in every functional state is the defining characteristic of arrestins. Flexibility, or plasticity, of proteins is often described as structural disorder, in contrast to the fixed conformational order observed in high-resolution crystal structures. However, protein-protein interactions often involve highly flexible elements that can assume many distinct conformations upon binding to different partners. Existing evidence suggests that arrestins are no exception to this rule: their flexibility is necessary for functional versatility. The data on arrestins and many other multi-functional proteins indicate that in many cases, "order" might be artificially imposed by highly non-physiological crystallization conditions and/or crystal packing forces. In contrast, conformational flexibility (and its extreme case, intrinsic disorder) is a more natural state of proteins, representing true biological order that underlies their physiologically relevant functions.
- Impaired β-arrestin recruitment and reduced desensitization by non-catechol agonists of the D1 dopamine receptor. [Journal Article]
- NCNat Commun 2018 Feb 14; 9(1):674
- Selective activation of dopamine D1 receptors (D1Rs) has been pursued for 40 years as a therapeutic strategy for neurologic and psychiatric diseases due to the fundamental role of D1Rs in motor funct...
Selective activation of dopamine D1 receptors (D1Rs) has been pursued for 40 years as a therapeutic strategy for neurologic and psychiatric diseases due to the fundamental role of D1Rs in motor function, reward processing, and cognition. All known D1R-selective agonists are catechols, which are rapidly metabolized and desensitize the D1R after prolonged exposure, reducing agonist response. As such, drug-like selective D1R agonists have remained elusive. Here we report a novel series of selective, potent non-catechol D1R agonists with promising in vivo pharmacokinetic properties. These ligands stimulate adenylyl cyclase signaling and are efficacious in a rodent model of Parkinson's disease after oral administration. They exhibit distinct binding to the D1R orthosteric site and a novel functional profile including minimal receptor desensitization, reduced recruitment of β-arrestin, and sustained in vivo efficacy. These results reveal a novel class of D1 agonists with favorable drug-like properties, and define the molecular basis for catechol-specific recruitment of β-arrestin to D1Rs.
- Site-specific polyubiquitination differentially regulates parathyroid hormone receptor-initiated MAPK signaling and cell proliferation. [Journal Article]
- JBJ Biol Chem 2018 Feb 14
- G protein-coupled receptor (GPCR) signaling and trafficking are essential for cellular function and regulated by phosphorylation, β-arrestin, and ubiquitination. The GPCR parathyroid hormone receptor...
G protein-coupled receptor (GPCR) signaling and trafficking are essential for cellular function and regulated by phosphorylation, β-arrestin, and ubiquitination. The GPCR parathyroid hormone receptor (PTHR) exhibits time-dependent reversible ubiquitination. The exact ubiquitination sites in PTHR are unknown, but extend upstream of its intracellular tail. Here, using tandem MS, we identified Lys-388 in the third loop and Lys-484 in the C-terminal tail as primary ubiquitination sites in PTHR. We found that PTHR ubiquitination requires β-arrestin and does not display a preference for β-arrestin1 or 2. PTH stimulated PTHR phosphorylation at Thr-387/Thr-392 and within the Ser-489/Ser-493 region. Such phosphorylation events may recruit β-arrestin, and we observed that chemically or genetically blocking PTHR phosphorylation inhibits its ubiquitination. Specifically, Ala replacement at Thr-387/Thr-392 suppressed β-arrestin binding and inhibited PTHR ubiquitination, suggesting that PTHR phosphorylation and ubiquitination are interdependent. Of note, Lys-deficient PTHR mutants promoted normal cAMP formation, but exhibited differential mitogen-activated protein kinase (MAPK) signaling. Lys-deficient PTHR triggered early onset and delayed ERK1/2 signaling compared with wild-type PTHR. Moreover, ubiquitination of Lys-388 and Lys-484 in wild-type PTHR strongly decreased p38 signaling, whereas Lys-deficient PTHR retained signaling comparable to unstimulated wild-type PTHR. Lys-deficient, ubiquitination-refractory PTHR reduced cell proliferation and increased apoptosis. However, elimination of all 11 Lys residues in PTHR did not affect its internalization and recycling. These results pinpoint the ubiquitinated Lys residues in PTHR controlling MAPK signaling and cell proliferation and survival. Our findings suggest new opportunities for targeting PTHR ubiquitination to regulate MAPK signaling or manage PTHR-related disorders.
- hMENA is a key regulator in endothelin-1/β-arrestin1-induced invadopodial function and metastatic process. [Journal Article]
- PNProc Natl Acad Sci U S A 2018 Feb 08
- Aberrant activation of endothelin-1 receptors (ET-1R) elicits pleiotropic effects relevant for tumor progression. The network activated by this receptor might be finely, spatially, and temporarily or...
Aberrant activation of endothelin-1 receptors (ET-1R) elicits pleiotropic effects relevant for tumor progression. The network activated by this receptor might be finely, spatially, and temporarily orchestrated by β-arrestin1 (β-arr1)-driven interactome. Here, we identify hMENA, a member of the actin-regulatory protein ENA/VASP family, as an interacting partner of β-arr1, necessary for invadopodial function downstream of ET-1R in serous ovarian cancer (SOC) progression. ET-1R activation by ET-1 up-regulates expression of hMENA/hMENAΔv6 isoforms through β-arr1, restricted to mesenchymal-like invasive SOC cells. The interaction of β-arr1 with hMENA/hMENAΔv6 triggered by ET-1 leads to activation of RhoC and cortactin, recruitment of membrane type 1-matrix metalloprotease, and invadopodia maturation, thereby enhancing cell plasticity, transendothelial migration, and the resulting spread of invasive cells. The treatment with the ET-1R antagonist macitentan impairs the interaction of β-arr1 with hMENA and inhibits invadopodial maturation and tumor dissemination in SOC orthotopic xenografts. Finally, high ETAR/hMENA/β-arr1 gene expression signature is associated with a poor prognosis in SOC patients. These data define a pivotal function of hMENA/hMENAΔv6 for ET-1/β-arr1-induced invadopodial activity and ovarian cancer progression.
- G protein pre-assembly rescues efficacy of W6.48toggle mutations in neuropeptide Y2receptor. [Journal Article]
- MPMol Pharmacol 2018 Feb 07
- Ligand binding and pathway-specific activation of G protein-coupled receptors (GPCRs) is currently study with great effort. Individual answers may depend on the nature of the ligands and the effector...
Ligand binding and pathway-specific activation of G protein-coupled receptors (GPCRs) is currently study with great effort. Individual answers may depend on the nature of the ligands and the effector pathway. Recently, we have presented a detailed model of NPY bound to the Y2R (Kaiser et al., 2015). Accordingly, the C-terminal part of the peptide binds deeply in the transmembrane bundle and brings the side chain of the most essential Y36in close proximity to W6.48Here, we investigate the role of this interaction for ligand binding and activation of this receptor. BRET sensors were used for detailed investigation of effector coupling, and led to the identification of pre-assembly of the Y2R-Gicomplex. It further confirmed ligand-dependent recruitment of arrestin3. Using equally sensitive readouts for Giactivation and arrestin recruitment as well as quantification with operational models of agonism allowed us to identify a strong inherent bias for Giactivation over arrestin3 recruitment for the wild type receptor. By systematic mutagenesis, we found that W6.48does not contribute to the binding affinity, but acts as allosteric connector to couple ligand binding to Gi-activation and arrestin3 recruitment. However, even mutagenesis to a small threonine did not lead to a complete loss of signaling. Interestingly, signaling was restored to wild type levels by ligands that contain a naphtylalanine as the C-terminal residue instead of Y36Steric and polar contributions of W6.48for the activation of the receptor are discussed in the context of different mechanisms of G protein coupling and arrestin recruitment.
- T Cell Expression of C5a Receptor 2 Augments Murine Regulatory T Cell (TREG) Generation and TREG-Dependent Cardiac Allograft Survival. [Journal Article]
- JIJ Immunol 2018 Feb 07
- C5aR2 (C5L2/gp77) is a seven-transmembrane spanning receptor that binds to C5a but lacks motifs essential for G protein coupling and associated signal transduction. C5aR2 is expressed on immune cells...
C5aR2 (C5L2/gp77) is a seven-transmembrane spanning receptor that binds to C5a but lacks motifs essential for G protein coupling and associated signal transduction. C5aR2 is expressed on immune cells, modulates various inflammatory diseases in mice, and has been shown to facilitate murine and human regulatory T cell (TREG) generation in vitro. Whether and how C5aR2 impacts in vivo TREGgeneration and pathogenic T cell-dependent disease models have not been established. In this article, we show that murine T cells express and upregulate C5aR2 during induced TREG(iTREG) generation and that the absence of T cell-expressed C5aR2 limits in vivo iTREGgeneration following adoptive transfer of naive CD4+T cells intoRag1-/-recipients. Using newly generated C5aR2-transgenic mice, we show that overexpression of C5aR2 in naive CD4+T cells augments in vivo iTREGgeneration. In a model of TREG-dependent cardiac allograft survival, recipient C5aR2 deficiency accelerates graft rejection associated with lower TREG/effector T cell ratios, whereas overexpression of C5aR2 in immune cells prolongs graft survival associated with an increase in TREG/effector T cell ratios. T cell-expressed C5aR2 modulates TREGinduction without altering effector T cell proliferation or cytokine production. Distinct from reported findings in neutrophils and macrophages, TREG-expressed C5aR2 does not interact with β-arrestin or inhibit ERK1/2 signaling. Rather, cumulative evidence supports the conclusion that C5aR2 limits C5aR1-initiated signals known to inhibit TREGinduction. Together, the data expand the role of C5aR2 in adaptive immunity by providing in vivo evidence that T cell-expressed C5aR2 physiologically modulates iTREGgeneration and iTREG-dependent allograft survival.
- The essential role of G protein-coupled receptor (GPCR) signaling in regulating T cell immunity. [Journal Article]
- IIImmunopharmacol Immunotoxicol 2018 Feb 12; :1-6
- CONCLUSIONS: GPCR signaling is critical in regulating T cell immunity.
New Search Next
- The mechanisms behind decreased internalization of angiotensin II type 1 receptor. [Review]
- VPVascul Pharmacol 2018 Feb 06
- The internalization of angiotensin II type 1 receptor (AT1R) plays an important role in maintaining cardiovascular homeostasis. Decreased receptor internalization is closely related to cardiovascular...
The internalization of angiotensin II type 1 receptor (AT1R) plays an important role in maintaining cardiovascular homeostasis. Decreased receptor internalization is closely related to cardiovascular diseases induced by the abnormal activation of AT1R, such as hypertension. However, the mechanism behind reduced AT1R internalization is not fully understood. This review focuses on four parts of the receptor internalization process (the combination of agonists and receptors, receptor phosphorylation, endocytosis, and recycling) and summarizes the possible mechanisms by which AT1R internalization is reduced based on these four parts of the process. (1) The agonist has a large molecular weight or a stronger ability to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5) P2), which can increase the consumption of PtdIns (4,5) P2. (2) AT1R phosphorylation is weakened because of an abnormal function of phosphorylated kinase or changes in phospho-barcoding and GPCR-β-arrestin complex conformation. (3) The abnormal formation of vesicles or AT1R heterodimers with fewer endocytic receptors results in less AT1R endocytosis. (4) The enhanced activity and upregulated expression of small GTP-binding protein 4 (Rab4) and 11 (Rab11), which regulate receptor recycling, and phosphatidylinositol 3-kinase increase AT1R recycling. In addition, lower expression of AT1R-associated protein (ATRAP) or higher expression of AT1R-associated protein 1 (ARAP1) can reduce receptor internalization.